Book of Abstracts. Albany 2019: the 20Th Conversation

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS 2019, VOL. 37, NO. S1, 1–92 https://doi.org/10.1080/07391102.2019.1604468

Book of Abstracts. Albany 2019: The 20th Conversation

Abstracts

1. The structure and dynamics of 2. Cryo-EM and drug discovery biomolecules in their native states Sriram Subramaniam Joachim Frank Djavad Mowafaghian Centre for Brain Health, 2215 Wesbrook Mall, Department of Biochemistry and Molecular Biophysics, Columbia University of British Columbia, Vancouver, BC, V6T 1Z3, Canada University, New York, NY 10032, USA [email protected] [email protected] Cryo-EM has transitioned rapidly in the last few years from being The aim of structural biology is to explain life processes in amethodthatwasonlycapableofhandlingasmallsubsetof terms of macromolecular interactions in the cell. These inter- cryo-EM worthy specimens, to one that is useful for analysis of a actions typically involve more than two partners, and can very large spectrum of protein complexes (Subramaniam, 2019). run up to dozens. A full description will need to characterize This transition of cryo-EM from being a technology that was billed all structures on the atomic level, and the way these struc- as a tool to analyze large and/or highly symmetric specimens, to tures change in the process. Because of the crowded envir- one that can successfully tackle a range of proteins and protein onment of the cell, such characterization is presently (but complexes of broad general interest has been transformative. The see below) only possible when the group of interacting mol- structures of an impressive number of proteins, small and large, ecules (often organized into processive ‘molecular machines’) sometimes with extensive conformational spread, have been suc- is isolated and studied in vitro. While X-ray crystallography cessfully analyzed by cryo-EM. Many of these protein complexes has provided structures of a large number of molecular may never be coaxed to produce well-ordered crystals for study structures, the need for crystals diffracting to high resolution by X-ray crystallography. It is important to recognize that in has severely limited the number of supramolecular assem- almost every instance, these selected successes in the application blies and the range of conformers that can be studied with of cryo-EM rest on decades of advances in biochemistry, biophys- this technique. Single-particle cryo-electron microscopy is ics and protein science that laid the necessary groundwork. Nor about to fill this gap, allowing functional processes to be can we overlook the fact that the landscape of macromolecular studied in great detail without imposing restraints on the entities that are still intractable to analysis by cryo-EM remains structures. There are many examples now for this expansion immense. Yet, the future looks bright, and there is every reason of Structural Biology toward a full characterization of a func- to hope that an increasingly complex array of biological assem- tional process. Future developments of single-particle cryo- blies will be tackled by cryo-EM. EM include the study of short-lived intermediates in a none- Work in my laboratory has focused on the application of quilibrium system by time-resolved techniques, and the char- cryo-EM to small dynamic protein assemblies, with particular acterization of continuous structural changes using data emphasis on its use for drug discovery and therapeutic applica- mining from large ensembles of molecule images. It is very tions, and for the development of image processing methods to interesting and promising that another technique of cryo-EM, improve resolution (Banerjee et al., 2016; Chittori et al., 2018; cryo-electron tomography of FIB-milled cell sections, has Guo et al., 2017; Kang et al., 2018; Meyerson et al., 2016). In my started to contribute information about processes even presentation, I will discuss recent examples where we have used within the cellular context. Distinct ribosome states, for cryo-EM in this context to study metabolic enzyme complexes, instance, have already been identified and localized by sub- ion channels, nucleic-acid protein complexes and intact viruses. tomogram averaging. With this advance, we get closer to the fulfillment of the most ambitious aim of Structural Biology, References the visualization and interpretation of molecular interactions Banerjee, S., Bartesaghi, A., Merk, A., Rao, P., Bulfer, S. L., Yan, Y., Green, in situ. N., … Subramaniam, S. (2016). 2.3 Å resolution cryo-EM structure of 2 BOOK OF ABSTRACTS. ALBANY 2019: THE 20TH CONVERSATION

human p97 and mechanism of allosteric inhibition. Science, 351(6275), 871–875. 4. Distance sensitive D-loop dynamics Chittori, S., Hong, J., Saunders, H., Feng, H., Ghirlando, R., Kelly, A. E., … and near-atomic resolution of F-actin Subramaniam, S. (2018). Structural mechanisms of centromeric chromosome recognition by the kinetochore protein CENP-N. Science, phalloidin structure by cryoEM 359(6373), 339–343. Guo, T., Bartesaghi, A., Yang, H., Falconieri, V., Rao, P., Merk, A., … Sanchaita Dasa , Peng Gec,d , Zeynep A. Oztug Subramaniam, S. (2017). Cryo-EM structures reveal mechanism and a Ã Ãa c,d inhibition of DNA targeting by a CRISPR-Cas surveillance complex. Durer , Elena E. Grintsevich ,Z.HongZhou and Ã a,b Cell, 171(2), 414–426. Emil Reisler Kang, Y., Kuybeda, O., de Waal, P. W., Mukherjee, S., Van Eps, N., Dutka, aDepartment of Chemistry and Biochemistry, UCLA, Los Angeles, P., … Xu, H. E. (2018). Cryo-EM structure of human rhodopsin bound CA, USA; bMolecular Biology Institute, UCLA, Los Angeles, CA, to an inhibitory G protein. Nature, 558(7711), 553–558. USA; cDepartment of Microbiology, Immunology and Molecular Meyerson, J. R., Chittori, S., Merk, A., Rao, P., Han, T. H., Serpe, M., … Genetics, University of California, Los Angeles (UCLA), Los Angeles, Subramaniam, S. (2016). Structural basis of kainate subtype glutamate CA, USA; dCalifornia NanoSystems Institute (CNSI), UCLA, Los receptor desensitization. Nature, 537(7621), 567–571. Angeles, CA, USA [email protected] Subramaniam, S. (2019). The cryo-EM revolution: Fueling the next phase. Contributed equally. IUCrJ, 6(Pt 1), 1–2. Ã Actin is indispensable for eukaryotic cells. Therefore, molecular details of F-actin structure and dynamics are essential for our understanding of its key cellular functions. It is well established 3. Deadly spiders and scary zombies— that nucleotide-bound state of F-actin (ATP/ADP-Pi or ADP) Not a Halloween story a near-atomic defines its dynamic properties, stability and interactions with regulatory factors. Previous studies also indicate that an resolution glance into the CNS innately flexible DNase I binding loop (D-loop, residues 40–50) plays a major role in such conformational dynamics. Recent Moran Shalev-Benami advances in cryoEM provide high-resolution information about Department of Structural Biology, Weizmann Institute of Science, nucleotide bound states of actin, however, the role of D loop in Rehovot, 7610001, Israel [email protected] monomer to polymer transition still remains elusive. Intriguingly, phalloidin, a ‘gold standard’ for actin staining in Synapses are specialized junctions between neurons that trans- vivo and in vitro, is also known to stabilize actin filaments and mit and compute information in the central nervous system affect the D-loop. Specifically, it can also convert polymeriza- (CNS). The establishment, properties, and dynamics of synapses tion-defective D loop mutants into stable polymers, thereby are governed by diverse trans-synaptic signaling molecules that bypassing D loop dependency. By utilizing a multidisciplinary communicate their signal via multifarious interactions with their approach of mutational disulfide crosslinking, light scattering synaptic partners. Mutations in the genes encoding these mole- measurements and cryoEM, we probe the structural mecha- cules have been associated with diverse neuropsychiatric and nisms that govern D-loop transitions in actin dynamics and neurodegenerative disorders thus highlighting their crucial how phalloidin stabilizes F-actin. Our biochemical data provides importance for normal brain function. Over the past few deca- a molecular ruler-based model of how intraprotomer distance des, tremendous efforts have been made to structurally char- between two D-loop residues facilitates a transition from G to acterize the trans-synaptic signaling molecules as well as their F-actin and vice versa. Additionally, we report the first 3.7 Å interacting partners. Nevertheless, their low expression levels resolution structure of Phalloidin bound F-actin in the ADP-Pi and high structural complexity has posed a great challenge to state. Our structure supports (i) the role of methylation of His traditional structural methods, such as NMR and X-ray crystal- 73 on actin in Pi binding, (ii) shows that phalloidin inhibits Pi lography. Advances in single particle electron cryo-microscopy (cryo-EM) now allow the capture of such complexed macromolecular assemblies in great details, providing snapshots of these fascinating molecules in action. Here we present the near-atomic resolution structures of two such synaptic components, the cannabinoid receptor 1 (CB1R), and teneurin, two transmembrane receptors that are primarily expressed in neurons and are considered to mediate various functions in syn- apse formation and maintenance. The structures provide a high-resolution glance into the receptors’ architectures and present structural insights into the interaction with their inter- and intra-cellular partners. Our results highlight cryo-EM as a highly effective alternative approach for studying challenging macromolecular machineries while providing a framework for Figure 1. CryoEM structure of ADP-BeFx-F-actin-phalloidin. (a) Overall structure elucidating the mechanisms of action of trans-synaptic signal- of ADP-BeFx-F-actin-phalloidin at 3.7 Å resolution, fitted with its atomic model. Red characters mark the relative position of actin subunits in the filament. (b) ing molecules that could in turn be used for design of future Representative regions (marked by amino acid numbers) of the density map fit- novel therapeutics. ted with their atomic models, showing the quality of the map (Figure 2). JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS VOL. 37, SUPPLEMENT, 2019 3

Figure 2. Phalloidin can rescue the polymerization of oxidized double D-loop mutant F-C41C45. (a) Yeast actin mutant C41C45, with two cyteines in the D-loop, was polymerized in the presence of 2mM MgCl2 as monitored by light scattering. Upon polymerization 20 lM CuSO4 was added to catalyze the disulfide bond formation between C41 and C45. Finally, the polymerization of destructed filaments was rescued by the addition of equimolar phalloidin. Arrows indicate the described addi- tions to the actin sample. (b) Electron micrographs of F-C41C45 before and after oxidation. Electron microscopy samples were taken from experiments ran in parallel to the one shown in A. For control filaments, 10 mM TCEP was added to ensure disulfide bond reduction. The scale bar in control image represents 0.2 mm. release from actin filaments via their direct interaction and (iii) My laboratory studies the structures of membrane proteins that defines phalloidin binding site on F-actin filaments and reveals are important in maintaining homeostasis in the brain. how it restricts the relative movement between the two protofi- Understanding structure (and hence function) requires scientists laments. Together, our results provide new molecular details of to build an atomic resolution map of every atom in the protein of F-actin structure and D loop dynamics. This structural and func- interest, that is, an atomic structural model of the protein of inter- tional information may be also useful for designing phalloidin est captured in various functional states. In 2013, we unveiled the derivatives and selecting best phalloidin matching conjugate method Microcrystal Electron Diffraction (MicroED) and demon- for staining different actin forms and structures (Figure 1). strated that it is feasible to determine high-resolution protein structures by electron crystallography of three-dimensional crys- Sanchaita obtained her PhD in 2011 from Wesleyan University tals in an electron cryo-microscope (CryoEM) (Nannenga, 2014; under Dr. Donald Oliver. Then she did post-doctoral research Shi, 2013). The CryoEM is used in diffraction mode for structural with Dr. Joachim Frank, Columbia, Steven Doxsey and David analysis of proteins of interest using vanishingly small crystals. Lambright, Univ of Mass. Currently she is an assistant project The crystals are often a billion times smaller in volume than what scientist with Dr. Emil Reisler at UCLA. is normally used for other structural biology methods like X-ray crystallography. In this seminar, I will describe the basics of this method, from concept to data collection, analysis and structure determination, and illustrate how samples that were previously 5. MicroED: conception, practice and unattainable can now be studied by MicroED. I will conclude by future opportunities highlighting how this new method is helping us understand major brain diseases like Parkinson’sdisease(Rodriguez,2015); Tamir Gonen helping us discover and design new drugs; shedding new light Howard Hughes Medical Institute and University of California, Los on chemical synthesis and small molecule chemistry; and show- Angeles, Los Angeles, CA 90095, USA [email protected] ing us unprecedented level of details with subatomic resolutions. 4 BOOK OF ABSTRACTS. ALBANY 2019: THE 20TH CONVERSATION

Funding to the 50S subunit during heat stress. Although HflX is recognized as a GTPase, several studies have shown that E. coli This research has been supported by funds from the Howard HflX is capable of hydrolyzing ATP as well and its N-terminal Hughes Medical Institute. domain 1 has recently been characterized as the ATPase domain. However, the functional role of its ATPase activity References remains unknown. Here, using biochemical assays and atomic force micros- Nannenga, B., Shi, D., Leslie, A. G. W., & Gonen, T. (2014). Continuous copy, we demonstrate for the first time, that E. coli HflX pos- rotation structure determination by MicroED. Nature Methods, 11 (9), sesses ATP-dependent RNA helicase activity and is capable of 927–930. Rodriguez, A. J., Ivanova, M., Sawaya, M. R., Cascio, D., Reyes, F., Shi, D., unwinding large subunit ribosomal RNA. A cryo-EM structure … Eisenberg, D. (2015). The toxic core of a-synuclein of Parkinson’s of the 50S-HflX complex in the presence of ATP and GTP disease: Structure from invisible crystals. Nature, 525 (7570), 486–490. (non-hydrolysable analogs) hinted at the mode of its action Shi, D., Nannenga, B., Iadanza, M. G., & Gonen, T. (2013). MicroED—Three as an RNA helicase, where a helical domain has a determin- dimensional electron crystallography of protein microcrystals. eLife2, ant role in RNA unwinding. We further show that, while – e01345: 1 e01317. heat-stress results in inactivation of the ribosome, HflX can restore heat-damaged ribosomes and, consequently, amelior- ate cell survivability. 6. RNA helicase activity of E. coli HflX is instrumental in rescuing heat- References inactivated 23S ribosomal RNA Coatham, M. L., Brandon, H. E., Fischer, J. J., Schummer,€ T., & Wieden, H. J. (2016). The conserved GTPase HflX is a ribosome splitting factor that binds to the E-site of the bacterial ribosome. Nucleic Acids Sandip Dey, Krishnamoorthi Srinivasan and Research, 44(4), 1952–1961. Jayati Sengupta Dey, S., Biswas, C., & Sengupta, J. (2018). The universally conserved Structural Biology & Bio-Informatics Division, CSIR—Indian GTPase HflX is an RNA helicase that restores heat-damaged Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata, Escherichia coli ribosomes. The Journal of Cell Biology, 217(7), – 700 032, India [email protected] 2519 2529. Zhang, Y., Mandava, C. S., Cao, W., Li, X., Zhang, D., Li, N., … Gao, N. (2015). HflX is a ribosome-splitting factor rescuing stalled ribosomes A recent study has revealed that the ribosome-associated under stress conditions. Nature Structural & Molecular Biology, 22(11), GTPase HflX acts as an anti-association factor upon binding 906–913. JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS VOL. 37, SUPPLEMENT, 2019 5

Understanding these changes could facilitate the design of 7. Supercoiling affects specific base supercoiling-dependent DNA nanostructures for gene therapy. accessibility to alter 3-D shape of Funding DNA minicircles This work was supported by NIH grant RO1GM115501.

Jonathan M. Fogg and Lynn Zechiedrich References Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030, USA [email protected] Fogg, J. M., Randall, G. L., Sumners, D. W. L., Pettitt, B. M., Harris, S. A., & Zechiedrich, L. (2012). Bullied no more: When and how DNA shoves proteins around. Quarterly Reviews of Biophysics, 45(03), 257–299. DNA supercoiling affects DNA metabolism yet much about Irobalieva, R. N.,Ã Fogg, J. M.,Ã Catanese, D. J., Sutthibutpong, T., Chen, M., how it does so is unknown (reviewed in Fogg et al., 2012). Barker, A. K., … Zechiedrich, L. (2015). Structural diversity of super- Using 336 bp DNA minicircles covering a range of positive to coiled DNA. Nature Communications, 12 6, 8440 (Ãco-first authors) negative supercoiling, we unveiled the first three-dimensional Randall, G. L., Zechiedrich, L., & Pettitt, B. M. (2009). In the absence of structures of supercoiled DNA using cryo-electron tomography writhe, DNA relieves torsional stress with localized, sequence-dependent structural failure to preserve B-form. Nucleic Acids Research, (Irobalieva et al., 2015). With supercoiling, DNA can form far 37(16), 5568–5577. more bent and contorted shapes than predicted. We sought to understand how the interplay of DNA sequence and supercoiling drives the formation of these shapes using coarse-grained 8. Understanding the functional simulations and biochemical probing. Base pair disruptions dynamics of the 26S proteasome by indicate regions of high bending, as localized denaturation nucleotides-proteasome (molecule dynamic simulations indicate from base flipping; Randall et al., 2009) creates flexible hinges. At the same time, interaction study sharp bending at the apices of highly writhed DNA circles leads a b to broken base pairs. Probing with nuclease Bal-31 revealed Rui Fang and Ying Lu a exposed bases as a function of supercoiling. Bal31 cleaved all Department of Molecular and Cellular Biology, Harvard University, b the negatively supercoiled 336 bp minicircles but the rate Cambridge, MA 02138, USA; Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA increased beyond a distinct negative supercoiling threshold. [email protected] This threshold shifted to more negative supercoiling for 672 bp minicircles with inherently less curvature, demonstrating the The 26S proteasome, consisting of a barrel-shaped proteo- relationship between bending and base accessibility. A sharp lytic 20S core complex and one or two 19S regulatory com- positive supercoiling threshold was required for Bal-31 cleavage plexes, is a macromolecular machine (2.5 MDa) responsible to occur. We mapped Bal-31 cleavage sites and, using coarse- for regulatory protein degradation in eukaryotic cells. The grained simulations, determined the DNA register of our cryo- proteasome has a hexameric ring of AAA ATPases at the þ electron micrograph images. Our data reveal three hotspots of bottom of the 19S proteasome which converts ATP’s chem- Bal-31 cleavage; two are located 180 apart along the DNA cir- ical energy to mechanical force to unfold protein substrates cumference, and another is seen only in minicircles with low and translocate the denatured polypeptide through the cen- negative supercoiling levels. The relative probability of Bal-31 tral pore into the 20S for degradation. Though genetic, bio- cleaving at either site varied as a function of supercoiling. chemical and structural studies have revealed great details Together these data reveal the interplay among sequence, super- about the proteasome, how the six ATPases in the 19S par- coiling and shape, resulting in conformational changes that ticle coordinate their ATP cycles to power substrate trans- should profoundly influence DNA interactions with proteins. location and conformational transitions of other parts of the proteasome remains elusive. To understand this question, we developed a fluorescent reporter with sub-nanomolar sensitivity to measure different aspects of proteasomal activities. In a competition assay, we found that ATP-cS inhibited substrate degradation much more strongly compared to ADP, and primarily affected the rate of substrate translocation. We then developed a single-molecule assay to study the kinetics of the nucleotides-proteasome interaction. By labeling nucleotides with fluorophores, we are able to detect single nucleotide binding and disassociation events under a TIRF microscope. The result shows that proteasomal ATPases are characterized by distinct nucleotide binding kinetics. Interestingly, the overall binding kinetics of ATP is similar to that of ADP and ATP-cS, while AMP binds rather weakly. To explain the different inhibitory effects of nucleotides in the competition assay, we devised a Markov-state model 6 BOOK OF ABSTRACTS. ALBANY 2019: THE 20TH CONVERSATION

representing all possible transition of the proteasomal ATPases. a Department of Systems Biology, Harvard Medical School, Boston, Simulation of this model suggests an entropy-driven mechanism MA 02115, USA; bState Key Laboratory for Artificial Microstructures underlying the inhibition by nucleotide inhibitors, which may be and Mesoscopic Physics, School of Physics, Peking University, adaptive under low ATP conditions. Our study provides novel Beijing, China; cDepartment of Cell Biology, Harvard Medical understanding of the working principles of proteasomal School, Boston, MA 02115, USA [email protected] ATPases, with the goal to obtain mechanistic insights by bridging the recent structural advances and biochemical observations. The 26S proteasome is a macromolecular machine (2.5-MDa) that mediates ubiquitin-dependent protein degradation in all eukaryotic species. The proteasome consists of a barrel-shaped 9. Structural and quantitative study 20S core complex flanked by two hexameric rings of AAA ATPases that harvest the energy from ATP hydrolysis to þ of the destruction complex in the unfold and translocate substrates through the central pore canonical wnt-signaling pathway into the degradation chamber. In a previous study, we determined how the ubiquitin configurations on substrates dictated Bai Luan, Wenzhe Ma, Hong Kang, Ying Lu and the degradation rate by the proteasome. Currently, we aim to Marc W. Kirschner understand how the 26S proteasome recognizes these ubiqui- Department of Systems Biology, Harvard Medical School, 200 tin configurations and commits the substrate to a processive Longwood Ave, Boston, MA 02115, USA degradation process. In a series of studies, we used modern [email protected] cryo-EM technology and classification algorithms to identify multiple conformations of the proteasomes, which provide The canonical Wnt/b-catenin signaling pathway plays pivotal important insights into these questions. To complement the roles in tumorigenesis. The cytosolic level of b-catenin is lack of kinetic information in structural analysis, we developed mainly regulated by a postulated molecular machine, which is a fluorescent reporter with sub-nanomolar sensitivity to called the b-catenin destruction complex. However, while the measure proteasomal activities under different conditions, term molecular machine usually connotes a discrete multipro- and established single-molecule assays to detect the transi- tein complex like the ribosome, this machine, while strongly ent interactions between nucleotides and the proteasome supported indirectly has never been purified or seen. The using a TIRF microscope. To synergize these results, we cre- destruction complex may be transient, and its postulated stoi- ated a rule-based model, inspired by comparative analysis of chiometry may never be realized at any given instant. The the proteasome structures, which simulates the dynamic instability of the components may lead to many different kin- transitions of a translocating proteasome on a complete con- etically determined structures. The variable stoichiometry formational landscape with unknown parameters that can be could also be very relevant physiologically. This complex con- obtained from kinetic studies. Suggested by the results, a tains a large scaffold, the adenomatous polyposis coli protein pathway of sequential loading/hydrolysis emerges with the (APC), which acts as an important tumor suppressor and has highest probability, and minor pathways with lower proba- been well characterized in colorectal cancer. APC mutants in bilities also exist which may become the major one and con- nearly all colorectal cancer have variable truncations, which sequently lower the degradation rates as in different could produce different subunit configurations between APC proteasomal mutants. In summary, our study promotes a and b-catenin, influencing the degradation of b-catenin down- deeper understanding of the functional dynamics of the pro- stream. To study the dynamic stoichiometry, we are develop- teasome driven by ATP hydrolysis, with the goal to obtain ing a quantitative single-molecule assay in the cell extracts of mechanistic insights by bridging the recent structural advan- both normal cells and colorectal cancer cells to observe the ces and biochemical observations. assembly process with millisecond time resolution. Ultimately, combining this kinetic information with biochemical crosslinking and cryo-electron microscopy, we hope to capture the transient state of the destruction complex of kinetically stable 11. Computational design of DNA subcomplexes. The purpose of the project is to illustrate the scaffold for optimization of CryoEM dynamic process of the destruction complex by combining structural information and quantitative single-molecule data, Kendar Serindaga,b, Kelly M. Thayerb,c,d, David L aiming to find the essential role of APC in colorectal cancer. Beveridgeb,c and David R. Langleyb,c,e aDepartment of Molecular Biology and Biochemistry, Wesleyan 10. The conformational landscape of University, Middletown, CT 06459, USA; bDepartment of Chemistry, Wesleyan University, Middletown, CT 06459, USA; the human 26S proteasome by Cryo- cMolecular Biophysics Program, Wesleyan University, Middletown, CT 06459, USA; dDepartment of Computer Science, Wesleyan EM structural analysis University, Middletown, CT 06459, USA; eArvinas, New Haven, CT 06511, USA Rui Fanga, Robin Chenb, Jason Hona, Jiayi Wub, Yuanchen Dongb, Yanan Zhub, Youdong Maob, Qi The ability of DNA to bind proteins and other molecules Ouyangb, Daniel Finleyc and Ying Lua makes it an ideal candidate for its use as a scaffold to which JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS VOL. 37, SUPPLEMENT, 2019 7 biomolecules can be attached. In this study, circular DNA fibrils can also be formed by several nonpathogenic proteins scaffolds of varying sizes have been designed. Curvature in like serum albumins and hen egg-white lysozyme under suit- the DNA ring is induced by the bacterial DNA binding protein able conditions in vitro. This suggests aggregation to be an Integration Host Factor (IHF). It is a heterodimer with an alpha inherent property of proteins. Although amyloid fibrillation helical body and two protruding beta sheet arms. These arms causes many fatal diseases, effective therapeutic treatments interact within the minor groove of DNA, and proline residues are still obscure. Various crown ethers have been shown to within the arms stabilize the DNA bending. Molecular dynamic retard fibrillation effectively (Banik et al., 2016; Tian et al., simulations were run to determine the mechanics and stability 2014). Here, we have studied the effect of crown ether, of the DNA ring itself, as well as the mechanics and stability of namely 15-crown-5, on amyloid fibrillation in lysozyme in IHF protein binding. Wet lab experiments are also being con- order to design effective anti-amyloidogenic agents that can ducted to create the physical construct. Applications for this target native proteins and offer advantageous prospect to scaffold include its use in cryo-electron microscopy (CryoEM), a develop therapeutics. Lysozyme is an antimicrobial enzyme new molecular imaging technique that enables researchers to present in various tissues as well as protective secretions determine the structure of biomolecules. CryoEM requires bio- which can form amyloid fibrils readily and hence serves an molecules to be at least 200 kDa and maintain an axis of sym- excellent model system for studying protein amyloidogenesis. metry in order to obtain an accurate image of the structure. In this work, hen egg-white lysozyme was taken as the model Thus, attaching biomolecules less than 200 kDa will enable protein system since it shares 60% sequence homology with them to be readily visualized by CryoEM. Additional functional- human lysozyme that is linked with many hereditary non- ity of this scaffold includes the facilitation and subsequent neuropathic systemic amyloidosis. Various assays were under- imaging of weak binding interactions. taken to ascertain the inhibitory influence of the crown ether on lysozyme fibrillation. The results of Thioflavin T fluorescence assay indicated that the crown ether can attenuate fibrillation effectively. This observation was further reiterated by a complementary Congo Red Assay in absorbance. The kinetics of fibrillation was also studied using the Thioflavin T fluorescence assay. Far-UV circular dichroism studies indicated that the lysozyme samples undergo a-to-b transition, upon amyloid fibrillation. From the far-UV circular dichroism studies it was also inferred that the b-sheet content of the protein was reduced in the presence of crown ether which suggested that amyloid fibrillation was inhibited. Fluorescence microscopy, Nile red fluorescence assay, 8-anilino-1-naphthalenesulfonica- cid binding assay, intrinsic (tryptophan) fluorescence studies along with steady-state fluorescence anisotropy also testified that the crown ether had significant fibril attenuating ability. Atomic force microscopy (AFM) imaging studies unequivocally established that fibril formation was reduced in the presence of the crown ether. Thus, the inhibitory effect of the crown ether, 15-crown-5, on amyloidosis may be exploited for designing better therapeutics for the treatment of amyloidogenesis-related diseases.

Acknowledgments

Financial assistance from the Department of Science & Technology and Biotechnology, Govt. of West Bengal (File No.: ST/P/S&T/15G-13/2018) is gratefully acknowledged. The author is grateful to Prof. Ranjan 12. Effect of crown ether, 15-crown-5, Chakrabarti, Hon’ble Vice-Chancellor, Vidyasagar University, for on lysozyme amyloid fibrillation his patronage.

Anirban Basu References Department of Chemistry and Chemical Technology, Vidyasagar University, Midnapore, 721 102, India Banik, D., Dutta, R., Banerjee, P., Kundu, S., & Sarkar, N. (2016). Inhibition [email protected] of fibrillar assemblies of l-phenylalanine by crown ethers: A potential approach toward phenylketonuria. The Journal of Physical Chemistry B, Amyloid fibrils are highly organized protein or peptide aggre- 120(31), 7662–7670. Tian, Y., Zhang, X., Li, Y., Shoup, T. M., Teng, X., Elmaleh, D. R., … Ran, C. gates with cross b-sheet rich secondary structure responsible (2014). Crown ethers attenuate aggregation of amyloid beta of for various pathological disorders such as type 2 diabetes, Alzheimer’s disease. Chemical Communication (Cambridge, UK), 50(99), Alzheimer’s, Huntington’s and Parkinson’s diseases. Amyloid 15792–15795. 8 BOOK OF ABSTRACTS. ALBANY 2019: THE 20TH CONVERSATION

and Joint Research Funds for Medical and Engineering from 13. Evaluation and validation Scientific Research at Shanghai Jiao Tong University. of synergistic effect of predicted amyloid-beta (Ab) inhibitor by deep References neural network Austen, B. M., Frears, E. R., & Davies, H. (2000). The use of Seldi ProteinChipTMArrays to monitor production of Alzheimer’s b-amyloid Aman Chandra Kaushik and Dong Quing Wei in transfected cells. Journal of Peptide Science, 6(9), 459–469. Kaushik, A. C., Kumar, A., Dwivedi, V. D., Bharadwaj, S., Kumar, S., Bharti, State Key Laboratory of Microbial Metabolism and School of life K., … Mishra, S. K. (2018). Deciphering the biochemical pathway and Sciences and Biotechnology, Shanghai Jiao Tong University, pharmacokinetic study of amyloid beta-42 with superparamagnetic Shanghai 200240, China [email protected] iron oxide nanoparticles (SPIONs) using systems biology approach. Molecular Neurobiology, 55(4), 3224–3236. Amyloid-beta 42 (Ab-42) protein has been established in numerous investigations as high-profile risk factor associated with onset and progression of Alzheimer’s disease (AD). Extracellular senile plaques accumulation, synaptic degener- 14. How nature harnesses entropy ation and intracellular neurofibrillary tangles (NFT) were to tune protein function recorded as essential features that facilitate onset of Ab-42 and results into AD. Hence, this study attempted a new Zachary A. Wooda, Nicholas D. Keula, Krishnadev screening technique to discover potential inhibitors against Orugantyb, Elizabeth T. Schaper Bergmane, Ab-42 by employing in silico deep neural network approach. Nathaniel R. Beattiea, Weston E. McDonalda, Renuka In this method, Pubchem compounds library was screened Kadirvelraja, Michael L. Grosse, Robert S. Phillipsc and d and concluded wgx-50 as potential inhibitor of Ab-42. Also, Stephen C. Harvey synergistic effect of wgx-50 gold nanoparticles (AuNP) aDepartment of Biochemistry and Molecular Biology, University of þ b induced significant inhibition of Ab-42 against wgx-50 as Georgia, Athens, GA, USA; Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA; control. Further, analysis of molecular docking, system biol- c Department of Chemistry, Washington University in St. Louis, St. ogy approach and time course simulation revealed synergis- Louis, MO, USA; dDepartment of Chemistry, University of Georgia, tic effect of wgx-50-AuNP complex as potential treatment for Athens, GA, USA; eDepartment of Biochemistry and Biophysics, AD. Additionally, we purposed the biological circuit for the University of Pennsylvania, Philadelphia, PA, USA [email protected] AD induced by Ab-42 that can be employed to monitor the effect of drugs on AD (Figure). How evolution shapes the conformational landscape of a protein to tune a specific function is poorly understood. Funding Protein evolution is constrained by the stability of the folded, native state. Despite this, many proteins contain intrinsically This work is supported by the Key Research Area Grant disordered (ID) peptide segments. In fact, 44% of human pro- 2016YFA0501703 from the Ministry of Science and teins contain ID segments >30 residues in length. The major- Technology of China, State Key Lab on Microbial Metabolism, ity of these segments have no known function and are often

Figure. JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS VOL. 37, SUPPLEMENT, 2019 9 removed to facilitate structural studies. Here, we show that are transcription factors (TFs), and the Src family kinases an ID segment can enhance the affinity of an effector bind- (SFKs), which are signaling enzymes. The goal here is to ing site by modifying the dynamics of an allosteric network. determine how AS and PTMs individually alter the out- The enhanced affinity does not depend on the sequence or comes of the signaling carried out by the various IDRs and charge of the ID segment. Instead, changes in effector bind- to determine whether AS and PTMs work together to bring ing affinity can be accurately predicted based on segment about differential cellular responses. We also present data length alone. Using a combination of transient state kinetics, indicating that a wide range of other signaling IDPs or sig- hydrogen-deuterium exchange mass spectrometry, thermal naling proteins containing IDRs also undergo both AS- and denaturation studies, computer simulation and crystal struc- PTM-based modifications, suggesting that these many pro- ture analysis, we show that the ID segment alters the energy teins likely take advantage of signal outcome modulations landscape of a folded protein to favor the allosteric response. that result from collaboration among these three features. Our evidence shows that the ID segment generates an Hence, we propose that the widespread cooperation of entropic force that can rectify the conformational ensemble IDPs, AS and/or PTMs substantially contributes to, or even of a protein to favor a specific functional state. Thus, the per- provides the basis for, the vast complexity of eukaryotic cell sistence of intrinsic disorder in the proteome may reflect the signaling systems. evolution of low complexity structural elements that can tune a specific protein function.

References 16. Multivalency regulates activity Keul, N. D., Oruganty, K., Bergman, E. T. S., Beattie, N. R., McDonald, in an intrinsically disordered W. E., Kadirvelraj, R., … Wood, Z. A. (2018). The entropic force gener- transcription factor ated by intrinsically disordered segments tunes protein function. Nature, 563(7732), 584. Sarah Clark, Kayla Jara, Patrick Reardon and Elisar Barbar Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR 97331, USA [email protected] 15. Intrinsically disordered proteins, alternative splicing, and post- Transcription factors contain multiple regulatory sites for either post-translational modifications or binding partners, translational modification (IDP-AS- and their activity is thus tuned by the combined action of PTM): a toolkit for these components. Recent studies have revealed a high developmental biology degree of intrinsic disorder in transcription factors, indicating that the inherent dynamical behavior harbored by these Keith Dunker structures is critical for these regulatory events to take place. Our developing understanding suggests that these intrinsic- Center for Computational Biology and Bioinformatics, Department of Biochemistry and Molecular Biology, Indiana University Schools ally disordered domains may provide a multivalent platform of Medicine and Informatics, Indianapolis, IN, 46202, USA for the recruitment of regulatory binding partners. An [email protected] example of a transcription factor with a long disordered domain is ASCIZ (ATMIN, ZNF822) which also has an Intrinsically, disordered proteins and regions (IDPs and IDRs) unusually high number of multivalent sites for the product lack well-defined tertiary structures, yet carry out various of its main target gene, the hub protein LC81. ASCIZ has a important cellular functions, especially those associated with folded N-terminal zinc finger domain for DNA binding and cell signaling and regulation. In eukaryotes, IDPs and IDRs an unusually long disordered C-terminal domain, which binds contain the preferred loci for both protein segments multiple copies of LC8: 11 in human and 7 in Drosophila pro- encoded by alternatively spliced pre-mRNA (AS) and many teins. Here, we integrate multiple approaches including NMR post-translational modifications (PTMs). Furthermore, AS and/ and single particle electron microscopy to elucidate the or PTMs at these loci generally alter the signaling outcomes structure, dynamics, thermodynamics and hydrodynamics of associated with these IDPs or IDRs. However, the prevalence the large disordered ASCIZ-LC8 complexes, that together of such functional modulations remains unknown. Also, the reveal a new model by which ASCIZ can maintain stable signal-altering mechanisms by which AS, and PTMs modu- pools of the hub protein LC8. We tested the main features of late function and the extent to which AS and PTMs collab- this model in cells using transcription activity assays which orate in their signaling modulations have not been well show a trend wherein mutant ASCIZ constructs with lower defined for particular protein examples. Here, we focus on LC8 occupancy display higher transcriptional activity, while three important signaling and regulatory IDR-containing constructs with higher LC8 occupancy have lower activity. protein families in humans, namely G-protein coupled We propose that a dynamic ensemble of complexes is receptors (GPCRs), which are transmembrane signaling pro- important for fine-tuning ASCIZ transcriptional activity, where teins, the nuclear factors of activated T-cells (NFATs), which stable, low occupancy complexes function to maintain a basal 10 BOOK OF ABSTRACTS. ALBANY 2019: THE 20TH CONVERSATION

self-perpetuating changes in protein conformations. This occurs most commonly in intrinsically disordered proteins that regulate information flow: chromatin modifying enzymes, transcription factors and RNA binding proteins. These conformations can be broadly defined as prions, although their structures do not usually match the cross- beta sheet amyloids of the archetypical prion PrP. However, like known prions, corresponding changes in protein function are heritable from one generation to the next without Figure 1. A collage of NMR spectra, electron micrograph and LC8/ASCIZ struc- any change to the genome. In this sense, such protein-based ture showing three LC8 dimers and two chains of ASCIZ, while the rest of the inheritance represents an extreme form of epigenetics. We complex remains disordered. Obtaining data of sufficient quality on a heterogeneous system of multiple conformations of IDP complexes was made possible have begun to characterize the biochemistry of these ele- by creative use of interdisciplinary approaches. ments and investigate their influence on disease, development and evolution. Lessons learned provide insight into mechanisms of pathological and beneficial protein aggregation alike, and how they might be modulated buffering transcription rate for LC8. A change in LC8 cellular therapeutically. concentration would shift this dynamic equilibrium to a higher or lower occupancy state without dramatically altering the level of transcription. High LC8 occupancy shuts 18. Seeing the structure of RNA- down transcription while low LC8 occupancy turns on tran- binding proteins in membraneless scription2. As LC8 is an essential regulator of dozens of cellular processes, an ability to maintain an LC8 ‘buffer’ is likely organelles and disease- important for cellular homeostasis. Although many other associated aggregates transcription factors are regulated by multisite phosphorylation or multiple binding events to different proteins we Nicolas Lux Fawzi find no examples of activity tuned by multivalent binding to Molecular Pharmacology, Physiology & Biotechnology, Brown the gene product in a negative autoregulatory role, which University, 70 Ship Street, Box G-E, Providence, RI, 02912-G, USA underscores the novelty and potential impact of this study [email protected] (Figure 1). Nicolas Fawzi, Brown University, leads a team using NMR spectroscopy, molecular simulation, microscopy and cell Funding assays to probe disordered protein domain assembly and function. Disordered domains of RNA binding proteins aggre- This research is supported by NSF MCB-1617019 and NIH GM gate in several neurodegenerative diseases and mediate for- 084276 and HEI 1S10OD018518. mation of functional, liquid, membraneless organelles. Nick’s group probes the structure of these protein (including TDP- References 43, FUS, hnRNPA2) within in vitro models of these organelles, the mechanistic structural changes due to disease-causing Barbar, E. (2008). Dynein light chain LC8 is a dimerization hub essential mutations, the ability of post-translational modification to in diverse protein networks. Biochemistry, 47(2), 503–508. Clark, S. A., Myers, J. B., King, A., Fiala, R., Novacek, J., Pearce, G., … alter assembly. Barbar, E. (2018). Multivalency Regulates activity in an intrinsically disordered transcription factor. eLife, 1, 7. pii: e36258. 19. The critical role of intrinsic disorder in creating 17. Remembering the past: a new bioactive materials form of protein-based inheritance Sarah E. Bondos Department of Molecular and Cellular Medicine, 440 Reynolds Daniel F. Jarosz Medical Building, Texas A&M Health Science Center, College Department of Chemical and Systems Biology, Stanford University, Station, TX 77843-1114, USA Stanford, CA, USA [email protected] The development of materials with diverse functional proper- During their lifetimes, individuals commonly experience tran- ties enables a broad range of applications. For materials sient changes in gene expression as a result of different composed of protein, well-established molecular biology environmental stimuli. These responses are often thought to techniques can theoretically be used to genetically fuse full- have little heritable influence once they decay. However, we length functional proteins to the self-assembling protein, cre- have recently discovered that such stimuli frequently induce ating a single amino acid chain capable of both forming JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS VOL. 37, SUPPLEMENT, 2019 11 materials and enacting the function of interest. However, in artificial molecular arrays assembled from modular DNA protein-based materials are typically assembled under dena- antijunction units. The array transformation is equivalent to a turing conditions, precluding incorporation of proteins in an molecular ‘Domino’: Once initiated at a few selected units, active state. Our lab discovered that the Drosophila mela- the transformation then propagates to neighboring units nogaster transcription factor Ultrabithorax (Ubx) self-assem- and eventually the entire array. The specific information bles into nanoscale to macroscale materials. The disordered pathways by which this transformation occurs can be con- regions of Ubx resemble the amino acid sequence of elastin, trolled by adding trigger strands to specific units, or by alter- and the mechanical properties of Ubx fibers resemble those ing the design of individual units, the connections between of elastin. Because Ubx materials rapidly form in mild, aque- units, and the geometry of the array. Beyond sophisticated ous buffers, a surprisingly wide variety of full-length mono- nanostructures and nanomachines, DNA nanotechnology has meric and multimeric proteins can be incorporated into found increasing capabilities in many applications, such as Ubx materials via gene fusion without harming materials fabrication of functional materials at nanoscale precision. As assembly or impairing the function of the appended pro- examples of DNA-based applications, I will also present some tein. Indeed, fusing Ubx is fused to angiogenic growth fac- of our most recent works on DNA-templated self-assembly of tors, creates fibers that control cell signaling and cell nanoparticles, and on DNA-based nanoscale drug deliv- behavior in vitro, and can instigate and guide blood vessel ery systems. formation in vivo. Finally, Ubx fibers retain the ability to bind specific DNA sequences, and Ubx monomers bound to DNA can still assemble into fibers. The intrinsically disor- References dered regions of Ubx allows incorporation of molecules up Ke, Y., Ong, L. L., Shih, W. M., & Yin, P. (2012). Three-dimensional struc- to three times the size of this protein. One-pot production tures self-assembled from DNA bricks. Science (New York, N.Y.), of functionalized Ubx materials provides a facile, scalable 338(6111), 1177–1183. Ong, L. L., Hanikel, N., Yaghi, O. K., Grun, C., Strauss, M. T., Bron, P., Lai- platform for customizing materials for a variety of Kee-Him, J., … , Yin, P. (2017). Programmable self-assembly of three- applications. dimensional nanostructures from 10,000 unique components. Nature, 552(7683), 72–77. Song, J., Li, Z., Wang, P., Meyer, T., Mao, C., & Ke, Y. (2017). Reconfiguration of DNA molecular arrays driven by information relay. 20. Complex self-assembly and Science, 357(6349), eaan3377. Wang, D., Song, J., Wang, P., Pan, V., Zhang, Y., Cui, D., & Ke, Y. (2018). transformation of DNA nanostructures Design and operation of reconfigurable two-dimensional DNA molecular arrays. Nature Protocols, 5, 693. Yonggang Ke Biomedical Engineering Department, Emory University and Georgia Institute of Technology, Atlanta, GA, 30322, USA [email protected] 21. Designer nucleic acid architectures A key challenge in nanotechnology is to design and fabricate for programmable self-assembly nanostructures and nanodevices, which can be used as general platforms for basic science research (e.g., material scien- Hao Yan ces, structural biology, molecular biology, etc.), and for Center for Molecular Design and Biomimetics, Biodesign Institute, practical applications. Owing largely to its programmable Arizona State University, Tempe, AZ 85287, USA design strategies, nucleic acids self-assembly, and in particu- [email protected] lar DNA self-assembly, has emerged as a powerful approach in programming self-assembly of custom-designed intricate DNA and RNA have emerged as an exceptional molecular nanostructures. building block for nano-construction due to its predictable The core mission of our lab (ke-lab.gatech.edu) is to conformation and programmable intra- and inter-molecular develop novel bottom-up self-assembly strategies to fully base pairing interactions. A variety of convenient design demonstrate the potential of DNA as a programmable nano- rules and reliable assembly methods have been developed material. Our most recent work focuses on making massive/ to engineer DNA nanostructures of increasing complexity. complex static DNA nanostructures and dynamic DNA devi- The ability to create designer DNA architectures with ces. In 2012, we invented a modular assembly strategy for accurate spatial control has allowed researchers to explore constructing complex 3 D shapes, up to 8 megadalton in novel applications in many directions, such as directed size, using short synthetic DNA oligos—‘DNA bricks’. We will material assembly, structural biology, biocatalysis, DNA discuss how we can use this method to construct fully computing, nano-robotics, disease diagnosis and drug addressable, three-dimensional GDa nanostructures with delivery. In this talk, I will discuss some of our work in rationally designed shapes. The second part of this talk will the field of structural nucleic acid nanotechnology, and focus on dynamic DNA nanomachines that can perform a presents some of the challenges and opportunities that range of controlled motions at nanoscale. Particularly, we exist in DNA and RNA-based molecular design and have demonstrated prescribed, long-range information relay programming. 12 BOOK OF ABSTRACTS. ALBANY 2019: THE 20TH CONVERSATION

We have self-assembled a 3D crystalline array and 22. Using DNA origami to decipher reported its crystal structure to 4 Å resolution. We can use spatial effects in biology crystals with two molecules in the crystallographic repeat to control the color of the crystals. Rational design of intermo- lecular contacts has enabled us to improve crystal resolution Bjorn€ Hogberg€ to better than 3 Å. We can now do strand displacement in Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm 171 77, Sweden [email protected] the crystals to change their color, thereby making a 3D- based molecular machine; we can visualize the presence of It is widely accepted that the biophysical context of ligands the machine by X-ray diffraction. and receptors has significant impact of downstream signal- The use of DNA to organize other molecules is central to ing, however, the concept is poorly understood due to diffi- its utility. Earlier, we made 2 D checkerboard arrays of metal- culties in controlling and analyzing the microenvironment on lic nanoparticles, and have now organized gold particles in the nanoscale. I will talk about how we think that DNA-nano- 3 D. Recently, we have ordered triplex components and a technology can be of great help in both learning the tactile semiconductor within the lattice. Thus, structural DNA nano- alphabet of cells by stimulation using protein decorated technology has fulfilled its initial goal of controlling the DNA-origami ‘nano-calipers’ and of help in understanding internal structure of macroscopic constructs in three dimen- the binding of antibodies. I will also briefly present our sions. A new era in nanoscale control awaits us. recent work on ‘3D-printing’ DNA origami wireframe structures, a method that provides a way to make origami more accessible to experiments in physiological salt conditions.

References

Benson, E., Mohammed, A., Gardell, J., Masich, S., Czeizler, E., Orponen, P., & Hogberg,€ B. (2015). DNA rendering of polyhedral meshes at the nanoscale. Nature, 523(7561), 441–444. Shaw, A., Hoffecker, I. T., Smyrlaki, I., Rosa, J., Grevys, A., Bratlie, D., … Hogberg,€ B. (2019). Binding to nanopatterned antigens is dominated by the spatial tolerance of antibodies, Nature Nanotechnology, 14(2), 184–190. Shaw, A., Lundin, A., Petrova, E., Ford€ os,} F., Benson, E., Al-Amin, A., … Teixeira, A. (2014). Spatial control of membrane receptor function using ligand nanocalipers. Nature Methods, 11(8), 841–846. Figure. The Central Concept of Structural DNA Nanotechnology: Combine Branched DNA with Sticky Ends to Make Objects, Lattices and Devices. 23. DNA is not merely the secret of life: semantomorphic chemistry in advanced materials 24. A novel indoline scaffold-based Nadrian C Seeman antibacterial compound designing New York University, New York, NY 10003, USA and pharmacological evaluation using We build branched DNA species that can be joined using chemoinformatics approach Watson-Crick base pairing to produce N-connected objects and lattices. We have used ligation to construct DNA topo- Aarushi Singh and Ramesh Chandra logical targets, such as knots, polyhedral catenanes, Drug Discovery & Development Laboratory, Department Borromean rings and a Solomon’s knot. of Chemistry, University of Delhi, Delhi 110007, India Nanorobotics is a key area of application. We have made [email protected] robust 2-state and 3-state sequence-dependent programmable devices and bipedal walkers. We have constructed 2- Antibiotic resistance is not only a global public health threat dimensional DNA arrays with designed patterns from many but also a huge economic burden to our society that different motifs. We have used DNA scaffolding to organize urgently needs to be addressed by improved antibiotics and active DNA components. We have used pairs of 2-state devi- continuing development of novel molecules to treat resistant ces to capture a variety of different DNA targets. We have bacterial infections (Kim et al., 2008; Kaplancikli et al., 2007). constructed a molecular assembly line using a DNA origami Nowadays combination therapies offer a competent layer and three 2-state devices, so that there are eight differ- approach to counteract antibiotic resistance in bacteria ent states represented by their arrangements. We have dem- (Singh et al., 2016, 2017). Better knowledge of mechanisms onstrated that all eight products can be built from this of antibiotic resistance has led to the finding of new alterna- system. Recently, we connected the nanoscale with the tives to antibiotic therapy. Hence, in this article, we report a microscale using DNA origami. novel series of indoline derivatives and their computational JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS VOL. 37, SUPPLEMENT, 2019 13

study as potent antimicrobials. The present study investi- using computational approach. Frontiers in Bioengineering and gates the indoline based derived library interaction with Biotechnology, 4(69) DNA gyrase B enzyme to be used as a potential antimicrobial drug. Computational approaches were employed to carry out the molecular interactions and pharmacological studies. In 25. Discovery of penicillin binding this study, we have compared indoline with its derivatives proteins (PBPs) inhibitors by blending and have found that compound 13 resulted in the strong virtual screening, molecular docking binding with the highest score ( 9.02 kcal/mol) in the À designed library where indoline showed ( 6.43 kcal/mol). and simulation studies À Furthermore, molecular dynamics simulation run also con- Mayurpankhi Buragohaina, Abha Vashishthab and firmed the strongest interaction of a compound and target b protein with less RMSD and RMSF deviation of the complex. Surabhi Johari a Notably, the compound was also found to possess the good Centre for Biotechnology and Bioinformatics Dibrugarh University, Dibrugarh, Assam, 786001, India; bSchool of Biosciences, Institute pharmacological properties and pharmacokinetic properties. of Management Sciences, University Courses, Ghaziabad, Uttar Pradesh, 201002, India [email protected] References

Kaplancikli, Z. A., Turan-Zitouni, G., Ozdemir, A., Revial, G., & Guven, K. KEYWORDS Methicillin; molecular docking; penicillin binding proteins; (2007). Phosphorus, Sulfur, and Silicon and the Related Elements, 182(4), resistance; triazine simulations 749–764. Kim, S.-W., Kuti, J. L., & Nicolau, D. P. (2008). Inhaled antimicrobial therapies An integrated protocol of virtual screening involving molecu- for respiratory infections. Current Infectious Disease Reports, 10(1), 29–36. lar docking, pharmacophore probing and simulations was Singh, V. K., Kumar, N., & Chandra, R. (2017). Structural insights of established to identify small novel molecules targeting cru- induced pluripotent stem cell regulatory factors Oct4 and its inter- cial residues involved in the penicillin binding proteins action with Sox2 and Fgf4 gene. Advances in Biochemistry and Biotechnology, J119. (PBPs) involved in bacterial cell wall biosynthesis. An excel- Singh, V. K., Kumar, N., Kalsan, M., Saini, A., & Chandra, R. A. (2016). lent approach was made utilizing ligand and structure-based Novel peptide thrombopoietin mimetic designing and optimization pharmacophore to identify common features and structure- 14 BOOK OF ABSTRACTS. ALBANY 2019: THE 20TH CONVERSATION based docking with respect to PBPs in leading to the development of novel inhibitors possessing new scaffolds. To our delight, multiple-substituted triazine derivatives series bear- ing a novel scaffold, of which structure is remarkably different from the existing b-lactam inhibitors, were designed. A structure-based pharmacophore mapping was developed to explore the binding sites of PBPs which were taken into consideration. Subsequently, virtual screening, ADMET searches Figure 1. A cartoon of a truncated gyrase-DNA complex highlighting the sites were at work to narrow down the proposed hits to be for- of action of antibiotics. warded as a potential drug likes candidates. Further, the binding patterns of the best-proposed hits were explored to successful. Their mechanism involves stabilization of a pro- understand the deeper insight for its structural optimization tein-linked transient double-strand break in DNA, which can by employing it on molecular dynamic simulations of 500ps. be lethal. However, resistance to fluoroquinolones is signifi- Selectivity profile for the most promising candidates was cant and alternative compounds are needed. Although ami- studied, revealing significantly C series molecules C15 was nocoumarin natural products (e.g., novobiocin) are potential found to be the most potent compounds among designed alternatives, these compounds have not achieved significant library. The proposed hits can be forwarded for further study clinical success. Working with pharma companies we have against PBPs involved in bacterial peptidoglycan cell wall investigated other compounds that stabilize the gyrase-DNA biosynthesis. cleavage complex, e.g., IPYs (Germe et al., 2018; Jeannot et al., 2018) and thiophenes (Chan et al., 2017)(Figure 1). References The potential of these and other compounds as future antibiotics will be discussed. Desai, N., Makwana, A., & Rajpara, K. (2016). Synthesis and study of 1,3,5- triazine based thiazole derivatives as antimicrobial agents. Journal of Saudi Chemical Society, 20, S334–S341. References Gonzales, P., Pesesky, M., Bouley, R., Ballard, A., Biddy, B., Suckow, M., … Dantas, G. (2015). Synergistic, collaterally sensitive b-lactam combina- Bates, A. D., & Maxwell, A. (2005). DNA Topology. Oxford: Oxford tions suppress resistance in MRSA. Nature Chemical Biology, 11(11), University Press. 855–861. Chan, P. F., Germe, T., Bax, B. D., Huang, J., Thalji, R. K., Bacque, E., … Ding, X. (2017). Thiophene antibacterials that allosterically stabilize DNA-cleavage complexes with DNA gyrase. Proceedings of the National Academy of Sciences of the United States of America, 114(22), E4492–E4500. Collin, F., Karkare, S., & Maxwell, A. (2011). Exploiting bacterial DNA 26. DNA topoisomerases as targets gyrase as a drug target: Current state and perspectives. Applied for antibacterial chemotherapy Microbiology and Biotechnology, 92(3), 479–497. Germe, T., Voros, J., Jeannot, F., Taillier, T., Stavenger, R. A., Bacque, E., … Bax, B. D. (2018). A new class of antibacterials, the imidazopyrazi- Anthony Maxwell nones, reveal structural transitions involved in DNA gyrase poisoning Department of Biological Chemistry, John Innes Centre, Norwich and mechanisms of resistance. Nucleic Acids Research, 46(8), Research Park, Norwich, NR4 7UH, UK [email protected] 4114–4128. Jeannot, F., Taillier, T., Despeyroux, P., Renard, S., Rey, A., Mourez, M., … Versluys, S. (2018). Imidazopyrazinones (IPYs): Non-quinolone bacterial DNA topoisomerases are enzymes that catalyze changes in topoisomerase inhibitors showing partial cross-resistance with quino- DNA topology (Bates & Maxwell, 2005). There are two types, lones. Journal of Medicinal Chemistry, 61(8), 3565–3581. I and II, differentiated by whether they catalyze reactions via Maxwell, A., Bush, N. G., Germe, T., & McKie, S. J. (2018). In Fong, I. W., D. single- or double-stranded breaks in DNA. They are essential Shlaes & K. Drlica (eds.), Antimicrobial resistance and implications for in all cells, having key roles in DNA replication, transcription the 21st century. Springer. Pommier, Y., Leo, E., Zhang, H., & Marchand, C. (2010). DNA topoisomer- and recombination. All topoisomerases are able to relax ases and their poisoning by anticancer and antibacterial drugs. supercoiled DNA, but DNA gyrase, essential in all bacteria, Chemistry & Biology, 17(5), 421–433. can also introduce negative supercoils in a reaction coupled to ATP hydrolysis (Collin, Karkare, & Maxwell, 2011). Due to their essential nature and the fact that they stabilize single- or double-stranded breaks in DNA, topoisomerases have become key drug targets for both antibacterial and anti-can- 27. Exploring peptidoglycan cer chemotherapy (Collin, Karkare, & Maxwell, 2011; Maxwell, biosynthesis and it’s inhibition Bush, Germe, & McKie, 2018; Pommier, Leo, Zhang, & Marchand, 2010)(Figure 1). Adrian Lloyd, Dom Bellini, Hector Newman and Building on structural and mechanistic data, we have Christopher Dowson focused on antibiotics targeted to bacterial gyrase. School of Life Sciences, University of Warwick, Coventry, CV47AJ, Fluoroquinolone drugs (e.g., ciprofloxacin) have been highly UK [email protected] JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS VOL. 37, SUPPLEMENT, 2019 15

Penicillin and the wider family of beta-lactams have 28. How bacteria and cancer cells remained the single most important family of antibiotics since their introduction in the early 1940s. Unfortunately regulate mutagenesis and their ability they are becoming less effective due to the emergence of to evolve resistance that now has an impact globally (E. P. Center for Disease Dynamics, 2015). Predominantly, resistance is medi- Susan M. Rosenberg ated by reducing the target drug concentration by catalysing Baylor College of Medicine, Houston, TX, USA their inactivation by beta-lactamases (Bush, 2015; Bush and Bradford, 2016; Drawz, Papp-Wallace, & Bonomo, 2014; Our concept of genomes is changing from one in which Ehmann et al., 2012; Lahiri et al., 2013), by increasing efflux, or the DNA sequence is passed faithfully to future generations reducing permeation (in Gram negative bacteria). The periplas- to another in which genomes are plastic and responsive to mic/extracytoplasmic targets of penicillin are a family of environmental changes. In contrast with classical assump- enzymes with a highly conserved catalytic activity involved in tions that mutations occur purely stochastically at constant, the final stage of bacterial cell wall biosynthesis: cross-linking gradual rates, microbes, plants, flies and human cancer cells of the structural polymer peptidoglycan (PG). These enzymes, possess mechanisms of mutagenesis that are upregulated named after their ability to bind penicillin, rather than their by stress responses. These generate transient, genetic-diver- catalytic activity are called penicillin-binding proteins (PBPs) sity bursts that can propel evolution, including evolution of (Frere & Page, 2014; Sauvage, Kerff, Terrak, Ayala, & Charlier, infectious disease and cancers, specifically when cells are 2008; Schweizer et al., 2017). Surprisingly, given the pivotal poorly adapted to their environments—that is, when importance of PBPs, we still have a very incomplete picture of stressed. Emerging molecular mechanisms of stress-indu- how PBPs interact with their natural substrates, precisely what cible mutagenesis vary but share common components that these substrates are, exactly how beta-lactam antibiotics inter- highlight the non-randomness of mutation: (1) regulation of fere with this process and the response of bacteria to generate mutagenesis in time by cellular stress responses, which pro- a further resistance mechanism, PBP-mediated resistance. mote mutations when cells are poorly adapted to their Here we discuss recent advances in the development of environments—when stressed; (2) limitation of mutagenesis new reagents assays and structures that are helping to in genomic space causing mutation hotspots and clusters, develop a rigorous biochemical structure functional analysis which may both target specific genomic regions and allow of how the targets of penicillin work, including a full kinetic concerted evolution (evolution requiring multiple muta- characterization, and the interplay between enzyme sub- tions). This presentation will focus on the molecular mech- strate and inhibitor. anism of stress-inducible mutagenic DNA break repair in E. coli as a model for stress-inducible mutagenesis that pro- References pels infectious disease, antibiotic resistance and other evolution. We consider its regulation by stress responses, Bush, K. (2015). A resurgence of b-lactamase inhibitor combinations effective against multidrug-resistant Gram-negative pathogens. demonstrate its formation of mutation hotspots near DNA International Journal of Antimicrobial Agents, 46(5), 483–493. breaks, and our discovery of a large gene network that Bush, K., & Bradford, P. A. (2016). Cold Spring Harbor Perspectives in underlies mutagenic break repair, most of which functions Medicine, 6 in stress sensing and signaling. We also show that muta- Drawz, S. M., Papp-Wallace, K. M., & Bonomo, R. A. (2014). New -lacta- b genesis is induced by the antibiotic ciprofloxacin, causing mase inhibitors: A therapeutic renaissance in an MDR world. Antimicrobial Agents and Chemotherapy 58(4), 1835–1846. resistance to other antibiotics, and demonstrate the stress- Ehmann, D. E., Jahic, H., Ross, P. L., Gu, R. F., Hu, J., Kern, G. … Fisher, response-dependent mutagenesis mechanism. Regulation of S. L., (2012). Avibactam is a covalent, reversible, non-b-lactam b-lacta- mutagenesis in time and genomic space may accelerate mase inhibitor. Proceedings of the National Academy of Sciences of the evolution including evolution of pathogens, cancers, and United States of America, 109(29), 11663–11668. E. P. Center for Disease Dynamics 2015 Journal (2015). drug resistance. Frere, J. M., & Page, M. G. (2014). Penicillin-binding proteins: Evergreen drug targets. Current Opinion in Pharmacology, 18, 112–119. Lahiri, S. D., Mangani, S., Durand-Reville, T., Benvenuti, M., Luca, F. D., Sanyal, G., & Docquier, J.-D. (2013). Structural insight into potent 29. Identifying and targeting broad-spectrum inhibition with reversible recyclization mechanism: Avibactam in complex with CTX-M-15 and Pseudomonas aeruginosa the Achilles heel of drug AmpC b-Lactamases. Antimicrobial Agents and Chemotherapy, 57(6), resistant bacteria 2496–2505. doi: 10.1128/AAC.02247-12. Sauvage, E., Kerff, F., Terrak, M., Ayala, J. A., & Charlier, P. (2008). The penicillin-binding proteins: Structure and role in peptidoglycan bio- Nagasuma Chandra synthesis. FEMS Microbiology Reviews, 32(2), 234–258. Indian institute of Science, Bangalore, India Schweizer, I., Bl€attner, S., Maurer, P., Peters, K., Vollmer, D., Vollmer, W. … Denapaite, D., (2017). New aspects of the interplay between peni- The emergence of drug resistant strains of M. tuberculosis cillin binding proteins, murM , and the two-component system ciarh of penicillin-resistant Streptococcus pneumoniae Serotype 19A iso- poses a major threat to public health, warranting urgent lates from Hungary. Antimicrobial Agents and Chemotherapy, 61(7), attention to the problem of tackling antimicrobial resistance. doi: 10.1128/AAC.00414-17. A number of studies have sought to understand the causes 16 BOOK OF ABSTRACTS. ALBANY 2019: THE 20TH CONVERSATION of drug resistance, and have led to the identification of sev- enzymes vary widely in their activity against the different eral mechanisms including mutations in key targets, upregu- beta-lactam classes (principally penicillins, cephalosporins lation of drug efflux pumps, etc. Yet, there is no and carbapenems) and their susceptibility towards inhibitors understanding of which mechanisms are operative in a given that include beta-lactam- (clavulanic acid) and non-beta-lac- condition, whether microbes explore multiple mechanisms tam (diazabicyclooctane; boronate) based agents. Beta-lacta- simultaneously, or if such mechanisms are influenced by mases also divide into two groups: active-site serine each other and lead to alterations in the cell in a synchron- enzymes (classes A, C and D) and zinc metallo-enzymes ized manner. Toward this, an understanding of the global (class B) that differ fundamentally in structure mechanisms leading to resistance becomes necessary. This in and mechanism. turn will provide a basis to develop novel therapeutic strat- Our research focuses on interactions of beta-lactamases egies for tackling drug resistance. with carbapenems, the most recently introduced beta-lac- To address this, we adopt a systems level analysis of the tams and key antibiotics for infections by Gram-negative biomolecular networks associated with the emergence or bacteria. Carbapenems escape hydrolysis by most serine sustenance of drug-resistant phenotypes, from which we beta-lactamases as the 6a-hydroxyethyl substituent and tau- decipher cellular alterations in an unbiased manner and tomerization within the fused pyrroline ring system are identify emergent vulnerabilities in resistant bacilli. We inte- together proposed to retard breakdown of the covalent grate genomic, transcriptomic and phenotypic data from the acylenzyme intermediate; carbapenem hydrolysis (carbape- model system M. smegmatis and generate an integrated gen- nemase activity) is restricted to a select few serine enzymes. ome-scale response network, and employ network-mining In contrast, metallo-beta-lactamases efficiently hydrolyze car- approaches to identify the highest differential activities bapenems. We have investigated carbapenem hydrolysis by operative in the drug-resistant strain. The corresponding pro- both serine carbapenemases and metallo-beta-lactamases, teins are seen to form a well-connected orchestrated subnet, combining high-resolution crystallographic methods and providing insights into newly emerged vulnerabilities in the high-level computational (molecular dynamics and quantum resistant bacilli. The networks suggest that multiple mecha- mechanics/molecular mechanics (QM/MM)) approaches to nisms are at play simultaneously to overcome the drug seek to identify the requirements for hydrolysis of these stress, of which response to oxidative stress predominates. substrates. I will further describe the interactions of both Through targeted screening we discover that the resistant serine and metallo-beta-lactamases with a range of inhibi- bacilli exhibit collateral sensitivity to several compounds that tors, extending from compounds in clinical use to experi- block antioxidant responses. We then test the shortlisted mental compound series aimed at expanding inhibition to compounds against M. tuberculosis and three different drug- include the metallo-beta-lactamases against which current resistant clinical isolates and study the mechanistic basis of agents are ineffective. the efficacies of these compounds. Three of the tested compounds were able to reverse resistance in mycobacteria. One of the identified compounds shows high promise as a key component of a re-purposable drug combination for treating 31. Kinetic and molecular drug-resistant tuberculosis. determinants of antibiotic permeation barriers

30. Interactions of b-lactamases with Helen I. Zgurskaya antibiotics and inhibitors aDepartment of Chemistry and Biochemistry, University of Oklahoma, Norman, OK 73019, USA a,b a Catherine L. Tooke , Philip Hinchliffe , Ramya The permeability barrier of Gram-negative cell envelopes is Salimraja, Karina Calvopina~ a, Viivi H.A. Hirvonenb, a b,c the major obstacle in the discovery and development of new Matthew B. Avison , Marc W. van der Kamp , antibiotics. In Gram-negative bacteria, these difficulties are Adrian J. Mulhollandb and James Spencera a exacerbated by the synergistic interaction between two bio- School of Cellular and Molecular Medicine, University of Bristol, chemically distinct phenomena, the low permeability of the Bristol BS8 1TD, UK; bSchool of Chemistry, University of Bristol, Bristol BS8 1TS, UK; cSchool of Biochemistry, University of Bristol, outer membrane and active multidrug efflux. We developed Bristol BS8 1TD, UK [email protected] an approach to separate the contributions of the two mechanisms in the activities of antibiotics and applied it in the dis- Beta-lactams remain the single most important antibiotic covery of efflux pump inhibitors and analyses of class. Beta-lactamases are hydrolytic enzymes that inactivate structure–activity relationships for active efflux and outer the antibiotic by degrading the scissile amide bond of the membrane permeation in different Gram-negative patho- four-membered beta-lactam ring, and are the most preva- gens. This presentation will be focused on species- and con- lent resistance mechanism in Gram-negative bacterial patho- text-dependent interplays between active drug efflux and gens responsible for a variety of opportunistic infections. To drug permeation and how they affect antibacterial activities date more than 4000 beta-lactamases have been identified of antibiotics, substrate specificities of efflux pumps and in environmental and clinical bacterial strains. These efflux inhibitors. JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS VOL. 37, SUPPLEMENT, 2019 17

References 32. A comparative computational Dasgupta, S., Mukherjee, S., & Mukhopadhyay, B. P. (2018). Recognition study for deamination of trans and of trans and gauche phenylethylamine conformers in the active site gauche conformers of of human monoamine oxidase B: A MD-simulation and DFT studies. Computational and Theoretical Chemistry, 1127, 44–51. phenylethylamine in presence Dasgupta, S., Mukherjee, S., Mukhopadhyay, B. P., Banerjee, A., & Mishra, D. K. (2018). Recognition dynamics of dopamine to human of lumiflavin Monoamine oxidase B: Role of Leu171/Gln206 and conserved water molecules in the active site cavity. Journal of Biomolecular Structure Subrata Dasgupta, Soumita Mukherjee and B.P. and Dynamics, 36(6), 1439–1462. Mukhopadhyay Department of Chemistry, National Institute of Technology, Durgapur, Durgapur 713209, West Bangal, India [email protected] 33. A comprehensive automated The deamination of phenylethylamine (PEA) and norepineph- computer-aided discovery pipeline rine (NOR) to corresponding aldehyde and ammonia by the from genomes to hit molecules enzyme human Monoamine oxidase (hMAO) is one of the most important concerns in neurobiochemistry due to its Ruchika Bhata,b, Rahul Kaushikb,c, Ankita Singhb, involvement in several neurological diseases and complica- Debarati DasGuptaa,b, Abhilash Jayaraja,b, Anjali tions. The enzyme has two isoforms with 70% sequence simi- Sonia,b, Ashutosh Shandilyaa,b, Vandana Shekharb, larity. Our earlier studies with dopamine-hMAO B showed Shashank Shekharb and B. Jayarama,b,c persistence of trans form at the active site cavity (Dasgupta, aDepartment of Chemistry, Indian Institute of Technology, Delhi, Mukherjee, Mukhopadhyay, Banerjee, & Mishra, 2018) b Hauz Khas, New Delhi, 110016, India; Supercomputing Facility for whereas MD —simulation followed by DFT studies for Bioinformatics & Computational Biology, Indian Institute of phenylethylamine(PEA)-hMAO B complex reveals existence of Technology, Delhi, Hauz Khas, New Delhi 110016, India c both trans and gauche conformation for protonated PEA and Kusuma School of Biological Sciences, Indian Institute of (Dasgupta, Mukherjee, & Mukhopadhyay, 2018). In this work Technology, Delhi, Hauz Khas, New Delhi, 110016, India [email protected]; [email protected] concerted hydride transfer mechanism for trans and gauche conformers of neutral-PEA with lumiflavin moiety (truncated Big data generation through sequencing of genomes and prosthetic group involved during catalysis) have been per- proteomes has led to over 2800 whole genomes and 84 formed by density functional theory (DFT) methods using million protein sequences, however, utilization of these data B3LYP/DFT-D3 functional with standard split valance basis set to generate lead molecules for curing diseases remains a 6-31 g. This computational work provides the first evidence challenge. We propose here, Dhanvantari, a comprehensive that could support the easier feasibility of deamination of software suite which automates the computational journey gauche conformer of hMAO substrates over trans-substrate. from genome to hit molecules via its various genomics, pro- The activation energy barriers for trans and gauche PEA are teomics and drug designing modules with possible entry at compared, in which gauche conformation is observed to be any module. The proposed software suite offers new oppor- preferred over trans conformer. The result indicates that the tunities and insights to ‘genome-based’ drug discovery along deamination of the neurotransmitters may depend on the con- with classical structure-based approaches to discover new formational preference of phenylethylamine (Figure 1). drug-like molecules. The pipeline helps in exploring new potential drug targets from genomic/proteomic data which Funding were earlier inaccessible and helps to find novel hits via This research has been financially supported by ICMR screening against million compounds or natural products or (Project No. ISRM/11 (25)/2017), Government of India. FDA approved drugs or even customized molecule libraries.

Figure 1. 18 BOOK OF ABSTRACTS. ALBANY 2019: THE 20TH CONVERSATION

Figure 1.

Case studies on Hepatitis B Virus and Hepatitis A Virus receptor of M. tb using AutoDock (v 4.0) and Hex. Molecular against their druggable proteins via this pipeline have led to Dynamic (MD) simulation of this docked receptor-ligand potent and novel inhibitors with low micro molar range complex using Gromacs (v.4.2) was then conducted. inhibitions in in vitro studies. The entire protocol proposed in Fluoroamine derivative of EGCG inhibited Mycobacterium Dhanvantari requires 6–12 h, however, individual steps get smegmatis, in silico (dock energy -10.9 kcal mol-1), in vitro completed within few minutes. The software suite is made and also inhibited the tyrosine kinase (TK) receptor Her2 and freely accessible as an online resource at http://www.scfbio- taxanes (TXL) microtubule receptor of cancer cells. The fluo- iitd.res.in/software/dhanvantari_new/Home.html with no add- roamine derivative also showed better inhibition of itional dependencies. Presently, there is no fully-automated Mycobacterium smegmatis than EGCG based on these studies. open source similar to Dhanvantari which can mine the infor- Further, MD simulation of the fluoroamine derivative sub- mation about the potential drug like molecules from the stantiated the docking results, since the receptor ligand com- genome as well as proteome information (Figure 1). plex showed greater stability than the receptor protein after 50 ns of MD simulation as evidenced from the RMSD, RMSF and energy profiles (Potential Energy, Kinetic Energy and References Total Energy) in line with observations of Gholami and Soni, A., Pandey, K. M., Ray, P., & Jayaram, B. (2013). Genomes to hits in Bordbar (2017). The anti-cancer properties of the fluoroamine silico—a country path today, a highway tomorrow: A case study of derivative evaluated in vitro in the Jurkat human leukemia Chikungunya. Current Pharmaceutical Design, 19(26), 4687–4700. cell line, showed almost a tenfold better inhibition than EGCG as reported in the literature. Taken together, these findings hold much promise for further study of the anti-TB 34. A fluoroamine derivative of and anti-cancer properties of the fluoroamine derivative. epigallocatechin gallate exhibits both, anti-cancer and anti-tuberculosis References properties. Insights from in silico and Gholami, S., & Bordbar, A. K. (2017). Putative binding sites of dopamine and arachidonoyl dopamine to beta lactoglobulin: A molecular docking and in vitro studies molecular dynamics study. Physical Chemistry Research, 5(2), 205–219. Hoagland, D. T., Liu, J., Lee, R. B., & Lee, R. E. (2016). New agents for the Samarendra Narayanan, Ramesh and treatment of drug resistant Mycobacterium tuberculosis. Advanced K.V. Dipti Mothay Drug Delivery Reviews, 102, 55–72. School of Sciences, Jain University, Jayanagar, Bangalore 560011, India [email protected]; [email protected] 35. A new approach to analyze Epigallocatechin gallate (EGCG), has been reported to have rotavirus transport mechanism in both anti-cancer and anti- mycobacterial properties. porous media by molecular modeling Fluorinated compounds are known to inhibit both TB and cancer (Hoagland et al., 2016). Few derivatives of EGCG were and molecular dynamics methods drawn structurally and subsequently chemically synthesized. A derivative with a fluorine and amine moiety was analyzed Elena M. Alvareda Migliaroa,b , Jorge Canterob, for inhibitory properties against (M. tb) and cancer using in Fernando Lopez Tortc, Matıas Victoriac, silico, and in vitro approaches. In silico approach included Margot Paulino Zuninib, Rodney Colinac and docking of the optimized ligand to the enoyl reductase (ER) Pablo A. Gamazoa JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS VOL. 37, SUPPLEMENT, 2019 19 aWater Department, CENUR, Universidad de la Republica, Salto, ORCID b Uruguay; Center for Structural Bioinformatics, DETEMA - Facultad Elena M. Alvareda Migliaro http://orcid.org/0000-0002-6065-5741 de Quımica, Universidad de la Republica, Montevideo, Uruguay; cVirology Laboratory, CENUR, Universidad de la Republica, Salto, Uruguay [email protected] References

It has always been believed that groundwater was a safe Gamazo, P., Victoria, M., Schijven, J. F., Alvareda, E., Lopez, T. L. F., Ramos, J., … Colina, R. (2018). Evaluation of bacterial contamination source, due to the ‘naturalfilter’ which takes place in porous as an indicator of viral contamination in a sedimentary aquifer in media. However, groundwater could be polluted with micro- Uruguay. Food and Environmental Virology,1–11. organism or chemical products from wastewater from septic Gamazo, P., Victoria, M., Schijven, J. F., Alvareda, E., Tort L. F, L., Ramos, tanks or leaking sewage pipes. In most cases people drink J., … Colina, R. (2016). Comparison of rotavirus and norovirus trans- this underground water without treatment and this could be port in standardised and natural soil-water systems, Abstract H33P-06 the source of viral gastroenteritis outbreaks in Salto city, presented at 2016 Fall Meeting, AGU, San Francisco, Calif., 11-15 Dec. Settembre, E. C., Chen, J. Z., Dormitzer, P. R., Grigorieff, N., & Harrison, located on the Northwestern region of Uruguay. The evalu- S. C. (2011). Atomic model of an infectious rotavirus particle. The ation of groundwater pollution in Salto by means of the EMBO Journal, 30(2), 408–416. presence and the incidence of human gastroenteric viruses like Rotavirus A has been studied recently. Furthermore, to understand the transport mechanisms of this virus, a study 36. Evidence for the N-Terminal of the kinetic aspects of the adsorption isotherm mecha- hypothesis for Alzheimer’s disease nisms by means of column assays have been performed by our group. These results showed that Rotavirus A was Georges Belfort detected in the Salto aquifer, and similar concentrations in Howard P. Isermann Department of Chemical and Biological Salto sewer effluent were measured. The aim of this study Engineering and the Center for Biotechnology and was to understand the main molecular characteristics respon- Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA sible for the Rotavirus A transport mechanism from natural soil-water systems that could affect it adsorption and inacti- Although the amyloid (abeta peptide, Ab) hypothesis is vation in porous media. We proposed: (1) A 100 ns explicit 25 years old, is the dominant model of Alzheimer’s Disease solvent MD simulation of the crystal structure of the rhesus (AD) pathogenesis, and currently guides the development of rotavirus VP4 sialic acid binding domain in complex with 2- potential treatments, it is still controversial. One possible rea- O-methyl-alpha-D-N-acetyl neuraminic acid (PDBid: 1KQR) son is a lack of a clear mechanistic path from the cleavage with NAMD program. (2) A study of the interaction energies products of the amyloid precursor protein (APP) such as sol- of solvent-protein. (3) An analysis of the interactions uble Ab monomer and a series of recently discovered soluble between the VP4 virus protein with an inorganic silica mem- molecular fragments to the deleterious effects on synaptic brane model by Stereed Molecular Dynamic (SMD) to explain form and function (Willem et al., 2015; Welzel et al., 2014). the structural and energetic features of protein membrane Both biophysical properties of these molecular entities and association. We obtained a validated model of VP4 with the the balance between production and clearance are consid- sialic acid ligand and we discussed the molecular interactions ered critical for AD pathogenesis. From a review of the of Rotavirus A in an inorganic environment (Figure 1). recent literature including aggregation kinetics (Murray et al., 2016) and structural morphology (Murray et al., 2016), A Funding b clearance (Murray et al., 2016), molecular simulations (Das, CSIC and National Research and Innovation Agency ANII Murray, & Belfort, 2015), long-term potentiation measure- (FMV_2_2011_1_6927 project) have supported this research. ments with inhibition binding, and the binding of a commer- cial monoclonal antibody, aducanumab, we hypothesize that

Figure 1. 20 BOOK OF ABSTRACTS. ALBANY 2019: THE 20TH CONVERSATION the N-terminal domains of neurotoxic Ab oligomers are Welzel, A. T., Maggio, J. E., Shankar, G. M., Walker, D. E., Ostaszewski, implicated in causing the disease. We call this the ‘N- B. L., Li, S., … Walsh, D. M. (2014). Secreted amyloid b-proteins in a Terminal Hypothesis for AD’. The supporting evidence related cell culture model include N-terminally extended peptides that impair synaptic plasticity. Biochemistry, 53(24), 3908–3921. to this hypothesis includes the discovery of the following: (i) Willem, M., Tahirovic, S., Busche, M. A., Ovsepian, S. V., Chafai, M., The first protective mutation of APP (A673T) or Ab (A2T) Kootar, S., … Daria, A. (2015). g-Secretase processing of APP inhib- against AD reduces BACE1 cleavage at the b-secretase pos- its neuronal activity in the hippocampus. Nature, 526(7573), ition (N-terminus of Ab) resulting in a drop in Ab concentra- 443–447. tion of about 40% (Jonsson et al., 2012). (ii) A causative mutation at the same location (A673V) or Ab (A2V) against AD results in an increase in Ab concentration of about 100%. 37. Calculation of protein–ligand and (iii) The A2T and A2V mutants of Ab1-42 increase the aggregation lag time prior to the onset of fibril formation com- protein–protein binding affinities pared with wild type Ab by a factor of 1.5 and 8, 1-42 using free energy perturbation theory respectively (Murray et al., 2016). (iv) Aggregate morphology of the N-terminal mutants (A2T and A2V) is altered compared Richard A. Friesner with wild type Ab (Murray et al., 2016). (v) Molecular 1-42 Department of Chemistry, Columbia University, 3000 Broadway, dynamic (MD) simulations show the importance of N-ter- New York, NY 10027, USA [email protected] minus on monomer folding and lowering of the formation of neurotoxic b-hairpin structures (Das et al., 2015). (vi) Long- Over the past several years, free-energy perturbation (FEP) term potentiation (LTP) deficit, which correlates with memory molecular dynamics simulation methods have been shown and learning, is reduced for the A2T Ab1-42 mutant, in com- to be capable of achieving useful prediction of protein–li- parison with the wild type and A2V Ab1-42 mutant (Murray gand binding affinities, with a root-mean-square error on et al., 2016). (vii) Blocking the N-terminus with sequence spe- the order of 1 kcal/mole. Recently, results of similar quality cific antibodies prevents LTP deficit whereas blocking the have been obtained for protein–protein binding affinities hydrophobic central core and C-terminus have minimal as well. At present, high-resolution structural data are effects. (viii) Fragments of APP induce LTP deficits only when required as a starting point for these calculations to suc- the N-terminus is included in the fragment (Willem et al., ceed. We will outline the methodological advances ena- 2015). (ix) A promising recombinant human monoclonal anti- bling reliable and accurate results to be obtained, and body, aducanumab, binds to the N-terminus of A oligomers. b discuss applications to structure-based drug discovery. (x) Recently, reported experimental structures also show a Finally, a brief discussion of new induced fit docking meth- flexible/exposed N-terminal region (Ab ) in the disease 1-14 ods, which will enable FEP to be effectively utilized in the relevant Ab fibril (Walti et al., 2016). Additionally, since 1-42 absence of a high-resolution crystal structure, will monomers are not neurotoxic while dimers, trimers etc. are, be presented. this suggests that at least two N-termini are necessary for LTP deficit induction. It then follows that the mechanism of toxicity could be due to multivalent interactions between the N-termini ( 2) of Ab oligomers with glutamate N-methyl 38. Cancer vaccine designing and D-aspartate receptors. Unpublished MD simulations demonstrate increased flexibility of the protective versus the causa- optimization using combined tive variants for N-termini in dimers. Taken together, these immunoinformatics and molecular collective findings strongly suggest that the N-terminus of dynamics simulation approach Ab oligomers could be causative for AD. Neeraj Kumar and Ramesh Chandra References Department of Chemistry, University of Delhi, Delhi 110007, India [email protected]; [email protected] Das, P., Murray, B., & Belfort, G. (2015). Alzheimer’s protective A2T mutation changes the conformational landscape of the Ab 1–42 monomer differently than does the A2V mutation. Biophysical Journal, 108(3), Cancer immunotherapy comes out to be an outstanding 738–747. treatment for cancer treatment and prevention. Cancer vac- Jonsson, T., Atwal, J. K., Steinberg, S., Snaedal, J., Jonsson, P. V., cines are responsible for generating immune response to Bjornsson, S., … Stefansson, K. (2012). A mutation in APP protects eliminate cancer cells, being given to cure cancer. In silico ’ against Alzheimer s disease and age-related cognitive decline. Nature, techniques has brought efficient way to develop vaccines, as 488(7409), 96–99. Murray, B., Sorci, M., Rosenthal, J., Lippens, J., Isaacson, D., Das, P., … these take less time to predict potential peptide epitope Belfort, G. (2016). A2T and A2V Ab Peptides exhibit different aggrega- nonamers (Buhrman et al., 2013; Neeraj et al., 2017). In this tion kinetics, morphology, structure and LTP inhibition. Proteins: study, we have designed a potent vaccine candidate for can- Structure, Function, and Bioinformatics, 84(4), 488–500. cer using immunoinformatics approaches. Cytotoxic T- Walti, M. A., Ravotti, F., Arai, H., Glabe, C. G., Wall, J. S., Bockmann, A., … Riek, R. (2016). Atomic-resolution structure of a disease-relevant lymphocyte epitope as a Peptide vaccine was designed to Abeta(1-42) amyloid fibril. Proceedings of the National Academy of elicit a desirable immune response against cancers, overex- Sciences of the United States of America. pressing melanoma-associated antigen-A11 (MAGE-A11). Potent JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS VOL. 37, SUPPLEMENT, 2019 21

Figure 1.

epitope was predicted using reliable algorithms and validated Three sets of carboline derived compounds were prepared with HLA-AÃ0201 and androgen receptor interaction (Dong et al by Pictet-Spengler cyclization. These tetrahydro b- and c-car- 2004; Henderson, Mossman, Nairn, & Cheever, 2005). bolines have CF3 group with an additional amino alkyl chains Our results showed a potent vaccine candidate ‘KIIDLVHLL’ (a- or d-position) and guanidine alkyl chains (a-position), of designed out of top ten epitope epitopes predicted using five varying length. Structure–activity relationship of these mole- different algorithms. Furthermore, epitope was validated with cules with calf thymus DNA was emphasized by fluorescence, molecular docking resulted in strong binding with HLA- ITC, FTIR and viscosity. Binding with DNA resulted in dra- AÃ0201 and androgen receptor with docking score -780.6 kcal/ matic enhancement and quenching in the fluorescence emis- mol and -641.06 kcal/mol respectively. Lead epitope showed sion. Gamma-carboline analogs showed maximum DNA the remarkable result with strong interactions (hydrogen binding followed by beta-carboline compounds with amino bonds and hydrophobic interactions), the radius of gyration alkyl chain and least with guanidine alkyl chain compounds. score of 23.0777 Å, world population coverage of 39.08% by It decreased with increasing chain length. The bindings were immune epitope database and TAP affinity IC50 value of entropically driven being more with guanidine alkyl chain 2039.65 nm. This study paves the way to potential vaccine can- analogs. Site preference and mode of binding with partial didate for the prevention of cancer (Figure 1). intercalation and external binding was supported by FTIR and viscosity. Cytotoxic potencies of the compounds were References tested on seven different cancer cell lines. The smallest alkyl chain analog attached to gamma position, Comp3, showed Buhrman, J. D., & Slansky, J. E. (2013). Improving T cell responses to modified peptides in tumor vaccines. Immunologic Research, 55(1–3), maximum cytotoxicity with GI50 6.2 mM, against HCT-116 34–47. causing apoptosis, followed by the guanidine alkyl chain Dong, H.-L., Li, Z.-S., Ye, J., Qu, P., Huang, Y.-Y., Wu, W., … Sui, Y.-F. compounds, but amino alkyl chain compounds to beta pos- (2004). Identification of HLA-A2-restricted CTL epitope encoded by ition showed poor cytotoxicity. the MAGE-n gene of human hepatocellular carcinoma. Cancer Biology These results may be of prospective use in a framework & Therapy, 3(9), 891–898. Henderson, R. A., Mossman, S., Nairn, N., & Cheever, M. A. (2005). Cancer to design novel carboline derivatives as antitumor drugs for vaccines and immunotherapies: Emerging perspectives. Vaccine, improved therapeutic applications in future. 23(17/18), 2359–2362. Neeraj, K., Chugh, H., Tomar, R., Tomar, V., Singh, V. K., & Chandra, R. (2017). Exploring the interplay between autoimmunity and cancer to Acknowledgment find the target therapeutic hotspots. Artificial Cells, Nanomedicine, and Biotechnology: An International Journal, 46 (4), 658–668. [10.1080/ I would like to acknowledge Prof. Valentine Nenajdenko and team from 21691401.2017.1350188] Moscow State University, Moscow, for the synthesis of the compounds and also grateful to DST-RFBR 2017-19 for funding of the research. 39. Carboline derivative compounds 40. Antioxidant enzyme catalase targeting DNA: synthesis, binding and activity of rat kidney cells nuclear in vitro cytotoxicty on human cancer fraction after the cisplatin and cell lines estradiol separate and joint action Kakali Bhadra Zhenya V. Yavroyan, Nune R. Hakobyan, Agapi G. Department of Zoology, University of Kalyani, Kalyani, 741235, West Bangal, India [email protected] Hovhannisyan, Gevorg A. Avagyan and Emil S. Gevorgyan KEYWORDS carboline derivative compounds; nucleic acid-small molecular Department of Biophysics, Yerevan State University, Yerevan, binding; FTIR; ITC; cell cytotoxicity; apoptosis Armenia [email protected] 22 BOOK OF ABSTRACTS. ALBANY 2019: THE 20TH CONVERSATION

Cisplatin is a widely used and highly effective cancer chemo- Our previous results showed the reliable changes in phos- therapeutic agent. Csplatin-induced cell death involves the pholipids in rat brain chromatin preparations after the in vivo activation of multiple pathways of apoptoses as well as oxidant action of cisplatin. It is impossible to exclude the significance stress activation. These same pathways contribute to the cyto- of chromatin lipids quantitative alterations for cisplatin anti- toxic actions of cisplatin on tumor cells. However, it must be tumor effects realization. Unfortunately the using of this noted that cisplatin provided severe undesirable side effects. The drug is greatly limited because of its severe side effects. The most common side effect of cisplatin is nephrotoxicity It is well results of recently investigations established that combin- known that oxidative stress has been recognized as an important ation of steroid hormone estradiol and cisplatin in chemo- factor that contributes to cisplatin nephrotoxicity. Nowadays therapy treatment schemes decreased cisplatin induced many efforts have been made to employ drugs and other medi- toxicities (Ghasemi et al., 2016). caments as candidate adjuvants to cisplatin to minimize this Taking intj foregoing consideration, it may be interest- adverse influence. Steroid hormones such as sex hormones estra- ing to explore the cisplatin and estradiol joint in vivo diol and progesterone are presently considered as the best adju- action on total quantities of rat brain chromatin phospholi- vant to cisplatin, are able to prevent intoxication (Grott et al., pids as well as changes of their individual frac- 2013;Nematbakhshetal.,2017). It is well known also, that cis- tions content. platin inhibits while estradiol promotes the activity of antioxidant Our results revealed reliably decrease (by 24%) of the enzymes. The aim of this research was to explore of catalase activ- total amount of phospholipids from rat brain chromatin ity in female rats kidney cells nuclear fraction after the cisplatin preparations after treatment with the antitumor drug cis- and estradiol separate and jointly action. As demonstrated our platin whereas the combinated injection of cisplatin and results cisplatin and estradiol provided opposite effects on cata- estradiol restored the baseline level. Joint use of cisplatin lase activity in case of separate application. In comparison with and estradiol lead to recovery the compliance rate of baseline cisplatin alone action leads to decrease the catalase phosphatidylcholine and phosphatidylethanolamine content. activity by 24%, whereas after the estradiol separate treatment Unlike this the quantity of sphingomyelin and phosphati- the activity of this enzyme increases by 26%. The combinated use dylinositol was increased in comparison to baseline as well of cisplatin and estradiol leads to partial recovery of catalase activ- as cisplatin treatment variants. Cisplatin and estradiol com- ity. In this case was recorded decrease of enzyme activity by 15%. binated treatment provides significant effect on cardiolipin Achieved results may be helpful for explaining of estradiol attenu- content. The absolute quantity of this phospholipid ating effects in case of its joint use with cisplatin. reduced by 38% instead of 15% in case of cisplatin alone treatment. References Changes in content of different phospholipids fractions caused by cisplatin in vivo action may play a definite role in Grott, M., Karakaya, S., Mayer, F., Baertling, F., Beyer, C., Kipp, M., & Kopp, nuclear lipid metabolic pathways. These changes should be H. G. (2013). Progesterone and estrogen prevent cisplatin-induced apoptosis of lung cancer cells. Anticancer Research, 33(3), 791–800. considered particular negative side effects during the basic Nematbakhsh, M., Pezeshki, Z., & Eshraghi Jazi, F. (2017). Cisplatin- antitumor effect of cisplatin. However, the changes of phos- induced nephrotoxicity; protective supplements and gender differen- pholipids in case of cisplatin and estradiol combinated treat- ces. The Asian Pacific Journal of Cancer Prevention, 18(2), 295–314. ment may be helpful for reducing of cisplatin toxicity and eliminating of its side effects.

References

41. Cisplatin and estradiol joint action Ghasemi, M., Nematbakhsh, M., Pezeshki, Z., Soltani, N., Moeini, M., & on quantities of rat brain chromatin Talebi, A. (2016). Nephroprotective effect of estrogen and progesterone combination on cisplatin-induced nephrotoxicity in ovariectom- phospholipids ized female rats. Indian Journal of Nephrology, 26(3), 167–175. 2 Kuvichkin, V. V. (2016). DNA-Lipids-Me þ complexes structure and their Nune Hakobyan, Zhenya Yavroyan, Agapi G. possible functions in a cell. Journal of Chemical Biology and Hovhannisyan and Emil S. Gevorgyan Therapeutics, 1(1), 1–6. Department of Biophysics, Yerevan State University, Yerevan, Armenia [email protected] 42. Click chemistry in silico and Nowadays, it is well known that nuclear lipids, including the chromatin bound ones, are important for regulating many essen- molecular simulation studies to tial cellular processes such as DNA replication, transcription and identify novel HIV-1 entry inhibitor gene expression. Recent advances demonstrated the involve- scaffolds targeting CD4-binding site ment of nuclear lipids in remodeling of chromatin and epigenetic regulation of gene expression. Furthermore, it was shown of the envelope gp120 protein that lipids of chromatin can regulate the chromatin structure via alteration of some enzymes activity, which accomplished histone Alexander M. Andrianova, Grigory I. Nikolaevb, Yuri V. modifications as well as inverse processes (Kuvichkin, 2016). Kornoushenkoa and Alexander V. Tuzikovb JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS VOL. 37, SUPPLEMENT, 2019 23

Irwin, J. J., Sterling, T., Mysinger, M. M., Bolstad, E. S., & Coleman, R. G. aInstitute of Bioorganic Chemistry, National Academy of Sciences (2012). ZINC: A free tool to discover chemistry for biology. Journal of of Belarus, Minsk 220141, Belarus; bUnited Institute of Informatics – Problems, National Academy of Sciences of Belarus, Minsk 220012, Chemical Information and Modeling, 52(7), 1757 1768. Belarus [email protected] Lipinski, C. A., Lombardo, F., Dominy, B. W., & Feeney, P. J. (2001). Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. In silico design of novel potential HIV-1 entry inhibitors able Advanced Drug Delivery Reviews, 46(1-3), 3–26. to block CD4-binding site of the envelope gp120 protein was carried out based on the click chemistry concept. In doing so, a Drug-Like subset of the ZINC database (Irwin, Sterling, Mysinger, Bolstad, & Coleman, 2012) was screened by the DataWarrior program to generate two virtual 43. Computational design of novel molecular libraries. Library 1 comprised small molecules 1,2,4-triazole-based compounds as with an azide group or an alkyne group and aromatic fragments critically important for the HIV-1 attachment to cellu- potential aromatase inhibitors lar receptor CD4. In library 2, all low-molecular compounds a b with an azide group or an alkyne group were collected. Alexander M. Andrianov , Grigory I. Nikolaev , Yuri V. a a The modular units from libraries 1 and 2 were then used as Kornoushenko and Sergei A. Usanov reagents to mimic the click chemistry reaction of azide- aInstitute of Bioorganic Chemistry, National Academy of Sciences b alkyne cycloaddition by the AutoClickChem program of Belarus, Minsk 220141, Belarus; United Institute of Informatics Problems, National Academy of Sciences of Belarus, Minsk 220012, (Durrant & McCammon, 2012). This resulted in a set of 1 Belarus [email protected] 655 301 hybrid molecules. 294 378 compounds that fully ’ ‘ ’ satisfied Lipinski s rule of five (Lipinski, Lombardo, Dominy, In women organism during the fertile phase, estrogen syn- & Feeney, 2001) were further screened by high-throughput thesis occurs mainly in the ovaries. However, the intensity of docking and molecular dynamics simulations to evaluate estrogen synthesis in the ovaries decreases in postmeno- the affinity of their binding to the target protein. As a pause associated with about a third of cases of breast can- result, five top hits (Figure 1) that exhibited strong attach- cer. At this phase, estrogens synthesized in the peripheral ment to the two well-conserved hotspots of the gp120 tissues using the cytochrome P450 enzyme complex, called CD4-binding site were selected for the final analysis. In ana- aromatase. This complex consists of the heme-containing logy to CD4, the identified compounds form hydrogen cytochrome P450 (CYP19A1) protein and flavoprotein bonds with Asp-368 and multiple van der Waals con- gp120 NADPH-cytochrome P450 reductase. Aromatase that is tacts with the gp120 residues that bind to Phe-43 CD4, encoded by a single large gene, CYP19A1, catalyzes conver- resulting in destruction of the critical interactions of gp120 sion of androgens to estrogens and exhibits biological activ- with Phe-43CD4 and Arg-59CD4. The complexes of the CD4- mimetic candidates with gp120 show relative conformity in both peripheral target tissues and in the mammary ational stability within the molecular dynamics simulations tumor tissues, providing a high level of estrogen concentra- and expose the high percentage occupancies of intermo- tion. In estrogen-dependent malignant neoplasms, estrogens lecular H-bonds, in line with the low values of binding free act as growth factors for tumor development. Therefore, energy. In this context, the identified compounds are con- inhibition of aromatase results in a decrease in the level of sidered as good scaffolds for the development of new func- estrogen in the organism and prevention of the growth and tional antagonists of viral entry with broad HIV-1 spread of cancer cells. neutralization. The third-generation aromatase inhibitors (AIs), which are now used as first-line therapy in the treatment of early- and advanced-stage breast cancer in postmenopausal women, References include two categories: the reversible nonsteroidal inhibitors Durrant, J. D., & McCammon, J. A. (2012). AutoClickChem: Click chemistry anastrozole and letrozole and the steroidal inhibitor exemes- in silico. PLoS Computational Biology, 8(3), e1002397 tane. Although these AIs are currently popular and effective

Figure 1. Chemical structures of the potential HIV-1 entry inhibitors. The atoms of hydrogen, oxygen and nitrogen forming hydrogen bonds in the dynamic structures of the complexes between the ligands and HIV-1 gp120 protein are numbered. 24 BOOK OF ABSTRACTS. ALBANY 2019: THE 20TH CONVERSATION in the treatment of postmenopausal estrogen receptor positive breast cancer, the search for novel drugs still 44. Computational modeling and remains necessary to avoid the risk of possible emerging engineering of allosteric regulatory resistances to available drugs as well as to reduce toxicity and undesirable side effects associated with a pro- mechanisms in signaling proteins: longed use. integration of multiscale simulations, In this study, computational development of novel tri- network biology and azole-based AIs was carried out followed by evaluation of their antitumor activity by tools of molecular modeling. In machine learning doing so, in silico design of potential AIs candidates fully a,b a satisfying the Lipinski’s ‘rule of five’ was performed using Gennady M. Verkhivker , Steve Agajanian , a a the concept of click chemistry. Complexes of these drug- Nathaniel Bischoff , Lindy Astl and Kristin a,c like molecules with the enzyme were then simulated by Blacklock a molecular docking and optimized by semiempirical quan- Department of Biomedical and Pharmaceutical Sciences, Schmid tum chemical method PM7. To identify the most promising College of Science & Technology and School of Pharmacy, Chapman University, Irvine, CA 92866, USA; bDepartment of compounds, stability of the PM7-based ligand/aromatase Pharmacology, University of California San Diego, San Diego, CA structures was estimated in terms of the values of binding 92093, USA; cDepartment of Chemistry and Chemical Biology, free energy and dissociation constant. As a result, eight Rutgers University, Piscataway, NJ 08854, USA hits that specifically interact with the aromatase catalytic [email protected] site and exhibit the high-affinity ligand binding were selected for the final analysis. All these compounds are The allosteric interactions allow for molecular communication shown to coordinate the iron atom of the aromatase and event coupling in signal transduction pathways and net- heme group and form multiple van der Waals contacts works. The overarching goal of understanding molecular with the critically important residues of the enzyme hydro- principles underlying recognition of protein kinase clients phobic pocket, such as Arg-115, Ile-133, Phe-134, Trp-224, and chaperone-based modulation of kinase activity is funda- hr-310, Val-370, Met-374, Leu-477 and Ser-478. Six of mental to activity of many signaling proteins. The synergistic eight compounds form hydrogen bond with Met-374 mim- roles of the Hsp90-Cdc37 chaperone machinery and protein icking the interaction of aromatase with the natural sub- kinases in biology and disease have stimulated extensive strate androstenedione. In addition, individual ligands are structural and functional studies of regulatory mechanisms. also involved in specific p- or T-stacking interactions with Allosteric interactions of the Hsp90 with cochaperones and the pyrrole rings of the enzyme heme group as well as protein kinase clients (left panel of Figure 1) derived from participate in hydrogen bonding with Thr-310, Leu-372, recent crystal structures (Verba & Agard, 2017) can determine Leu-477 and Ser-478. The selected AIs candidates show regulatory mechanisms and cellular functions of many signal- strong attachment to the enzyme active site, in line with ing proteins and cascades. We report the results of multiscale the low values of binding free energy and dissoci- simulations and network biology studies of the Hsp90 chap- ation constant. erones and a panel of protein kinases with an atomic-level Taken together, the data obtained suggest that the identi- analysis of regulatory dynamic changes and binding of these fied compounds may present good scaffolds for the develop- protein systems in signaling networks (Czemeres, Buse, & ment of novel potent drugs against breast cancer. Surely, the Verkhivker, 2017). The results of biophysical and computa- properties of these virtual compounds warrant further bio- tional biology studies combined with biochemical experi- logical characterization as cellular assays to confirm in vitro ments have been used to derive a network model of kinase their interesting in silico profile. regulation by Hsp90 chaperones (shown on the right panel

Figure 1. JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS VOL. 37, SUPPLEMENT, 2019 25 of Figure 1). Among our primary findings is the evidence differences responsible for changes in molecular recogni- that a small number of functional motifs may be utilized by tion. We perform pMD simulations on complexes with the chaperone and protein kinases to act as central regula- varying protein sequence and ligand identity to unravel tors of protein client binding in signaling networks. This structural and dynamic properties that underlie coupled work has offered atomistic insights into mechanisms of kin- changes in molecular recognition and resistance. We have ase activation by molecular chaperones orchestrated by dir- applied this pMD strategy to protein-ligand complexes for ection-specific cross-talk between key regulatory regions. The series of natural substrates, inhibitors and protease muta- developed platform of atomistic simulations and network tions. Combined with experimental potency data, we deter- biology approaches is also integrated with machine learning- mine which physical properties or molecular interactions, based models of allosteric regulation for oncogenic proteins including with water, are key to molecular recognition for in signaling cascades. a given system. These changes in structure and dynamics dictate the interdependency of molecular mechanism of resistance Funding and are principals that are generally applicable to other This research has been supported by institutional funding quickly evolving diseases where drug resistance is from Chapman University. quickly evolving.

References References

Czemeres, J., Buse, K., & Verkhivker, G. M. (2017). Atomistic simulations Leidner, F., Kurt Yilmaz, N., Paulsen, J., Muller, Y. A., & Schiffer, C. A. and network-based modeling of the Hsp90-Cdc37 chaperone binding (2018). Hydration structure and dynamics of inhibitor-bound HIV-1 with Cdk4 client protein: A mechanism of chaperoning kinase clients protease. Journal of Chemical Theory and Computation, 14(5), by exploiting weak spots of intrinsically dynamic kinase domains. 2784–2796. doi: 10.1021/acs.jctc.8b00097. Epub 2018 Apr 18. PLoS One, 12(12), e0190267. Matthew, A. N., Leidner, F., Newton, A., Petropoulos, C. J., Huang, W., Ali, Verba, K. A., & Agard, D. A. (2017). How Hsp90 and Cdc37 lubricate kin- A., … Schiffer, C. A. (2018). Molecular mechanism of resistance in a ase molecular switches. Trends in Biochemical Sciences, 42(10), clinically significant double-mutant variant of HCV NS3/4A protease. 799–811. Structure, 26(10), 1360–1372.e5. doi: 10.1016/j.str.2018.07.004. Epub 2018 Aug 23. Ozen,€ A., Lin, K. H., Kurt Yilmaz, N., & Schiffer, C. A. (2014). Structural basis and distal effects of Gag substrate coevolution in drug resist- 45. Constraining evolution—avoiding ance to HIV-1 protease. Proceedings of the National Academy of drug resistance: lessons from viruses Sciences of the United States of America, 111(45), 15993–15998. doi: 10.1073/pnas.1414063111. Epub 2014 Oct 29. Paulsen, J. L., Leidner, F., Ragland, D. A., Kurt Yilmaz, N., & Schiffer, C. A. Celia Schiffer (2017). Interdependence of inhibitor recognition in HIV-1 protease. Institute for Drug Resistance, University of Massachusetts Medical Journal of Chemical Theory and Computation, May 913(5), 2300–2309. School, Worcester, MA 01655, USA doi: 10.1021/acs.jctc.6b01262. Epub 2017 Apr 11. Prachanronarong, K. L., Ozen,€ A., Thayer, K. M., Yilmaz, L. S., Zeldovich, Drug resistance negatively impacts the lives of millions of K. B., Bolon, D. N., … Schiffer, C. A. (2016). Molecular basis for differ- patients and costs our society billions of dollars by limiting ential patterns of drug resistance in influenza N1 and N2 neuraminid- ase. Journal of Chemical Theory and Computation, 12(12), 6098–6108. the longevity of many of our most potent drugs. Drug Epub 2016 Nov 17. resistance can be caused by a change in the balance of Ragland, D. A., Nalivaika, E. A., Nalam, M. N., Prachanronarong, K. L., Cao, molecular recognition events that selectively weakens H., Bandaranayake, R. M., … Schiffer, C. A. (2014). Drug resistance inhibitor binding but maintains the biological function of conferred by mutations outside the active site through alterations in the target. To reduce the likelihood of drug resistance, a the dynamic and structural ensemble of HIV-1 protease. Journal of the – detailed understanding of the target’s function American Chemical Society, 136(34), 11956 11963. doi: 10.1021/ ja504096m. Epub 2014 Aug 18. is necessary. Soumana, D. I., Kurt Yilmaz, N., Ali, A., Prachanronarong, K. L., & Schiffer, Both structure at atomic resolution and evolutionarily con- C. A. (2016). Molecular and dynamic mechanism underlying drug straints on its variation is required. This rationale was derived resistance in genotype 3 hepatitis C NS3/4A protease. Journal of the from our lab’s experience with substrate recognition and American Chemical Society, 138(36), 11850–11859. doi: 10.1021/ drug resistance in HIV, HCV and Influenza. In particular, we jacs.6b06454. Epub 2016 Sep 2. have acquired a rich and versatile experimental dataset of viral proteases altered by the selective pressures of inhibitors. With these data, we are integrating alterations in both the pro- 46. De novo aggregation tein sequence and the inhibitor with changes in potency and we correlate this data to our co-crystal structures and our strat- of alzheimer’sAb25-35 peptides egy of parallel molecular dynamics (pMD) to elucidate molecu- in a lipid bilayer lar mechanisms of drug resistance. pMD is a strategy we have developed to collectively ana- Amy K. Smith and Dmitri K. Klimov lyze a series of MD simulations of similar yet distinct molecu- School of Systems Biology, George Mason University, Manassas, lar complexes to decipher conformational and dynamic VA 20110, USA [email protected] 26 BOOK OF ABSTRACTS. ALBANY 2019: THE 20TH CONVERSATION

One of the mechanisms of cytotoxicity attributed to College of Chemical Science and Technology and Pharmacy, Alzheimer’sAb peptides postulates that their aggregation Yunnan University, Kunming, 650091, P. R. China disrupts membrane structure causing uncontrollable perme- [email protected] 2 ation of Ca þ ions. To gain molecular insights into these processes, we have performed all-atom explicit solvent rep- Dopamine molecules, by combining different sub-types of lica exchange with solute tempering (REST) molecular dopamine receptors, regulate the motor function, cognitive dynamics simulations probing aggregation of the naturally activity, and emotions of living organisms, which are closely occurring Ab fragment Ab25-35 within the DMPC lipid related to the pathology and treatment of Parkinson’s dis- bilayer. To compare the impact produced on the lipid bilayer ease and schizophrenia (Ye et al., 2013; Li et al., 2017; Xie by Ab25-35 oligomers and monomers, we used as a control et al., 2017). In vivo, the production of dopamine molecules our previous simulations, which explored binding of Ab25-35 is by midbrain dopaminergic neuronal tissue. In the substan- monomers to the same bilayer. We found that compared to tia nigra tissue, dopamine molecules are produced in the monomeric species aggregation results in much deeper dopaminergic neuron cells of the anterior membrane, and insertion of Ab25-35 peptides in the hydrophobic core of the the produced dopamine molecules packaged by the vesicles lipid bilayer causing more pronounced disruption in its struc- are released into the intercellular space through the cell ture. Our study indicates that Ab25-35 peptides aggregate membrane. At the intercellular space, various dopamine by incorporating monomer-like structures with stable C-ter- receptors are present on the dopaminergic neuron cell mem- minal helix. As a result, the Ab25-35 dimer features unusual branes of the posterior membrane cells. These receptors can helix head-to-tail topology supported by a parallel off-regis- accept the binding by dopamine molecules to exert dopa- try interface. The head-to-tail topology readily affords further mine function. However, dopamine molecules placed in the growth of an aggregate by recruiting additional peptides. intercellular space had long been not cared. No one had Free-energy landscape of Ab25-35 binding and aggregation studied the actual part of the dopamine receptors for dopa- reveals that inserted dimers represent the dominant equilib- mine molecules to act. No one had studied for dopamine rium state augmented by two metastable states associated molecules how to work actually. And no one had studied the with surface bound dimers and inserted monomers. Analysis presence of dopamine molecular pathways throughout the of the free-energy landscape allows us to propose the path- dopamine receptors. We have used the dopamine third way and mechanism of Ab25-35 binding, aggregation and receptor crystal structure (Chien et al., 2010), to study the insertion into the lipid bilayer. Our all-atom explicit solvent complex structure of the dopamine molecule with third simulations provide the first, to our knowledge, all-atom receptor protein, to study and obtain the dopamine active description of equilibrium de novo Ab peptide aggregation pocket that exert the functions (Jin et al., 2011), and to study mediated by a lipid bilayer. and obtain dopamine molecular channels within the dopamine receptor structures (Li et al., 2017). There are two types of dopamine molecular channels within the dopamine receptor to be found by us, one is ‘dopamine functional channel’ and the other is ‘dopamine protective channel’. Then, we have further proved the existence of ‘dopamine functional channel’ and the existence of ‘dopamine protective channel’ by a combination of molecular dynamics experiments and animal experiments. Due to the presence of dopamine functional channel, the dopamine molecules released into the intercellular space can be divided into two categories. A class of dopamine molecules that can reach the functional sites through the dopamine functional channel can truly function as a signal transduction and regulation. These functional dopamine molecules are called ‘functional dopamine’. In addition, another class of dopamine molecules, which are nonfunctional dopamine molecules, will be reabsorbed or decomposed. Obviously, functional dopamine occupies a small fraction of total dopamine, similar to drinking water occupying a small fraction of total water. The relationship between functional 47. Dopamine channels with dopamine content and total dopamine content, we have applications studied to establish a formula to represent: G f T (1) D ¼ Â D Zhengqiong Zhang, Xiaowen Zhou, Zeren Xu, wherein GD is a functional dopamine content; TD is the total Chengqi Lu and Sichuan Xu dopamine content (expressed as a percentage); f is the JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS VOL. 37, SUPPLEMENT, 2019 27 dopamine functionalization factor, which is the ratio of total Jin, Y., Wang, Y., Bian, F. Y., Shi, Q., Ge, M. F., Wang, S., … Xu, S. C. dopamine converted to functional dopamine, normalized to (2011). Three-dimensional structure of dopamine 3-subtype receptor 1 as a value for normal healthy persons. with the active site residues for the binding of dopamine. Acta Physico-Chimica Sinica, 27, 2432–2446. Based on the above formula (1), we have clarified the Li, A., Xie, W., Wang, M., & Xu, S. C. (2017). Molecular dynamics of dopa- essence of Parkinson’s disease to be the lack of functional mine to transmit through molecular channels within D3R. Acta dopamine, and proposed the scientific pathology of Physico-Chimica Sinica, 33, 927–940. Parkinson’s disease that is a significant reduction in func- Xie, W., Wang, M., Li, A., & Xu, S. C. (2017). Molecular dynamics simula- tional dopamine to cause Parkinson’s disease. We can scien- tion of d-benzedrine transmitting through molecular channels within tifically name Parkinson’s disease as ‘Functional dopamine D3R. Journal of Biomolecular Structure and Dynamics, 35(8), 1672–1684. ’ deficiency syndrome . According to the scientific pathology Ye, N., Neumeyer, J. L., Baldessarini, R. J., Zhen, X. C., & Zhang, A. (2013). of Parkinson’s disease, all types of Parkinson’s disease can be Recent progress in development of dopamine receptor subtype- included and confirmed without misdiagnosis and missed selective agents: Potential therapeutics for neurological and psychi- diagnosis. According to the scientific pathology of atric disorders. Chemical Reviews, 113(5), PR123–PR178. Parkinson’s disease, new drugs for treating Parkinson’s disease can be found. Depending on the types of Parkinson’s disease, different therapy programs and new drugs can be used able to cure the disease of patients with early and mid- stage Parkinson’s disease, and feasibly better to control 48. Elucidating the molecular patients with the late advanced Parkinson’s disease for keep- interaction of noscapine with bovine ing better symptoms. Our research can make Parkinson’s dis- serum albumin (BSA): spectroscopic ease from a difficult, complex, incurable and uncontrollable disabling disease changed to be a simple, curable or control- and molecular dynamics approach lable disease, although it cannot be eradicated at present. Chhaya Chaudhary, Damini Sood and Ramesh Chandra Funding Department of Chemistry, University of Delhi, Delhi 110007, India [email protected] Support from the National Natural Science Foundation of China (21163024, 21563032). The interaction of the drug with serum albumin forms an integral part of the pharmacokinetic profile of the drug. The References present study employed various spectroscopic techniques i.e., fluorescence, UV-Visible and circular dichroism and Chien, E. Y., Liu, W., Zhao, Q., Katritch, V., Han, G. W., Hanson, M. A., Shi, L., & Stevens, R. C. (2010). Structure of the human dopamine D3 molecular dynamics approach to investigate the mechanism receptor in complex with a D2/D3 selective antagonist. Science, of interaction of an anticancer drug, Noscapine (Ns) with 330(6007), 1091–1095. Bovine Serum Albumin (BSA). Ns binding to BSA leads to the

Figure 1. 28 BOOK OF ABSTRACTS. ALBANY 2019: THE 20TH CONVERSATION quenching of intrinsic fluorescence of BSA, and the mechan- Recent news has warned the medical community that war ism was a static one. The CD spectra showed that Ns binding against pathogenic bacteria is yet not over, repeated breaking to BSA leads to a loss of alpha helicity of the protein. The FT-IR of different drug resistance in different ranges of pathogen has spectra further confirmed the changes in secondary structure been observed in the patient, especially in developing and of BSA upon interacting with Ns. The in vitro results were vali- underdeveloped countries. Many researchers, probably the dated with molecular docking and dynamics techniques. next to cancer are experimentally using many prong attack on Molecular docking showed strong interactions with a high the understanding the mechanism and developing new strat- score. Molecular dynamics simulation analysis also suggested egy of inhibitors for combating this phenomena, resurging the stable binding with lower deviation in RMSD and RMSF XDR, MDR, PDR. The extensive discussion to define these values through persistent long simulation run. The findings classes is available in the literature (Diacon, 2014; Iseman, thus obtained were in good relation with the spectroscopic 1993;Koser€ et al. 2015; Magiorakos, 2011). Research work data. These results suggest the optimal diffusion of Ns into the engaging with clinician, epidemiologist, biologists, microbiolo- bloodstream for the treatment of cancer (Figure 1). gist, etc. in this field is emerging to highlight the complexity of the problem. However, many chemicals have been developed References to combat the MDR/XDR Tb yet not been fully successful for a Checchi, P. M., Nettles, J. H., Zhou, J., Snyder, J. P., & Joshi, H. C. (2003). long time. Tuberculii, bacteria have shown to have multiple Microtubule-interacting drugs for cancer treatment. Trends in spontaneous mutations at a predictable rate and independent Pharmacological Sciences, 24(7), 361–365. of different drug administrations. Such phenomena are preva- Lu, Y., Chen, J., Xiao, M., Li, W., & Miller, D. D. (2012). An overview of lent to many bacterial and more common to viral disease tubulin inhibitors that interact with the colchicine binding site. Pharmaceutical Research, 29(11), 2943–2971. which leads researchers to develop multiple target Naik, P. K., Lopus, M., Aneja, R., Vangapandu, S. N., & Joshi, H. C. (2012). drug designing. In silico inspired design and synthesis of a novel tubulin-binding anti- Present discussion will open up the future of understand- cancer drug: Folate conjugated noscapine (Targetin). Journal of ing of disease, its inter relationship within biochemical path- Computer-Aided Molecular Design, 26(2), 233–247. way and effect of protein–protein interaction identified as target for combating persistence stage in bacteria.

49. Entrapment of flow of substrate References to diversion pathway: strategy Diacon, A. H. (2014). Multidrug-resistant tuberculosis and culture conversion to combat persistent tuberculosis with bedaquiline. New England Journal of Medicine, 371, 8. nejm.org Iseman, M. D. (1993). Treatment of multidrug-resistant tuberculosis. New England Journal of Medicine, 329, 784–791. Indira Ghosh Koser,€ C. U., Javid, B., Liddell, K., Ellington, M., Feuerriegel, J., Niemann, S., … School of Computational & Integrative Sciences, Jawaharlal Nehru Tor€ ok,€ M. E., et al. (2015). Drug-resistance mechanisms and tuberculosis University, New Delhi, India drugs. Lancet, 385(9965), 305–307. doi:10.1016/S0140-6736(14)62450-8. Magiorakos, A.-P. (2011). Multidrug-resistant, extensively drug-resistant The post genomic era has provided the leap into the quantita- and pan drug-resistant bacteria: An international expert proposal for tive biology and introduced biologists into the ‘omes’ and ‘ics’ interim standard definitions for acquired resistance. Clinical Microbiology and Infection, doi:10.1111/j.1469-0691.2011.03570.x world. For biologists, it is utmost important to know the biol- Prakash, O., & Ghosh, I. (2006). Developing an antituberculosis com- ogy of disease and the mechanism of action of medicines. But pounds database & data mining in the search of a motif responsible no longer are these contained in the domain of physiology for the activity of a diverse class of Antituberculosis agents. Journal of and medicinal chemistry. The importance of reorganizing our Chemical Information and Modeling, 46, 17–23. thinking process is mostly reflected in the merger and acquisi- Singh, V. K., & Ghosh, I. (2006). Kinetic modeling of tricarboxylic acid cycle and glyoxylate bypass in Mycobacterium tuberculosis, and its tion of Biotechnology, Bioinformatics and Chemoinformatics application to assessment of drug targets. Theoretical Biology and companies by/with large pharmaceutical companies recently. Medical Modelling, BMC Journal, 3(3), 27. The development in the field of molecular modeling and Vinekar, R., & Ghosh, I. (2009). Determination of phosphorylation sites for simulation has played the leading role in the drug design NADP-specific isocitratedehydrogenase from Mycobacterium tubercu- – technology in pharmaceutical industry. Starting from losis. Journal of Biomolecular Structure and Dynamics, 26(6), 663 895. Genomics, alignment of genes of interest amongst the species, three-dimensional structure prediction from the knowledge of sequence of amino acids, prediction of active sites, 50. Identification of potent and novel searching for lead compounds using database and structure- phosphatidylinositol 3-Kinase catalytic based design, prediction of binding efficiency prior to the experiment and lead optimization using physico-chemical subunit alpha inhibitors: a structure- properties of the compounds are a few to cite. One such based drug design approach case study using in silico attempt towards the designing from known antituberculosis chemicals a set of novel inhibi- Geet Madhukar and Naidu Subbarao tor and finding the drug target(s) using pathway simulation School of Computational and Integrative Sciences, Jawaharlal and analysis (Prakash & Ghosh, 2006; Singh & Ghosh, 2006; Nehru University, New Delhi, India Vinekar & Ghosh, 2009) will be discussed in brief. [email protected]; [email protected] JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS VOL. 37, SUPPLEMENT, 2019 29

Head and neck squamous cell carcinoma (HNSCC), one of the most common causes of deaths due to cancers in Asian Countries and the sixth most common cause of cancer globally is a heterogeneous group of upper aerodigestive tract malignancies (Saleh et al., 2018). Phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) is reported to be commonly altered in many human tumors including HNSCC (Wen & Grandis, 2015). Phosphatidylinositol-3-kinases (PI3Ks) are lipid kinases involved in the regulation of cell survival, growth and metabolism. PI3 Kinase phosphorylates PI(3,4)P2 (PIP2) converting it to PI(3,4,5)P3 (PIP3). Alterations such as mutation, gene amplification and overexpression of PIK3CA are commonly reported in HNSCC (Garcıa-Escudero et al., 2018). This leads to irregulated cell growth due to improper activation of p110a enzymatic activity. The present study aims to find out Figure 1. Proposed pharmacophore model. the potent and novel inhibitors of PIK3CA isoform of PI3Ks. Extensive database screening from NCI, Life Chemical (Kinase Focused Library Similarity search, Kinase Screening Library, LC Kinase docking) and ChEMBL (KinaseSARfari) were performed. Pharmacophore modeling and atom based 3D-QSAR have Computational approaches were employed to carry out a been developed on N-acylglycino- and hippurohydroxamic Structure-Based Drug Design study. Screened compounds from acid derivatives, which are known potential inhibitors of ure- NCI and KinaseSARfari showed superior results and surpassed ase. This is followed by virtual screening and ADMET (absorp- the benchmark compounds with huge differences. Top com- tion, metabolism, excretion and toxicity) studies on a large pounds of NCI (NSC 671560, NSC 729878) and KinaseSARfari library of known drugs in order to get lead molecules as (ChEMBL43781) reported GLIDE Gscore of 15.73 and À Helicobacter pylori urease inhibitors. A suitable three-featured 17.55 kcal/mol, respectively whereas benchmark compounds À pharmacophore model comprising one H-bond acceptor and from ChEMBL43781 had maximum GLIDE Gscore of two H-bond donor features (ADD.10) has been found to be 10.36 kcal/mol. Also, NVP-BYL719 (PDB:4JPS) the only reported À the best QSAR model (Figure 1). An external library of com- selective inhibitor of PIK3CA has GLIDE Gscore 8.16 kcal/mol. À pounds ( 3000 molecules), prefiltered using Lipinski’s rule To validate our results the top ranking compounds were docked of five, has been further screened using the pharmacophore using GOLD and the results were consistent. Along with GLIDE model ADD.10. By analyzing the fitness of the hits with and GOLD scores, the binding affinity of the protein-ligand com- respect to the pharmacophore model and their binding plex was also scored using X-Score program and found consist- interaction inside the urease active site, four molecules have ent with the previous results. A total of 10 compounds from all been identified as extremely good urease inhibitors. Two of the screened libraries were selected and are currently under in- these have significant potential and should be taken up for vitro investigation, for further validation of our findings. further drug designing process. References

Garcıa-Escudero, R., Segrelles, C., Duenas,~ M., Pombo, M., Ballestın, C., Alonso-Riano,~ M., … Lorz, C. (2018). Overexpression of PIK3CA in head and neck squamous cell carcinoma is associated with poor out- 52. Identification of potential come and activation of the YAP pathway. Oral Oncology, 79, 55–63. Saleh, K., Eid, R., Haddad, F. G. H., Khalife-Saleh, N., & Kourie, H. R. (2018). inhibitors for AroG against New developments in the management of head and neck cancer— Mycobacterium tuberculosis impact of pembrolizumab. Therapeutics and Clinical Risk Management, 14, 295–303. Wen, Y., & Grandis, J. R. (2015). Emerging drugs for head and neck can- Nalamolu Ravina Madhulitha, Katari Sudheer Kumar, cer. Expert Opinion on Emerging Drugs, 20(2), 313–329. Chiranjeevi Pasala, Sivaranjani Pakala and Amineni Umamaheswari Bioinformatics Centre, Department of Bioinformatics, Sri 51. Identification of novel urease Venkateswara Institute of Medical Sciences University, Tirupati inhibitors: pharmacophore modeling, 517507, Andhra Pradesh, India [email protected] virtual screening and molecular Infections by opportunistic bacteria have significant contribu- docking studies tions to morbidity and mortality in humans. Tuberculosis has many manifestations affecting bone, central nervous system Richa Arora, Upasana Issar and Rita Kakkar and many other organ systems. Tuberculosis is primarily a Computational Chemistry Laboratory, Department of Chemistry, pulmonary disease that is initiated by the deposition of University of Delhi, Delhi 110 007, India Mycobacterium tuberculosis (M. tb) contained in aerosol drop- [email protected] lets onto lung alveolar surfaces. Developing potent inhibitors 30 BOOK OF ABSTRACTS. ALBANY 2019: THE 20TH CONVERSATION is an important strategy to tackle the pathogenecity of M. tb References strains. Fourteen crystal structures of Phospho-2-dehydro-3- Katari, S. K., Natarajan, P., Swargam, S., Kanipakam, H., Pasala, C., & deoxyheptonate aldolase (AroG) are available in the protein Umamaheswari, A. (2016). Inhibitor design against JNK1 through e- data bank (PDB). AroG of M. tb possess 3 co-crystal structures pharmacophore modeling docking and molecular dynamics simula- (2B7O, 3NV8 and 3PFP) with substrate (PEP) and inhibitor tions. Journal of Receptor and Signal Transduction Research, 36(6), (O35) in the PDB. Among the two diverse co-crystal struc- 558–571. Madhulitha, N. R., Pradeep, N., Sandeep, S., Hema, K., & Chiranjeevi, P. tures (3NV8 and 3PFP), the best resolution structure (3NV8) (2017). E-Pharmacophore model assisted discovery of novel antago- was considered for structure based drug design to propose nists of nNOS. Biochemistry & Analytical Biochemistry, 6, 307. antagonists through swiss similarity screening, dockings and doi:10.4172/2161-1009.1000307 molecular dynamics simulations. O35 and PEP of AroG were Pasala, C., Chilamakuri, C. S. R., Katari, S. K., Nalamolu, R. M., Bitla, A. R., & screened against swiss similarity search of 31 databases com- Umamaheswari, A. (2018). Epitope-driven common subunit vaccine design against H. pylori strains. Journal of Biomolecular Structure and prising 31,11,77,426 compounds obtained 2858 structural Dynamics. doi: 10.1080/07391102.2018.1526714. hits. The hits were processed through rigid receptor docking (RRD), quantum polarized ligand docking (QPLD) and binding free energies were calculated by Prime-MM/GBSA approach (Madhulitha et al., 2017) with AroG. RRD followed by MM- GBSA calculations to the generated library of AroG resulted 53. In silico assessment of 29 compounds, upon comparison with crystal ligands 11 progesterone receptor ligand binding compounds were scored better. To define the leads, 11 com- domain using molecular dynamic: pounds were subjected to QPLD and binding free energy calculations revealed 5-leads on comparison with PEP and O35, a new insight into breast lead1 possess the least binding free energy and better bind- cancer treatment ing affinity. Lead1 and PEP have QPLD XP Gscore of 8.490 À a a and 6.487 kcal/mol, DGscore of 51.80 and 35.209 kcal/ Vahid Zarezade , Marzie Abolghasemi , Fakher À À À b,c a mol, respectively. The stability of AroG-lead1 complex was Rahim and Ali Veisi better than AroG-PEP complex in natural physiological condi- aBehbahan University of Medical Sciences, Iran; bHealth Research tions using Desmond v5.3 for 100 ns molecular dynamics Institute, Thalassemia and Hemoglobinopathies Research Centre, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran; simulations (Katari et al., 2016; Pasala et al., 2018) revealed cMetabolomics and Genomics Research Center, Endocrinology and the least potential energy ( 179483.298 kcal/mol), protein À Metabolism Molecular-Cellular Sciences Institute, Tehran University backbone RMSD (3.1183 Å), protein backbone RMSF of Medical Sciences, Tehran, Iran [email protected] (1.1317 Å), lead1 RMSD (2.8421 Å), lead1 RMSF (0.9607 Å) with 15,031 protein-ligand contacts. The proposed lead1 possess Progesterone receptor (PR), also known as a member of the favorable ADME/T properties and acts as the best AroG steroid/nuclear receptor (NR) superfamily, regulates a com- antagonist of M. tb (Figure 1). plex network of distinct target genes. Ligand binding to a PR leads to a conformational change in the receptor, which activates PR to bind to DNA and regulates the transcription (Brennan & Lim, 2015). Loss of PR expression is associated Acknowledgments with worse overall prognosis and survival among patients with Breast cancer (BC) (Abdel-Hafiz & Horwitz, 2012). NRM is highly acknowledged to ICMR for sanctioning SRF (ISRM/11(21)/ 2017). Authors are thankful to DBT, Ministry of Science and Technology, Although considerable research has been devoted to BC, Government of India, New Delhi for supporting the work through rather less attention has been paid to PR analysis. In the fol- BTISnet BIF program (No. BT/BI/25/037/2012). lowing research, an extensive in silico analytical study was

Figure 1. JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS VOL. 37, SUPPLEMENT, 2019 31

Figure 1. Predicted binding modes of the top-ranked standard drug to the models of PR. (a) A 2-dimensional form conformation of Levonorgestrel docked to PR. (b) A 3-dimensional form conformation of Levonorgestrel docked to PR. In 3-D format, PR is green and magenta color, while for ligands (Levonorgestrel), carbon atoms are cyan; oxygen atoms are red and nitrogen atoms are blue.

performed to the computational measurement of PR modula- Brennan, M., & Lim, B. (2015). The actual role of receptors as cancer tion by a large number of diverse ligands. In this study, we markers, biochemical and clinical aspects: Receptors in breast cancer. performed molecular dynamic (MD) simulation in two Advances in Experimental Medicine and Biology, 867, 327–337. doi: 10.1007/978-94-017-7215-0_20. phases. In the first phase, the prepared conformation of PR was used as the initiating structure for MD simulation. The constituents of the simulation system were protein and water. In the 2nd phase, the acquired conformation of PR from the first phase of MD besides the docked ligand was 54. In silico designing of potential engaged in the MD simulation process. In the docking noscapine analog and results, we found a hydrogen-bond between this compound and Arg94 in the PR. Moreover, we docked 12 standard pharmacological evaluation using drugs to the PR, which found best docking g score in chemoinformatics approach Levonorgestrel with highest binding energy ( 8.35 kcal/mol) À and lowest ki (0.758 mM). This drug showed interaction with Vartika Tomar, Neeraj Kumar and Ramesh Chandra His31, Asn33, Thr34, Lys35, Pro36, Asp37, Thr38, Ser39, Ser41, Drug Discovery & Development Laboratory, Department of Leu42, Asp109, Leu110, Ile111, Leu112 and Arg116 in PR. To Chemistry, University of Delhi, Delhi 110007, India show the conformational alignment between best docked [email protected] natural compound to best docked standard drugs we super- imposed these compounds corresponds to PR (Figure 1). Noscapine and its derivatives have been explored for its interaction with tubulin protein, to be used as strong anticancer agent. A detailed computational analysis have been References performed to determine the noscapine analog to inhibit the Abdel-Hafiz, H. A., & Horwitz, K. B. (2012). Control of progesterone receptor tubulin protein with high specificity, overcoming the issues transcriptional Synergy by sumoylation and desumoylation. BMC of non-specificity and side effects associated with potential Molecular Biology, 13 (Mar 22) article N0. 10: doi:10.1186/1471-2199-13-10 interaction. In this study, noscapine analogs were designed

Figure 1. 32 BOOK OF ABSTRACTS. ALBANY 2019: THE 20TH CONVERSATION using the state of art methodology employing alkyl groups, Atherosclerosis is one of the top, multi-factorial diseases in order to enhance the potency of the drug to bind target majorly responsible for the cause of deaths that makes the tubulin receptor (potential target for cancer treatment). life-threatening clinical events such as acute coronary syn- We have performed the molecular docking and molecular dromes and stroke. Alongside, the widespread emergence of dynamics simulation. Also, pharmacological evaluations were bacterial resistance to existing antibiotics has emphasized also performed to assess the pharmacokinetic properties of the need to develop novel therapeutics to treat atheroscler- the lead compound. Our results illustrated that compound-1 osis with the identification of new targets and novel thera- possess the strongest interaction with the target tubulin pro- peutics (Hema, 2015). The common offending organisms tein with higher binding free energy score of 8.49 kcal/mol include Chlamydiophila pneumoniae, Poryphromonas gingiva- À among the whole library and noscapine ( 7.86 kcal/mol). lis, Helicobacter pylori, Enterobacter hormaechei, Prevotella À Our results were confirmed by the molecular dynamics simu- nigrescens, Prevotella intermedia, Streptococcus sanguinis, lation study of lead compound tubulin complex with lower Tannerella forsythia, Aggregatibacter actinomycetemcomitans RMSD and atomic fluctuation values. Importantly, lead com- D11S-1, Aggregatibacter actinomycetemcomitans D7S-1 and pound was found to good pharmacokinetic properties and Escherichia coli (Hema, 2015). Over expression of human IL-6 showed the potential lead to be used as potential cancer drives the atherosclerosis disease progression hence, therapeutics (Figure 1). regarded as a prominent target in the present study to design novel antagonists (Figure 1). Kalai et al., 2014 reported that inhibiting the binding affinity of human IL-6 References and gp-130 effectively blocks the signaling pathway involved Conde, C., & Caceres, A. (2009). Microtubule assembly, organization and in atherosclerosis progression and also quoted that the resi- dynamics in axons and dendrites. Nature Reviews Neuroscience, 10(5), dues Ile 29, Ile 32 and Leu 33 Arg 30, Arg 182 are majorly 319–332. responsible for the binding of human IL-6 and gp-130 (Kalai Lu, Y., Chen, J., Xiao, M., Li, W., & Miller, D. D. (2012). An overview of tubulin inhibitors that interact with the colchicine binding site. et al., 1997). The proposed lead 1 had good binding affinity, Pharmaceutical Research, 29(11), 2943–2971. good docking scores and similar orientation with lead 1 Naik, P. K., Lopus, M., Aneja, R., Vangapandu, S. N., & Joshi, H. C. (2012). were in well conformity with the binding orientation of gp- In silico inspired design and synthesis of a novel tubulin-binding anti- 130 (Figure 2). The additional five hydrogen bonds were cancer drug: Folate conjugated noscapine (Targetin). Journal of formed with the binding site residues Tyr 31, Leu 33, Glu Computer-Aided Molecular Design, 26(2), 233–247. 110, Gln 111, Leu 178 and hydrogen bond with Lys 27 was observed in 98% of trajectories, Tyr 31 was observed in 96% 55. In silico identification of leads and Arg 182 was observed with 79% trajectories strengthen the stability of lead 1 with human IL-6 with better binding targeting interleukin-6 against affinity. The consistent energy, five water-mediated interac- pathogenesis of atherosclerosis tions, RMSD and RMSF of the complex was also within the limit (Figure 3). Hence, the proposed lead can act as poten- a,d b tial antagonists for inhibiting the human IL-6 in signaling Kanipakam Hema Ã, Sandeep Swargam , Natarajan Pradeepc and Amineni Umamaheswarid pathway in the progression of atherosclerosis. The lead mol- aFunctional Genomics Unit, CSIR-IGIB, New Delhi 110007, India; ecule would hold as a promising antagonist in drug discov- bInstitute of Molecular Medicine, Jamia Hamdard University, New ery if synthesized and tested in animal models. Delhi 110062, India; cDepartment of Biotechnology and Medical Engineering, NIT, Rourkela, Odisha, 769008, India; dBioinformatics Acknowledgments Centre, Department of Bioinformatics, SVIMS University, Tirupati, Andhra Pradesh, 517507, India [email protected] This research has been supported by DST-INSPIRE, Govt. of India, for the Presenting author: [email protected] SRF. The authors are thankful to DBT, ministry of science and technol- Ã ogy, Govt. of India for providing all facilities to carry out the work.

Figure 1. Figure 2. Figure 3. JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS VOL. 37, SUPPLEMENT, 2019 33

References Hoechst and DNA. As a result of our study, the binding of Hoechst 33258 to the Dickerson–Drew sequence can be bet- Hema, K., Priyadarshini, V., Pradhan, D., Munikumar, M., Sandeep, S., Pradeep, N., … Umamaheswari, A. (2015). Identification of putative ter understood and the results presented may be used in drug targets and vaccine candidates for pathogens causing athero- designing of novel DNA binding drugs with useful biological sclerosis. Biochemistry & Analytical Biochemistry, 4,1–9. applications. Hema, K., Vani Priyadarshini, I., Sandeep, S., Pradeep, N., Chiranjeevi, P., & Umamaheswari, A. (2015). Subunit vaccine design against pathogens causing atherosclerosis. Journal of Biomolecular Structure and References Dynamics, 33(suppl. 1) Kalai, M., Montero-Julian, F. A., Grotzinger,€ J., Fontaine, V., Teng, M.-K., Usman, N., Frederick, C. A., & Wang, A. H.-J. (1988). The Vandenbussche, P., Deschuyteneer, R., … Content, J. (1997). Analysis molecular structure of the complex of Hoechst 33258 and the DNA of the human interleukin-6/human interleukin-6 receptor binding dodecamer d(CGCGAATTCGCG). Nucleic Acids Research, 16(6), interface at the amino acid level: Proposed mechanism of interaction. 2671–2690. Blood, 89(4), 1319–1333.

56. In silico studies of interaction of 57. In-silico assessment of adenosine hoechst 33258 within minor groove receptor ligand binding domain: a of B-DNA new insight into adenosine receptor based therapeutics Upasana Issar, Richa Arora and Rita Kakkar a b Computational Chemistry Laboratory, Department of Chemistry, Navina Panneerselvan , Radha Madendran and University of Delhi, Delhi 110 007, India Malathi Rangunathana [email protected] aDepartment of Genetics, Dr.ALM PG IBMS, University of Madras, Taramani 600113, Chennai; bDepartment of Bioinformatics, Vel’s DNA is the most sought after drug design target in the University, Velan Nagar, Pallavaram 600117, Chennai league of anticancer drugs. Drugs interfere with the replica- [email protected] tion and transcription of DNA, thereby causing cell death. Hoechst 33258 is a prominent minor groove binder that dis- Extracellular adenosine acts as a local modulator with a gen- plays specific binding towards the adenine-thymine (AT) rich erally cytoprotective function in the body. During ischemia region of the minor groove of B-DNA (Teng, Usman, or inflammation conditions adenosine levels are elevated. Frederick, & Wang, 1988). Molecular modeling studies have Among the four adenosine receptors (ARs), A2b AR been carried out in order to completely understand the expressed highly in angiogenesis during wound healing and interaction of various rotameric, tautomeric and ionization tumor growth; its role in genetic and pharmacological stud- states of Hoechst 33258 (existing at physiological pH) with ies are known to an extend (Du et al., 2015), however the the Dickerson-Drew dodecamer d(CGCGAATTCGCG)2 binding ability of A2b with anti-angiogenic plant compounds sequence. The electrostatic and hydrogen bonded interac- are not well known (Jacobson & Gao, 2006). In order to valid- tions (Figure 1) are found to be the major forces responsible ate the usefulness of this receptor model, docking analysis of for the Hoechst-DNA complexation. QM/MM studies reveal several selective and nonselective compounds were carried important geometrical and energetic aspects of minor out to find the binding affinities and selectivity of ligands to groove binding. The high complexation energy (-549.67 kcal adenosine binding region of A2b AR. Plant compounds like 1 molÀ ) signifies that a stable complex is formed between Chrysin, Apigenin, Theobromine, Theophylline, Genistein, Resveratrol, Pentoxifylline and synthetic compounds DAPT, SU5416, SQ22536, ZM 241385 and MSR 1754 that are known to be AR targets. Our results illustrated that A2b AR possess the strongest interaction to Chrysin with higher binding free energy score of 8.09 kcal/mol with 6-OH bond interactions À at ALA82, PHE84, VAL85, ALA60, ALA64 compared to positive control of ZM241385 with 8.70 kcal/mol binding energy. À Other plant molecules like resveratrol make three electrostatic interaction with GLN6, GL4, LEU2 with binding affinity of 7.04 kcal/mol of OH bond; Theobromine and theophyl- À line similarly showed lower binding affinity of 6.448 kcal/ À mol and -6.41 kcal/mol. Apigenin shows 6.76 kcal/mol with À 6-OH bond at THR5, LYS269, TRP270 electrostatically; Genistein shows 6.33 kcal/mol binding affinity made of 5- À OH bond with LEU2 (one bonding distance), GLN6 (four bonding distance); and pentoxyflline with 6.81 kcal/mol À Figure 1. Hydrogen bonding between Hoechst 33258 and DNA. (OH bonding) distance of 2.3 at THR139. Among the 34 BOOK OF ABSTRACTS. ALBANY 2019: THE 20TH CONVERSATION

Figure 1.

synthetic molecule DAPT with 8.35 kcal/mol binding energy needs in the clinics. We have therefore selected UDP-N- À making two strong OH at ASP7 two bond distance of 2.1 acetylglucosamine–N-acetylmuramyl-(pentapeptide) pyro- and 2.0; next SU5416 with -7.218 kcal/mol with TRP270 and phosphoryl-undecaprenol N-acetylglucosamine transferase LYS269 OH bond. Furthermore, molecular stimulation ana- (MurG), one of the promiscuous common targets for multiple lysis might be carried out to find binding ability of the com- H. pylori strains identified in our previous study (Pasala et al., pounds tested. Based on the study result, the selected plant 2018). Cell wall-related MurG is of special interest, since, it is compounds like chrysin might considered as novel and sig- involved in peptidoglycan biosynthesis with the potential nificant compound that can be modeled and characterized interaction between other proteins, unique to the bacteria, for auxiliary therapeutic implement (Figure 1). has no mammalian counterpart, and agents inhibiting its synthesis have the potential to become broad-spectrum ther- Funding apeutics. In the study, the selected target was allowed to Modeller9v20 to generate 100 models with incorporating This research has been supported by UGC-BSR award No: F- substrate Uridine-Diphosphate-N-Acetylglucosamine (UD1) 7-115/2007. using a template (PDB: 3S2U). After validations, the third model was deposited at Protein Model DataBase References (PM0081627). The reviewed previous ligands of MurG were allowed to hierarchical-clustering. Subsequently, the obtained Du, X., Ou, X., Song, T., Zhang, W., Cong, F., Zhang, S., & Xiong, Y. (2015). five diverse ligands from five clusters with clustering strain Adenosine A2B receptor stimulates angiogenesis by inducing VEGF 1.261and DU1 were subjected to SwissSimilarity web-tool to and eNOS in human microvascular endothelial cells. Experimental perform ligand-based virtual screening of several libraries of Biology and Medicine, 240(11), 1472–1479. Jacobson, K. A., & Gao, Z. G. (2006). Adenosine receptors as therapeutic 3 billion small molecules. The retrieved 4177 similar contours targets. Nature Reviews Drug Discovery, 5(3), 247. were further assigned to lead-optimization campaign includes, rigid receptor docking (Glide parameters), quantum-polarized ligand docking (quantum mechanics) and induced-fit docking (80 replications with charge calculations) 58. Integration of binding potency coupled with Molecular Mechanics/Generalized Born Surface Area methods to estimate relative binding affinity (DGbind) of estimations and stability assessments the previous, screened compounds and DU1. As a result, for therapeutic design against MurG seven compounds were observed with better DGbind free energies than top diverse ligand BMS-190134 ( 63.12 kcal/ of H. pylori À mol) and DU1 ( 49.33 kcal/mol). Moreover, interaction stabil- À ity of DU1 and the best lead1 ( 67.85 kcal/mol) with MurG Pasala Chiranjeevi, Katari Sudheer Kumar, À was dynamically assessed for 50 ns and 150 ns chemical time Nalamolu Ravina Madhulitha, Sharon Priya Alexander and Amineni Umamaheswari respectively (Katari et al., 2016; Pasala et al., 2018b). Consequently, the consistency of lead1-MurG was found with Bioinformatics Centre, Department of Bioinformatics, SVIMS University, Tirupati 517507, Andhra Pradesh, India lower deviations, fluctuations and fair compactness than [email protected] DU1-MurG. The stable interactions with lead1 have been maintained throughout the simulations period and correlated The adverse effects and widespread emergence of resistant with docking analysis thereby that contacts might be vital Helicobacter pylori strains to the existing drugs is becoming a hotspots for catalytic activity of MurG. Therefore, as the pro- serious public health concern. As a result there is a real and posed 7 leads fulfilling all the criteria from different aspects pressing demand to develop new therapeutics; however, it of drug design study, these could be promiscuous thera- remains one of the major challenges to science and unmet peutic agents against H. pylori (Figure 1). JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS VOL. 37, SUPPLEMENT, 2019 35

Figure 1.

Acknowledgments design against H. pylori strains. Journal of Biomolecular Structure and Dynamics. doi:10.1080/07391102.2018.1526714. (In press) PC acknowledges to ICMR, New Delhi, for supporting with the Senior Research Fellowship (3/1/3/JRF-2014/HRD-8). Authors are highly thankful to DBT, Ministry of Science and Technology, Govt. of India for providing Bioinformatics infrastructure facility to SVIMS Bioinformatics Centre (BT/ BI/25/037/2012 (BIF-SVIMST)). 59. Interaction of noscapine with human serum albumin (HSA): a spectroscopic and molecular References modeling approach

Katari, S. K., Natarajan, P., Swargam, S., Kanipakam, H., Pasala, C., & Umamaheswari, A. (2016). Inhibitor design against JNK1 through e- Heerak Chugh and Ramesh Chandra pharmacophore modeling docking and molecular dynamics simula- Department of Chemistry, University of Delhi, New Delhi 110007, tions. Journal of Receptors and Signal Transduction Research, 36(6), India [email protected] 558–571. Pasala, C., Chilamakuri, C. S. R., Katari, S. K., Nalamolu, R. M., Bitla, A. R., & Noscapine, an opium derived anticancer agent manifests Umamaheswari, A. (2018). An in silico study: Novel targets for poten- therapeutic applications and is compliant to beneficial modi- tial drug and vaccine design against drug resistant H. pylori. Microbial Pathogenesis, 122, 156–161. fications. In this paper, we study its interaction with human Pasala, C., Chilamakuri, C. S. R., Katari, S. K., Nalamolu, R. M., Bitla, A. R., & serum albumin (HSA) by different spectroscopic methods Umamaheswari, A. (2018). Epitope-driven common subunit vaccine including UV–vis, fluorescence, circular dichroism and 36 BOOK OF ABSTRACTS. ALBANY 2019: THE 20TH CONVERSATION thermal denaturation and molecular docking. Binding and Technology, Ahvaz Jundishapur University of Medical Sciences, c thermodynamics parameters support a feasible interaction of Ahvaz, Iran; Department of Radiology, Shariati Hospital, Tehran d Nos-HCl with HSA. Further, type of interactions between Nos- University of Medical Sciences, Tehran, Iran; Neuroimaging Network (NIN), Universal Scientific Education and Research HCl and HSA were also elucidated. The energy transfer e Network (USERN), Tehran, Iran; Health Research Institute, between HSA and Nos-HCl was analyzed by Forster mechan- Thalassemia and Hemoglobinopathies Research Centre, Ahvaz ism and lifetime measurements. Additionally, it was shown Jundishapur University of Medical Sciences, Ahvaz, Iran; that Nos-HCl binds HSA in subdomain IIIA which constitute fMetabolomics and Genomics Research Center, Endocrinology and the drug binding Sudlow’s Site II. Preliminary results for bind- Metabolism Molecular-Cellular Sciences Institute, Tehran University ing site were deciphered via the site marker competitive of Medical Sciences, Tehran, Iran experiments which were then confirmed using molecular docking analysis. Posterior cingulate cortex (PCC) is a paralympic cortical structure that is located in the middle of default mode network (DMN) that has a fundamental role in connecting different regions of the DMN (Hampson, Driesen, Skudlarski, Gore, & Constable, 2006). The aim of this meta-analysis was to assess the metabolomics content changes in PCC of the patients with Alzheimer disease (AD) comparing to healthy controls (HC). We performed a comprehensive search through eight international indexing databases, including PubMed, Scopus, Cochrane library, CINAHL, ISI Web of Science, Science Direct from inception, Embase and PROSPERO from 1980 to 2018. Two authors independently extracted data and classified methods for analysis. Overall, a total of 3,067 relevant studies were initially found of which 18 studies comprising 1,647 cases, (658 (40%) males and 941 (60%) females, 921 (55.9%) HC and 678 (44.1%) AD cases). The analyses showed significant differences between AD patients and HC in term of

References

Empey, D. W., Laitinen, L. A., Young, G. A., Bye, C. E., & Hughes, D. T. D. (1979). Comparison of the antitussive effects of codeine phosphate 20 mg, dextromethorphan 30 mg and noscapine 30 mg using citric acid-induced cough in normal subjects. European Journal of Clinical Pharmacology, 16(6), 393–397. Mahmoudian, M., & Rahimi-Moghaddam, P. (2009). The anti-cancer activity of noscapine: A review. Recent Patents on anti-Cancer Drug Discovery, 4(1), 92–97. Pasquier, E., & Kavallaris, M. (2008). Microtubules: A dynamic target in cancer therapy. IUBMB Life, 60(3), 165–170.

60. Magnetic resonance spectroscopy- based metabolomics analysis of posterior cingulate in patients with alzheimer’s disease: a systematic review study

Kiarash Shirbandia,b, Mohammad Davoodic, Elmira Hosseinid, Fakher Rahime,f and Babak Arjmandf a Neuroimaging Network (NIN), Universal Scientific Education and Figure 1. Overview of the role of various metabolites engaged in the metabolic Research Network (USERN), Ahvaz, Iran; bDepartment of Radiologic pathway of Alzheimer disease (AD). JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS VOL. 37, SUPPLEMENT, 2019 37

NAA (NAA: N-Acetyl Aspartate), mI (myo-Inositol), Glu and cheminformatics techniques to describe the differential (Glutamine) and Glx (Glutamate & Glutamine) concentrations. pharmacological and antagonistic activities of a selected frag- Also, metabolite ratios including NAA/Cr, Cho/Cr and mI/Cr ment, SB640 and its parent compound, Anguinomycin D were significantly different. Hence, there were no significant towards their target protein, Exportin Chromosome Region differences in term of Cr and Cho levels and mI/NAA ratio Maintenance 1 (CRM1), involved in pro-carcinogenic chemo- between AD and HC groups. Our meta-analysis showed therapeutic resistance. Our findings revealed that the fragment metabolite changes in the PCC can be used as a marker for exhibited improved pharmacokinetics with minimal toxicities early diagnosis of the AD. Although NAA biomarker is con- and off-target activities compared to the parent compound. sidered to be the most important metabolite and mI is most Furthermore, reduction into a smaller fragment enabled opti- sensitive for early diagnosis of the AD, Cho/Cr ratio as a crit- mal positioning and binding with crucial residues at the pro- ical biomarker can be of interest to researchers (Figure 1). tein target site which in turn accounted for a more prominent CRM1 inactivation as compared to the parent compound that had minimal structural effects due to motion and dynamical References constraints caused by its long polyketide tail. Our findings Hampson, M., Driesen, N. R., Skudlarski, P., Gore, J. C., & Constable, R. T. therefore indicate that the ‘size does not matter’ and that (2006). Brain connectivity related to working memory performance. reduction of complex bioderived compounds to fragments Journal of Neuroscience, 26(51), 13338–13343. could be an essential strategy for improving potency and minimizing associable adverse drug reactions (Figure 1). 61. Minimizing toxicity and maximizing the potency of bioderived References compounds by synthetic Crane, E. A., & Gademann, K. (2016). Capturing biological activity in natural product fragments by chemical synthesis. Angewandte Chemie fragmentation—a computational International Edition, 55,2–23. ‘proof-of-concept’ Olotu, F. A., Munsamy, G., & Soliman, M. E. (2018). Does size really matter? Probing the efficacy of structural reduction in the optimization of bioderived compounds—a computational “proof-of-concept”. Fisayo A. Olotu, Geraldene Munsamy and Computational and Structural Biotechnology Journal, 16, 573–586. Mahmoud E. Soliman Molecular Bio-computation and Drug Design Laboratory, School of Health Sciences, University of KwaZulu Natal, Westville Campus, 62. Mutant p53 REs and the Durban 4001, South Africa [email protected] mechanism of gain of function Despite the potency embedded in natural products with in cancer regards to multiple disease treatment, the numerous challenges of toxicities and undesirable biological ‘off-targeting’ Jessy Safieh, Demitrij Golovenko and Tali E. Haran have limited its clinical transition. More recently, the syn- Faculty of Biology, Technion, Haifa, Israel [email protected] thetic reduction of complex natural products into simpler fragments has been identified as a viable strategy to develop p53 functions as a sensor of cellular stress signals. A variety next generation leads with improved potencies and minimal of stress signals stabilize its structure and bring about an toxic effects. Therefore, to validate the efficacy of this increase in its concentration, with the end result of a variety method, we employed combinatorial molecular modeling of functional outcome preventing cancer. However, p53 itself

Figure 1. 38 BOOK OF ABSTRACTS. ALBANY 2019: THE 20TH CONVERSATION is mutated in 50% of all cancer cases. p53 core domain is In response to cellular stress signals, the tumor suppressor the main target for mutations in human tumors. 95% of the p53 acts as transcription factor by binding as a tetramer to a mutations identified in human tumors are found in this region. wide range of DNA sites, thereby activating numerous genes This underscores the importance of sequence-specific DNA that are critical for cancer prevention (Vogelstein, Lane, & binding by the core for p53 functions. Gain-of-function muta- Levine, 2000). Its main functional domain, the DNA binding tions are mutations that switch p53 into a tumor-promoting core domain (p53DBD), is accompanied by structured tetra- oncogene, by endowing the mutant protein with new abilities, merisation domain and intrinsically disordered N- and C-ter- activating new target genes, that can contribute to various mini. p53 binds specifically to double-stranded DNA stages of tumor progression. Multiple mechanisms were pro- response elements (REs) which are composed of two deca- posed to account for different mutant p53 gain-of-function meric half-sites of the general form RRRCWWGYYY (R A, G; ¼ activities. A major transcriptional mechanism for gain-of-func- W A, T; Y C, T) (el-Deiry, Kern, Pietenpol, Kinzler, & ¼ ¼ tion of mutant p53 is binding of mutant p53 through cooper- Vogelstein, 1992). Our previous studies on crystal structures ation with other transcription factors. We will present a novel of p53DBD in complexes with consensus DNA sites revealed model for mutant p53 gain-of-function in cancer that recon- non-canonical Hoogsteen (HG) base pairs at the A/T doublets ciles many observations on the gain-of-function phenomenon. within conserved CATG motifs at the half-site centers, We will also present binding data demonstrating that the role flanked by GGG/CCC (Kitayner et al., 2010). In the HG geom- played by DNA structure in mutant p53/DNA interaction fol- etry, the A bases are rotated around their glycosidic bonds lows the rules established for wild-type p53/DNA interactions. by nearly 180 relative to that of the common Watson-Crick (WC) geometry, to form alternative hydrogen bonds (Figure 1(a)). As a result of the HG geometry, the backbone atoms 63. How the tumor suppressor p53 on opposite DNA strands are closer by 2 Å in the region of recognizes its DNA response elements HG base pairs compared to that of WC base pairs, affecting the shape of the DNA helix. This finding led to the proposal Dmitrij Golovenkoa,b, Oksana Degtjarika, Tali E. that the unique DNA shape enhances the stabilization of the Haranb, Haim Rozenberga and Zippora Shakkeda p53DBD-DNA complex (Kitayner et al., 2010). aDepartment of Structural Biology, Weizmann Institute of Science, To characterize the effect of DNA shape on p53-DNA Rehovot, 76100, Israel; bDepartment of Biology, Technion–Israel interactions in terms of structural and biochemical aspects, Institute of Technology, Haifa, 32000, Israel we used a novel approach to ‘lock’ base-pairing into WC or [email protected] HG geometry. The method relies on designing REs where the central A/T doublets are replaced by modified nucleotides in

Figure 1. Schematic representation of Watson-Crick and Hoogsteen base pairs for natural and modified variants. Hydrogen bonds between donor and acceptor groups indicated by blue bars, and repulsive interactions by red bars. JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS VOL. 37, SUPPLEMENT, 2019 39

References

el-Deiry, W. S., Kern, S. E., Pietenpol, J. A., Kinzler, K. W., & Vogelstein, B. (1992). Definition of a consensus binding site for p53. Nature Genetics, 1(1), 45–49. Golovenko, D., Brauning, B., Vyas, P., Haran, T. E., Rozenberg, H., & Shakked, Z. (2018). New Insights into the role of DNA shape on its recognition by p53 Proteins. Structure (London, England: 1993), 26(9), 1237–1250. Kitayner, M., Rozenberg, H., Rohs, R., Suad, O., Rabinovich, D., Honig, B., & Shakked, Z. (2010). Diversity in DNA recognition by p53 revealed by crystal structures with Hoogsteen base pairs. Nature Structural & Molecular Biology, 17, 423–429. Vogelstein, B., Lane, D., & Levine, A. J. (2000). Surfing the p53 network. Nature, 408(6810), 307–310.

64. Structural studies on reactivation of tumor-related p53 mutants by methylene quinuclidinone (MQ), the active drug spontaneously formed from APR-246

Oksana Degtjarika, Dmitrij Golovenkoa, Yael Diskin- Posnerb, Lars Abrahmsen c, Haim Rozenberga and Zippora Shakkeda aDepartment of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel; bDepartment of Chemical Research Support, Weizmann Institute of Science, Rehovot 76100, Israel; cAprea Therapeutics AB, Solna, 17165, Sweden [email protected]

The tumor suppressor p53 is also known as the ‘guardian of Figure 2. Structures of p53DBD tetramers bound to DNA REs (20 bp long) with 4 base pairs ‘locked’ into Hoogsteen geometry (A) or Watson-Crick geometry the genome’ due to its ability to act as a transcription factor (B) (highlighted by thick lines). that regulates the expression of a range of target genes in response to genotoxic stress, leading to DNA repair, cell cycle a way that avoids introduction of bulky groups and preserves arrest or apoptosis, all essential to prevent cancer (Vogelstein, the A/T patterns at the minor-groove edges of the modified Lane, & Levine, 2000). The function of p53 can be compro- base-pairs in both geometries. Thus, to shift the equilibrium mised by mutations that lead to p53 inactivation and thus to toward the HG geometry, A bases were replaced by 2-oxo-A cancer development. The majority of these mutations are (2OA), and to enforce the WC geometry, A and T bases were located at the DNA binding core domain of p53 (p53DBD). replaced by Inosine (I) and 5-methyl-C (5mC), respectively Among them, six mutational ‘hotspots’ at residues: R175, G245, (Figure 1(b,c)). In this manner, the selection between the two R248, R249, R273 and R282 (Figure 1), were shown to occur at forms, HG or WC, is determined by the balance between high frequency in human cancer (Olivier et al., 2002). The cor- attractive and repulsive interactions, as manifested by the responding amino-acid replacements (shown in red) can either high-resolution crystal structures of the two types of p53- affect p53 binding to DNA and referred as DNA-contact DNA complexes, displaying the expected conformational dif- mutants: R273H/C, R248Q/W; or cause conformational changes ferences between the two helices (Figure 2(a,b)). A detailed in p53DBD and referred as structural mutants: R175H, G245S, analysis of the various structures combined with DNA bind- R249S and R282W. Several crystal structures of the p53DBD of ing and kinetic studies demonstrated that complexes with such mutants have been reported (Eldar, Rozenberg, Diskin- the unusual HG geometry are stabilized relative to those Posner, Rohs, & Shakked, 2013; Suad et al., 2009). with all-WC base pairs. We also showed that two natural PRIMA-1 and APR-246/PRIMA-1MET are small molecules high-affinity REs, related to DNA-damage response and that are converted into the biologically active compound, incorporate CATG motifs, are also predisposed to adopt the methylene quinuclidinone (MQ) which reactivates mutant unique DNA shape induced by HG base pairs (Golovenko p53 by binding covalently to cysteines in the p53DBD et al., 2018). (Lambert et al., 2009). APR-246 is presently undergoing clin- To conclude, the combined structural and biochemical ical trials (https://www.clinicaltrials.gov). In our study, we data demonstrate that in addition to direct readout made by investigate the reactivation mechanisms of mutant p53 by MQ direct interactions between the protein and DNA, indirect from a series of high-resolution crystal structures of wild-type readout rendered by shape readout plays a major role in p53 and several hotspot mutants bound to MQ in the absence DNA recognition by proteins. and/or the presence of DNA. Our data show that MQ can bind 40 BOOK OF ABSTRACTS. ALBANY 2019: THE 20TH CONVERSATION

Figure 1. Six hotspot mutation sites in p53DBD bound to DNA. p53 binds to its DNA target as a tetramer (dimer of dimers). Here is a view of a p53DBD dimer (grey) bound to DNA (blue), showing the six hotspot sites (red). The view is along the DNA helix. The figure is based on coordinates from PDB code 2ac0 (Kitayner et al., 2006). to several cysteine residues located at the surface of the core [email protected] domain at positions: 124, 182, 229, 275 and 277. A detailed ÃPresenting author: Email: [email protected] comparison between the structures of specific p53 mutants before and after binding to MQ, reveals the role played by MQ Matrix metalloproteinase-14 (MMP-14) or membrane type in stabilizing p53 and its interaction with DNA, thus providing MMP-1 is an extra cellular matrix (ECM) protease activates a structural framework for the design of new molecules for progelatinase A (MMP-2) involved in proteolysis of collagen, specific targeting of cysteines in p53 mutants. gelatin, elastin, laminin, fibronectin and integrins for cancerous cell to invade and metastasise. About 2,756 inhibitors References from CLRI-MMpi database were retreived, optimized and minimized through LigPrep using OPLS3 forcefield. The Eldar, A., Rozenberg, H., Diskin-Posner, Y., Rohs, R., & Shakked, Z. (2013). ligands were passed through virtual screening workflow Structural studies of p53 inactivation by DNA-contact mutations and (VSW) protocol, where the ligands were refined and docked its rescue by suppressor mutations via alternative protein-DNA interactions. Nucleic Acids Research, 41(18), 8748–8759. by high throughput virtual screening [HTVS-10%], standard Kitayner, M., Rozenberg, H., Kessler, N., Rabinovich, D., Shaulov, L., Haran, precision [SP-10%] and extra precision [XP-100%] over the T. E., & Shakked, Z. (2006). Structural basis of DNA recognition by p53 Grid of MMP-14 (5H0U) using grid-based ligand docking with tetramers. Molecular Cell, 22(6), 741–753. energetics (GLIDE) and binding free energy (DG) was esti- Lambert, J. M., Gorzov, P., Veprintsev, D. B., Soderqvist, M., Segerback, D., mated by Prime-MM/GBSA for 27 dock complexes Bergman, J., … Bykov, V. J. (2009). PRIMA-1 reactivates mutant p53 by covalent binding to the core domain. Cancer Cell, 15(5), 376–388. (Chiranjeevi, Swargam, Pradeep, Hema, & Kumar, 2016). The Olivier, M., Eeles, R., Hollstein, M., Khan, M. A., Harris, C. C., & Hainaut, P. best bound MMpi database inhibitors (MMpI1999248, (2002). The IARC TP53 database: New online mutation analysis and MMpI2009210 and MMpI200383) were screened against recommendations to users. Human Mutation, 19(6), 607–614. SwissSimilarity databases comprising 0.3 Billion compounds Suad, O., Rozenberg, H., Brosh, R., Diskin-Posner, Y., Kessler, N., Shimon, resulted 2756 structural analogs. 28 compounds were L. J., … Shakked, Z. (2009). Structural basis of restoring sequence- specific DNA binding and transactivation to mutant p53 by suppres- obtained from VSW, fourteen compounds were better scored sor mutations. Journal of Molecular Biology, 385(1), 249–265. (XP G and DG) than the best three MMpi. These were Vogelstein, B., Lane, D., & Levine, A. J. (2000). Surfing the p53 network. redocked by quantum polarized ligand docking (QPLD) and Nature, 408(6810), 307–310. resulted 5 leads; the best lead (lead 1) possesses QPLD XP Gscore of 8.711 kcal/mol and DG score of - 67.035 kcal/ À À mol, but the best MMpi (MMpI1999248) possesses QPLD XP 65. Potent MMP-14 antagonist design Gscore of -6.092 kcal/mol and DG score of -66.538 kcal/mol (Madhulitha, Pradeep, Sandeep, Hema, & Chiranjeevi, 2017). through screening, docking and The best lead and the best MMpi (MMpI1999248) MMP-14 dynamics studies complexes were simulated for 100 ns and the dynamics were assessed for stability by analyzing root-mean-square-deviation (RMSD), root-mean-square-fluctuation (RMSF), total Katari Sudheer KumarÃ, Pasala Chiranjeevi, Nalamolu Ravina Madhulitha, Vankadoth Umakanth energy (T.E.), potential energy (P.E.), protein-ligand contacts Naik and Amineni Umamaheswari (hydrogen bond, pi-cation, hydrophobic, metal co-ordination Bioinformatics Centre, Department of Bioinformatics, SVIMS and water bridges), radius of gyration and torsions University, Tirupati 517507, Andhra Pradesh, India (Katari et al., 2016). Lead 1-MMP-14 complex showed T.E. and JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS VOL. 37, SUPPLEMENT, 2019 41

P.E. of 64,432.622 kcal/mol and 78,558.567 kcal/mol lesser 100 ns MD simulations suggested that lead 1 could act as a À À than MMpI1999248-MMP-14 complex with 64,405.771 kcal/ potent antagonist by blocking the functional activity of À mol and 78,552.540 kcal/mol. Lead 1 with respective to pro- MMP-14, MMp-2 activation, ECM degradation, cancer cell À tein and lead 1 with respective to lead 1: average RMSD was invasion as well as metastasis. 2.16 Å and 0.68 Å, average RMSF was 0.99 Å and 0.38 Å lesser than the MMpI1999248 with respective to protein and with respective to itself: average RMSD was 5.50 Å and 2.75 Å and Acknowledgments average RMSF of 2.26 Å and 1.85 Å were observed during KSK is highly acknowledged to DBT for sanctioning JRF and SRF (BT/BI/ 100 ns molecular dynamics (MD) simulations. The overall 25/037/2012). Authors are thankful to DBT, Ministry of Science and molecular contacts formed by MMP-14-lead 1 (9,745) are Technology, Government of India, New Delhi for supporting the work comparatively higher than MMP-14-MMpI1999248 (9,482) in through BTISnet BIF program (No. BT/BI/25/037/2012). 42 BOOK OF ABSTRACTS. ALBANY 2019: THE 20TH CONVERSATION

References aBioinformatics Centre, Department of Biotechnology and Medical Engineering, National Institute of Technology, Rourkela, Odisha Chiranjeevi, P., Swargam, S., Pradeep, N., Hema, K., & Kumar, K. S. b (2016). Inhibitor design for VacA toxin of Helicobacter pylori. Journal 769008, India; Bioinformatics Centre, Department of Bioinformatics, Sri Venkateswara Institute of Medical Science of Proteomics and Bioinformatics, 9, 220–225. doi: 10.4172/ c jpb.1000409. University, Tirupati 517507, Andhra Pradesh, India; NIN-TATA Katari, S. K., Natarajan, P., Swargam, S., Kanipakam, H., Pasala, C., & Centre for Excellence in Public Health Nutrition, ICMR-National Institute of Nutrition, Jamai-Osmania (Post), Hyderabad 500007, Umamaheswari, A. (2016). Inhibitor design against JNK1 through e- d pharmacophore modeling docking and molecular dynamics simula- Telangana, India; Jamia Hamdard Institute of Molecular Medicine, Jamia Hamdard University, Hamdard Nagar, New Delhi 110062, tions. Journal of Receptor and Signal Transduction Research, 36(6), e 558–571. India; Functional genomics unit, CSIR-IGIB, New Delhi 110007, Madhulitha, N. R., Pradeep, N., Sandeep, S., Hema, K., & Chiranjeevi, P. India [email protected] (2017). E-Pharmacophore model assisted discovery of novel antagonists of nNOS. Biochemistry & Analytical Biochemistry, 6, 307. Alzheimer’s disease (AD), the utmost common and progres- doi:10.4172/2161-1009.1000307 sive neurodegenerative disease characterized by aggregation of b-amyloid plaques triggered by mitochondrial oxidative 66. Scaffold hopping strategy on the stress also. Raising substantial evidences evokes that diluted antioxidant activity correlates with overproduction of free route discerning novel glutathione radicals and peroxides. Which extends the alterations in peroxidase agonists membrane permeability damaging the mitochondrial respiratory chain, subsequently amplifying the neuronal dysfunction Natarajan Pradeepa, Manne Munikumarc, Sandeep there by triggering neurodegeneration. Glutathione peroxi- Swargamd, Kanipakam Hemae, Katari Sudheer dases (GPx1-2) is abundant antioxidant enzyme that catalyze kumarb, Amineni Umamaheswarib, Praveen Kumar reduction of hydrogen peroxide to water. Increased free radi- Guttulaa and Mukesh Kumar Guptaa cals and absurdity in potentiality to detoxify the reactive

Figure 1.

Figure 2. JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS VOL. 37, SUPPLEMENT, 2019 43

Figure 3. intermediates associated with diluted activity of GPx has made modeling, multiple docking strategies and molecular dynamic simula- it as a possible attractive therapeutic target for AD interventions to discover of novel antagonists of BACE1. Journal of tion. Five existing agonists were subjected to geometry based Biomolecular Structure and Dynamics, 33(supp1), 129–130. Wang, L., Deng, Y., Wu, Y., Kim, B., LeBard, D. N., Wandschneider, D., … virtual screening against in-house library having more than 21 Abel, R. (2017). Accurate modeling of scaffold hopping transforma- million small molecules. The resulted 1273 hits were subjected tions in drug discovery. Journal of Chemical Theory and Computation, to rigid receptor docking (RRD) and quantum polarized ligand 13(1), 42–54. docking (QPLD) succeeded with binding free-energy (DG) calculations, resulted best ligands (2 for GPx1 and 1 for GPx2). The best ligands and existing agonists were subjected to scaf- 67. The distribution of 3,5,30- fold hopping against fragment libraries resulted 644 com- triiodothyronine between the pounds, which are subjected to multi-level multiple docking strategies to derive the best leads (12 for GPx1; 14 for GPx2) transport systems of blood and nuclei with favorable pharmacokinetic properties (Figure 1). Further, of the tissues GPx and the best lead complexes were subjected to 50 ns MD simulations to analyze the stability (Figure 2) as well as the Margarita I. Garipova, Alfira I. Shigapova, Rashit G. intactness (Figure 3) of the best leads towards the receptor. Farkhutdinov, Vadim V. Fedyaev, Julia M. Sotnikova The proposed leads, obtained from scaffold hopping of exist- and Alfira B. Yakupova ing agonists showed favorable binding orientation with Department of Biology, Bashkir State University, Z. Validy, 32, Ufa increased binding affinity towards GPx, by enhancing the sulf- 450076, Russian Federation [email protected] hydryl groups availability to ROS and peroxide intermediates ensures the anti-oxidant activity. The intensifying GPx levels It is known that hydrophobic hormones can be transporting with the proposed leads enriches the anti-oxidant effect. Thus, in blood by two mechanisms: in serum proteins complex the results emphasis that the proposed novel leads would be and in internal volume of erythrocytes (Dolomatov et al., effective in treating oxidative stress mediated AD therapeutics. 1999; Eliseeva et al., 2009; Garipova & Usmanova, 2013; Garipova & Dazko, 2015; Garipova, Morugova, Kireeva, References Ibragimov, & Baranova, 2010; Gimatdinova et al., 2011). It is Natarajan, P., Priyadarshini, V., Pradhan, D., Manne, M., Swargam, S., interesting to answer the question of how the intracellular Kanipakam, H., … Amineni, U. (2016). E-pharmacophore-based virtual hormone concentration is related to it is concentration in screening to identify GSK-3b inhibitors. Journal of Receptor and Signal each of hormone transporting systems. There distribution of Transduction Research, 36(5), 445–458. Pradeep, N., Munikumar, M., Swargam, S., Hema, K., Sudheer Kumar, K., & 3,5,30-triiodothyronine (T3) between the transport systems of Umamaheswari, A. (2015). 197 Combination of e-pharmacophore blood and nuclei of leucocytes, lung and liver tissues of rat 44 BOOK OF ABSTRACTS. ALBANY 2019: THE 20TH CONVERSATION were examined. The 3,5,3’-triiodothyronine concentrations in Departments of Systems Biology and Biochemistry and Molecular blood plasma, hemolysate and nucleoplasm prepared from leu- Biophysics, Columbia University, NY 10032, USA cocytes, lung and liver of 10 laboratory non-linear male rats [email protected] were determined by enzyme immunoassay. The 3,5,30-triiodothyronine concentration in blood plasma was averaged The presentation will describe the application of protein 6.33 ± 0.34 pmol/L. The T3 concentration in erythrocyte internal structure to predict protein–protein and protein-ligand inter- volume (hemolysate) was reliably less 2.94 pmol/L ( 8, 34, actions on a genome-wide scale. Protein networks and sig- À ¼ h 0.001).This indicates relative depletion of erythrocyte tri- naling pathways are constructed and described in structural ¼ iodothyronine depot. T3 concentrations in tissue nucleus were terms yielding multiple potential targets for drug interven- dramatically more, than in blood hormone transporting systems. tion. Drug leads are generated via structure alignment of tar- The triiodothyronine concentration in leukocytenucleiwas 2.57 get proteins with proteins that form a complex with small times more than in blood plasma (16.24 ± 0.98 pmol/L). Inlung molecules where the structure is known. An integrated soft- nuclei T3 concentration was 12.23 ± 0.87 pmol/L and in liver ware package allows for the multi-directional navigation nucleoplasm 16.68 ± 1.03 pmol/L. Perhaps, triiodothyronine con- between small molecules, proteins and networks/pathways centration is associated with biosynthetic activity of the tissue: using large genomic, structural and chemical databases. correlation coefficient between T3 and RNA concentrations in nucleoplasm was 0.57 (h 0.003). Funding ¼ The distribution of triiodothyronine between blood This research has been supported by NIH (5-R01-GM030518-38). plasma, internal volume of erythrocytes and blood leukocyte nuclei of healthy donors were studied. It’s shown, that in References blood plasma T3 concentration is 4.14 ± 0.6 pmol/L, that is 17.6% of total hormone content in blood sample. The tri- Garzon, J. I., Deng, L., Murray, D., Shapira, S., Petrey, D., & Honig, B. iodothyronine concentration in erythrocytes was (2016). A computational interactome and functional annotation for the human proteome. eLife, 5, e18715. 6.05 ± 0.4 pmol/L, that is, 25.8% of total hormone content. It Hwang, H., Dey, F., Petrey, D., & Honig, B. (2017). Structure-based predic- is noteworthy, that the T3 concentration in leucocyte nucleo- tion of ligand–protein interactions on a Genome-wide Scale. plasm averaged 13.3 ± 0.6pmol/L, that is more than three Proceedings of the National Academy of Sciences of the United States of times higher than the hormone concentration in blood America, 114(52), 13685–13690. plasma. Thus, both in the nuclei of laboratory rats tissues Hwang, H., Petrey, D., & Honig, B. (2016). A hybrid method for protein–- protein interface prediction. Protein Science, 25(1), 159–165. and healthy donors T3 concentration in nuclei significantly Zhang, Q. C., Petrey, D., Deng, L., Qiang, L., Shi, Y., Thu, C. A., … Honig, exceeds the T3 concentration in both blood hormone transport- B. (2012). Structure-based prediction of protein–protein interactions ing systems, which indicates the mechanisms of active transport on a Genome-wide Scale. Nature, 490(7421), 556– 560. Zhang, Q. C., Petrey, D., Garzon, J. I., Deng, L., & Honig, B. (2012). PrePPI: and accumulation of 3,5,30-triiodothyronine in the tissue nuclei. A structure-informed database of protein–protein interactions. Nucleic References Acids Research, 41(D1), D828–D833.

Dolomatov, S. I., Pishak, V. P., Slipenuk, T. S., Meshishen, I. F., & Okopnaya, T. V. (1999). Erythrocyte capacity to depose thyroid hor- 69. Applying quantitative single mones: Regulatory function of physicochemical factors in vitro. Voprosy Medicinskoy Chimii, (6), 572–577. molecule localization microscopy Eliseeva, O. S., Kireeva, N. A., Pershina, A. S., & Garipova, M. I. (2009). Investigation of the insulin interaction with the erythrocytes surface. to probe the mechanism of Vestnik OGU, N (6), 476–478. nucleocytoplasmic transport Garipova, M. I., & Usmanova, R. R. (2013). Isolation and partial characterization of a general hormone transporting blood protein complex. Journal of Biomolecular Structure and Dynamics, 31(supp 1), 118. Vol. No. Kathleen M. Lennon, Devin L. Wakefield, Matthew S. Garipova, M. I., & Dazko, O. I. (2015). Two different hormone transporting sys- Brehove, Ottavia Golfetto, Sunetra Biswas and Tijana tems inhuman blood: Features of peptide hormone transport in human Jovanovic-Talisman blood. Journal of Biomolecular Structure and Dynamics, 33(S1), 162. Vol. No. Department of Molecular Medicine, Beckman Research Institute, Garipova, M. I., Morugova, T. V., Kireeva, N. A., Ibragimov, R. I., & Baranova, City of Hope, CA 91010, USA [email protected] M. V. (2010). Affinity preparation and insulin-binding human blood serum protein diversity investigation. Voprosy Medicinskoy Chimii, (8), 40–44. Gimatdinova, E. V., Hayrullina, R. M., Garipova, M. I., Sotnikova, U. M., & Transport between the nucleus and cytoplasm is tightly regu- Veselov, S. U. (2011). Diagnostic and prognostic opportunities of pro- lated by nuclear pore complexes (NPCs). While these intricate calcitonin and c-reactive protein in different infectious and inflamma- molecular machines obstruct passage of nonspecific biomole- tory process with children. Fundamental Research 10(2), 280–282. cules, cargo-laden transport proteins are efficiently transported across the nuclear membrane. NPCs are essential for transcrip- 68. Using structure to identify tion, signaling and other fundamental cellular processes. As protein–protein and drug protein such, errors in the nucleocytoplasmic transport can cause cellular dysregulation. Dysregulated transport has been observed in interaction networks both neurodegenerative disorders and cancer. Despite its critical role in health and disease, specific mechanism(s) of nucleocyto- Barry Honig plasmic transport currently remain unresolved. Understanding JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS VOL. 37, SUPPLEMENT, 2019 45 the NPC transport mechanism(s) has been hindered by the (1) behavior of these assemblies and their selective permeability complexity of NPC geometry, (2) disordered nature of barrier with respect to cargo-carrying transport proteins can be under- elements called FG-Nups and (3) challenge of measuring milli- stood based on minimal complexity models relying on the second transport events at nanometer scales in the intricate cel- statistical physics of molecular assemblies on the nanoscale. lular environment. To probe potential transport mechanisms, Due to the unstructured nature of the proteins in the NPC we combined biochemical approaches with advanced quantita- passageway, it does not possess a molecular ‘gate’ that transi- tive single molecule localization microcopy (Golfetto et al., tions from an open to a closed state during translocation of 2018). We measured both binding and diffusion of transport individual cargoes. Rather, its passageway simultaneously con- proteins on oriented FG-Nup monolayers. Our ultimate goal is tains multiple transport proteins carrying different cargoes in to obtain critical information about both physiological and both directions. Although this feature increases NPC through- pathological NPC transport at the single molecule level. put, it remains unclear how the NPC maintains selective and efficient bi-directional transport under such crowded conditions. I will present of a coarse-grained computational model Funding of the NPC transport and will discuss various proposed solu- This research has been supported by STOP Cancer tions to the crowding problem in light of the model results Foundation and Beckman Research Institute. and the available experimental data (Figure 1). Funding References This research has been supported by National Science and Golfetto, O., Wakefield, D. L., Cacao, E. E., Avery, K., Kenyon, V., Jorand, Engineering Research Council of Canada through the NSERC R., … Jovanovic-Talisman, T. (2018). A platform to enhance quantita- Discovery Grant Program. tive single molecule localization microscopy. Journal of the American Chemical Society, 140(40), 12785. References 70. Self-regulating mechanisms of bi- Jovanovic-Talisman, T., & Zilman, A. (2017). Protein transport by the nuclear pore complex: Simple biophysics of a complex biomachine. directional transport through the Biophysical Journal, 113 (1), 6–14. nuclear pore complex Vovk, A. (2016). Simple biophysics underpins collective conformations of the intrinsically disordered proteins of the nuclear pore complex. eLife, e10785 T. Zheng, C. Gu and A. Zilman Department of Physics, Institute for Biomaterials and Biomedical Engineering, University of Toronto, 60 St George st, Toronto, ON, 71. Statistical mechanical model Canada M1M 2P7 [email protected] of transport receptor binding in the Nuclear pore complex (NPC) is a biomolecular ‘nanomachine’ nuclear pore complex that controls nucleocytoplasmic transport in eukaryotic cells. The key component of the functional architecture of the NPC is Rob D. Coalson the assembly of the polymer-like intrinsically disordered pro- Department of Chemistry, University of Pittsburgh, Pittsburgh, PA, USA teins that line its passageway and play a central role in the NPC transport mechanism. Due to paucity of experimental The nuclear pore complex (NPC) is a large protein complex methods capable to directly probe the morphology and the that controls the flow of proteins and mRNA into and out of dynamics of this assembly in intact NPCs, much of our know- the nuclei of eukaryotic cells. A major component of the NPC ledge about its properties derives from in vitro experiments consists of natively unfolded nucleoporin (nup) proteins, interpreted through theoretical and computational modeling. which are anchored on the inside of a cylindrical pore scaf- Remarkably, despite their molecular complexity, much of the fold. They form a polymer brush that assists in the regulation

Figure 1. 46 BOOK OF ABSTRACTS. ALBANY 2019: THE 20TH CONVERSATION of cargo flow from nucleus to cytoplasm. Large molecules of cargo-carrying transport receptors (karyopherins or Kaps, that enter/leave the nucleus must attach themselves to spe- specifically importins and exportins) that traverse the NPC. cial receptor proteins that ferry the cargo molecules in/out Otherwise, the FG Nups are thought to comprise a barrier that of nucleus. Via hydrophobic contacts between the receptor hinders large nonspecific molecules from entering the pore. protein and the nup filaments, the receptor proteins bind Still after two decades of research, the NPC modus operandi weakly and reversibly to the nup filaments and make their remains unclear because the FG Nups have eluded direct struc- way through the pore, via a mechanism that is at present tural visualization inside the pore. Also, little is known as to unknown. The complexity of the in vivo NPC motivates con- how multivalent Kap-FG Nup interactions promote rapid NPC struction of coarse-grained models that can capture some of translocation. In my talk, I will highlight our efforts to unravel its essential features while retaining computational tractabil- the emergent physical principles that underlie such remarkable ity. We have developed such a model, focusing particularly on biological function. Unexpectedly, our results show that the FG the interactions of this polymer brush with solution phase Nups are necessary but insufficient for establishing the NPC nanoparticles that are attractive to brush monomers (mimick- barrier. Rather, a surprising finding is that Kaps are essential for ing the attraction of receptor proteins to hydrophobic seg- both NPC barrier and transport function. In consolidating these ments of nup filaments in the NPC). These attractions cause results, I will discuss how Kaps might exert control over NPC the nanoparticles to infiltrate into the polymer brush and alter function - as opposed to the general view that NPCs exert con- its brush morphology. We have developed a Self-Consistent trol over the transport of Kaps. Field Theory model to analyze the equilibrium properties of the brush-nanoparticle system in an approximate but compu- tationally efficient manner (Opferman, Coalson, Jasnow, & Funding Zilman, 2013). In addition, we have performed large scale This work is supported by the Swiss National Science Foundation. coarse-grained Molecular/Langevin Dynamics simulations to explore the same properties (Nasrabad, Jasnow, Zilman, & Coalson, 2016; Ozmaian, Jasnow, Nasrabad, Zilman, & Coalson, References 2018). A variety of results pertaining to collapse/expansion of Kapinos, L. E., Huang, B., Rencurel, C., & Lim, R. Y. H. (2017). Karyopherins the brush upon nanoparticle infiltration will be presented. regulate nuclear pore complex barrier and transport function. The Their relevance to the in vivo mechanism of NPC operation will Journal of Cell Biology, 216(11), 3609–3624. be stressed (Vovk et al., 2016). Sakiyama, Y., Mazur, A., Kapinos, L. E., & Lim, R. Y. H. (2016). Spatiotemporal dynamics of the nuclear pore complex transport barrier resolved by high-speed atomic force microscopy. Nature References Nanotechnology, 11(8), 719–723. Schleicher, K. D., Dettmer, S. L., Kapinos, L. E., Pagliara, S., Keyser, U. F., Nasrabad, A. E., Jasnow, D., Zilman, A., & Coalson, R. D. (2016). Precise Jeney, S., & Lim, R. Y. H. (2014). Selective transport control on molecu- control of polymer coated nanopores by nanoparticle additives: lar velcro made from intrinsically disordered proteins. Nature Insights from computational modeling. Journal of Chemical Physics, Nanotechnology, 9(7), 525–530. 145, 064901. 1-5. Schoch, R. L., Kapinos, L. E., & Lim, R. Y. H. (2012). Nuclear transport recep- Opferman, M. G., Coalson, R. D., Jasnow, D., & Zilman, A. (2013). The tor binding avidity triggers a self-healing collapse transition in FG- morphology of polymer brushes infiltrated by attractive nanoinclu- nucleoporin molecular brushes. Proceedings of the National Academy of sions of various sizes. Langmuir, 9, 8584–8591. Sciences of the United States of America, 109(42), 16911–16916. Ozmaian, M., Jasnow, D., Nasrabad, A. F., Zilman, A., & Coalson, R. D. (2018). Effects of cross-linking on partitioning of nanoparticles into a polymer brush: Coarse-grained simulations test simple approximate ‘ ’ theories. Journal of Chemical Physics, 148(2), 024902. 1-12. 73. A double tale of evolutionary Vovk, A., Gu, C., Opferman, M. G., Kapinos, L. E., Lim, R. Y. H., Coalson, accretion in the structure of R. D., Zilman, D. … (2016). Simple biophysics underpins collective conformations of the intrinsically disordered proteins of the nuclear biological networks pore complex. eLife, 5, 10785. 1–29. Gustavo Caetano-Anolles, Fizza Mughal and M. 72. The nuclear pore complex: Fayez Aziz Department of Crop Sciences, Illinois Informatics Institute, paradoxes and possibilities University of Illinois, Urbana, IL 61801, USA [email protected]

Roderick Y. H. Lim The evolution of structure in biology is driven by accretion Biozentrum and the Swiss Nanoscience Institute, University of and change (Caetano-Anolles, Caetano-Anolles, & Caetano- Basel, Basel, Switzerland [email protected] Anolles, 2018). Accretion brings together disparate parts to form bigger wholes. Change provides opportunities for Nuclear pore complexes (NPCs) mediate the traffic of diverse growth and innovation. Networks describe how parts associ- signal-specific cargoes between the cytoplasm and nucleus ate with each other to form integrated systems. Here we in eukaryotic cells. This involves numerous intrinsically disor- explain the structure of biological networks with a biphasic dered proteins known as phenylalanine-glycine nucleoporins (bow-tie) theory of module creation embodied in a ‘double (FG Nups) that lie physically tethered inside each NPC. tale’ of evolutionary accretion (Mittenthal, Caetano-Anolles, & Importantly, the FG Nups facilitate the selectivity and speed Caetano-Anolles, 2012). In a first phase, parts are weakly JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS VOL. 37, SUPPLEMENT, 2019 47

Figure 1. Log-log plots of the clustering coefficient C(k) as a function of the number of links k for enzyme and subnetwork projections of metabolic enzyme-subnetwork bipartite networks as they grow in evolutionary time, billions of years ago (Gya). The scaling is the hallmark of hierarchical modularity. It increases in evolution and is stronger at lower levels of metabolic organization. linked and associate variously. As they diversify, they com- a Theoretical Division, Los Alamos National Laboratory, Los Alamos, pete with each other and are selected for performance. The NM; bDepartment of Physics, University of Cagliari, Cagliari, Italy; emerging interactions constrain their structure and associa- cDepartment of Pathology and Cancer Center, University of New tions. This causes parts to self-organize into modules with Mexico, Albuquerque, NM; dDepartment of Chemistry and tight linkage. In a second phase, variants of the modules Biochemistry, University of Oklahoma, Norman, OK evolve and become new parts for a new generative cycle of [email protected] higher-level organization. The paradigm predicts the rise of hierarchical modularity in evolving networks at different I will discuss two examples where we have used machine timescales and complexity levels, which we confirm with learning on molecular dynamics simulations and experimen- phylogenomic and molecular simulation data. Analyses of evolving networks describing the emergence of metabolism, tal data to address the complexity of cell membranes that the rise and diversification of the proteome, the evolution of presents a formidable challenge to conventional biophys- the ribosome, and nanosecond-level change in protein loop ical approaches. dynamics consistently reveal an increase of hierarchical Recently, we suggested a previously unknown molecular modularity and scale-free behavior as networks unfold in mechanism for T-cell activation based on the preferential evolutionary time (Figure 1). As expected, evolutionary con- binding and localization of the intrinsically disordered cyto- straints on network structure are stronger at lower levels of plasmic signaling tails, which are easily modified by the pres- biological organization. Remarkably, the phylogenomic data- ence of different lipid species and by the physical state of driven ‘double tale’ of evolutionary accretion was already the membrane. Moreover, in a biological membrane with recounted in P. Strasb. Gr. Inv. 1665-6, a 2,000-year-old liquid-ordered (Lo) and liquid-disordered (Ld) domains, we papyrus roll from the ancient city of Panopolis in Upper show that specific lipids may play a significant role in immu- Egypt attributed to Empedocles and archived at Strasbourg’s noreceptor signaling and similar mechanisms may be pos- National University Library (Janko, 2004). sible in a broader context across other signaling pathways such as RAS. We introduce an unsupervised machine algorithm based on the non-negative matrix factorization com- Funding bined with custom clustering for analysis of MD simulations. Specifically, we implement this algorithm to detect and This research has been supported by USDA NIFA award H- describe the lateral lipid segregation in a biological mem- 1014249 and several Blue Waters supercomputer allocations. brane mimic, a ternary lipid mixture with Lo and Ld domains. References The widespread emergence of multi-drug resistance is one of the most serious barriers to effective treatment of Caetano-Anolles, G., Caetano-Anolles, K., & Caetano-Anolles, D. (2018). Evolution of macromolecular structure: A ‘double tale’ of biological bacterial infections in both public health and biothreat accretion and diversification. Science Progress, 101(4), 360–383. scenarios. Gram-negative bacteria in particular represent Janko, R. (2004). Empedocles, On nature I 233–364: A new reconstruction of unique challenges for antibiotic design due to the com- P. Strasb. Gr. Inv. 1665–6. Zeitschrift Papyrologie Epigraphik, 150,1–26. bined effects of their low permeability outer membranes Mittenthal, J. E., Caetano-Anolles, D., & Caetano-Anolles, G. (2012). and their nonspecific efflux pumps. Specifically, we have Biphasic patterns of diversification and the emergence of modules. Frontiers in Genetics, 3, 147 shown that the low permeability of the two-membrane cell envelope in P. aeruginosa and the insufficient chemical diversity of potential antibiotic compounds present a chal- 74. Membranes and machine learning: lenge to antibiotic discovery. We employ both traditional machine learning techniques and a novel, fragment-based optimizing the transport of signals approach to identify the key descriptors and molecular frag- and drugs across membranes ments within a compound governing permeability and avoidance of efflux, as well as their combined effects in the Rachael Mansbacha, Cesar A Lopez a, Paolo wild type. This approach allows high-throughput screening Ruggeroneb, Bridget S Wilsonc, Nick Hengartnera, of functionally relevant fragments and offers an alternative Helen I Zgurskayad, Boian Alexandrova and S. way to search the chemical space for novel anti- Gnanakarana biotic candidates. 48 BOOK OF ABSTRACTS. ALBANY 2019: THE 20TH CONVERSATION

the peptide is recognized as foreign. In recent years person- 75. Feature selection in alized treatment approaches such as cancer immunotherapy biomolecular models based on the knowledge of MHC selectivity of a particular patient started to emerge. Detection of peptides bound to Julie Mitchell MHC on the tumor cell surface, containing cancer driver Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, mutations (neoantigens) is essential for efficiency of the TN, USA [email protected] treatment, and therefore, it is important to understand mechanisms, which drive MHC-peptide complex formation. Protein–protein interactions regulate many essential bio- Over the years many complexes have been crystallized and logical processes and play an important role in health and several approaches were developed for bound peptide struc- disease. The process of experimentally characterizing protein ture prediction. However, existing docking methods are not residues that contribute the most to protein–protein inter- suitable for a large scale structural analysis of multiple pepti- action affinity and specificity is laborious. Thus, developing des ( 104) due to large execution times, thus faster and models that accurately characterize hotspots at protein–pro- more efficient modeling techniques are required. Machine tein interfaces provides important information about how to Learning approaches have demonstrated promising results in drug therapeutically relevant protein–protein interactions. In protein structure and ligand binding prediction. Here we pre- this work, we combined the KFC2a protein–protein inter- sent an ultra-fast peptide-MHC docking method based on action hotspot prediction features with Rosetta scoring func- 3 D Convolutional Neural Network (CNN) scoring and con- tion terms and interface filter metrics. A 2-way and 3-way strained inverse kinematics peptide sampling. Our algorithm forward selection strategy was employed to train support is suitable for docking of multiple peptide-MHC complexes vector machine classifiers, as was a reverse feature elimin- and can provide insights for selectivity and preferential bind- ation strategy. From these results, we identified subsets of ing of different MHC alleles and facilitate structure based KFC2a and Rosetta combined features that show improved binding analysis (Figure 1). performance over KFC2a features alone. The forward selection algorithm also helped elucidate the biophysical principles that determine whether a given amino acid is a binding hot spot. 77. A revisiting of the RESP charge derivation model

Pavel Banasa,b, Michal Janeceka, Petra Kuhrov€ aa, 76. Ultra-fast modeling of Michal Otyepkaa,b and Jirı Sponera,b peptide–MHC interactions with aRegional Centre of Advanced Technologies and Materials, Department of Physical Chemistry, Faculty of Science, Palacky 3D convolutional neural net University, tr. 17 listopadu 12, Olomouc, 771 46, Czech Republic; bInstitute of Biophysics of the Czech Academy of Sciences, Mikhail Ignatov, Evangelos Coutsias and Kralovopolska 135, Brno, 612 65, Czech Republic Dima Kozakov [email protected] Laufer Center for Physical and Quantitative Biology, Stony Brook University, Stony Brook, NY, USA The representation of the electrostatic interactions by Coulombic interactions between the atom-centered partial Association of a peptide with Major Histocompatibility charges is a fundamental part of the molecular mechanics Complex (MHC) is crucial for adaptive immune system in ver- and empirical force field methods. The broad success of the tebrates. Upon binding to an MHC molecule, the peptide is AMBER force field family originates mainly in the RESP presented to T-cells, which triggers an immune response, if charge model (Bayly, Cieplak, Cornell, & Kollman, 1993) that

Figure 1. JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS VOL. 37, SUPPLEMENT, 2019 49 derives the partial charges to reproduce the electrostatic field around the molecules. 78. Evaluation of the current state In this study we revisited the RESP charge derivation of AMBER DNA force fields model in order to improve its description of the electrostatic potential around the molecules and thus the descrip- Marie Zgarbovaa, Michal Otyepkaa, Jirı Sponera,b and tion of the electrostatic interactions in the force field. In Petr Jureckaa particular, we re-optimized the atomic radii used for defin- aRegional Centre of Advanced Technologies and Materials, ition of the grid points for evaluation of the electrostatic Department of Physical Chemistry, Faculty of Science, Palacky field around the molecule. Some grid points introduced by University, tr. 17 listopadu 12, Olomouc, 771 46, Czech Republic; b the standard RESP procedure using Singh-Merz-Kollman Institute of Biophysics of the Czech Academy of Sciences, Kralovopolska 135, Brno, 612 65, Czech Republic radii (Singh and Kollman, 1984) are deeply buried into the [email protected] electron density, especially in case of aromatic molecules such as nucleobases, so that they significantly bias the fit- Force field-based modeling of nucleic acids has witnessed ted charges in an artificial way. In addition, we redefined increasing interest in the past few years. Currently, several the restraining scheme, in which we replaced the hyper- force field modifications for DNA simulations based on ff99 bolic restraints toward zero charge values introduced by parameters are available in the AMBER suite of programs. Bayly et al. by weighted parabolic restraints toward CM5 Knowing how various conformations are described by avail- charges (Marenich, Jerome, Cramer, & Truhlar, 2012). These able empirical force fields is crucial for molecular modeling restraints are able to selectively eliminate the poor statis- of biomolecules, including DNA. tical of the ESP charges of the atoms buried in the mol- Several refinements of torsion potential (glycosidic poten- ecule. On the other hand, they are negligibly small tials —vOL3, vOL4; backbone potentials —efOL1 and bOL1) whenever the ESP charges are statistically well-defined. The have been suggested in our laboratory. All these refinements redefined charges are able to reproduce the electrostatic were derived using methodology that includes conform- potential around the molecules more accurately than ation-dependent solvation effects and are available in OL15 standard RESP charges, especially for aromatic systems, parameter package. These modifications significantly and are able, e.g., to entirely eliminate underestimation of improved description of not only the canonical B-DNA struc- the base pairing interactions between nucleobases (Banas ture but also the noncanonical systems, such as Z-DNA and et al., 2012), which precludes accurate description of the guanine quadruplexes. Here, we report benchmark simula- nucleic acids by empirical force fields. tions of various representative DNAs as well as a comparison of several (bsc0, bsc1 and OL15) force fields. Furthermore, a comparative study of all 136 unique tetranucleotide steps Funding using MD simulations with our latest OL15 force field will be The authors gratefully acknowledge the support by the presented. We will provide a comprehensive analysis of the effect of BII substates on all B-DNA helical parameters and Ministry of Education, Youth and Sports of the Czech show how to reliably compare values from experiment Republic and Czech Science Foundation 18-25349S and by and simulation. the Operational Programme Research, Development and Education—European Regional Development Fund, project no. CZ.02.1.01/0.0/0.0/16_019/0000754. Funding The authors gratefully acknowledge the support by the References Ministry of Education, Youth and Sports of the Czech Republic and Czech Science Foundation no. 17-16107S and Banas, P., Mladek, A., Otyepka, M., Zgarbova, M., Jurecka, P., Svozil, D., … Sponer, J. (2012). Can we accurately describe the struc- by the Operational Programme Research, Development and ture of adenine tracts in B-DNA? Reference quantum-chemical Education—European Regional Development Fund project computations reveal overstabilization of stacking by molecular no. CZ.02.1.01/0.0/0.0/16_019/0000754. mechanics. Journal of Chemical Theory and Computation, 8(7), 2448–2460. Bayly, C. I., Cieplak, P., Cornell, W. D., & Kollman, P. A. (1993). A well- References behaved electrostatic potential based method using charge restraints Zgarbova, M., Sponer, J., Otyepka, M., Cheatham, T. E., III, Galindo- for deriving atomic charges: The RESP model. The Journal of Physical Murillo, R., & Jurecka, P. (2015). Refinement of the sugar-phosphate Chemistry, 97(40), 10269–10280. backbone torsion beta for AMBER force fields improves the descrip- Marenich, A. V., Jerome, S. V., Cramer, C. J., & Truhlar, D. G. (2012). tion of Z- and B-DNA. Journal of Chemical Theory and Computation, Charge Model 5: An extension of hirshfeld population analysis for the 11, 5723–5736. accurate description of molecular interactions in gaseous and con- Zgarbova, M., Jurecka, P., Lankas, F., Cheatham, T. E., III, Sponer, J., & densed phases. Journal of Chemical Theory and Computation, 8(2), Otyepka, M. (2017). Influence of BII backbone substates on DNA 527–541. Twist: A unified view and comparison of simulation and experiment Singh, U. C., & Kollman, P. A. (1984). An approach to computing electro- for all 136 distinct tetranucleotide sequences. Journal of Chemical static charges for molecules. Journal of Computational Chemistry, 5(2), Information and Modeling, 57, 275–287. 129–145. 50 BOOK OF ABSTRACTS. ALBANY 2019: THE 20TH CONVERSATION

Zgarbova, M., Jurecka, P., Banas, P., Havrila, M., Sponer, J., & Otyepka, 79. Force fields in trouble—what M. (2018). A- to B-DNA transition in AMBER force fields and its coupling to sugar pucker. The Journal of Physical Chemistry B, 121, could our DNA and RNA potentials 2420–2433. do better? Zgarbova, M., Jurecka, P., Sponer, J., & Otyepka, M. (2017). Noncanonical a/c backbone conformations in RNA and the accuracy of their a a a,b description by the AMBER force field. Journal of Chemical Theory and Petr Jurecka , Marie Zgarbova , Jirı Sponer and Computation, 14, 319–328. Michal Otyepkaa aRegional Centre of Advanced Technologies and Materials, Department of Physical Chemistry, Faculty of Science, Palacky University, 17. listopadu 12, Olomouc 77146, Czech Republic; bInstitute of Biophysics of the Czech Academy of Sciences, 80. Tuning the hydrogen-bonding Kralovopolska 135, Brno 612 65, Czech Republic [email protected] interactions of the AMBER RNA force field While canonical A-RNA and B-DNA nucleic acid structures seem to be modeled well by current AMBER force fields, the Petra Kuhrov€ aa, Vojtech Mlynskyb, Marie Zgarbovaa, description of non-canonical structures is less satisfactory. Miroslav Krepla,b, Giovanni Bussic, Robert B. Bestd, This has far-reaching consequences, such as problems with Michal Otyepkaa, Jirı Sponer a,b and Pavel Banasa,b simulations of the nucleic acids folding, which are due to aRegional Centre of Advanced Technologies and Materials, inaccurate description of the unfolded state ensemble. In our Department of Physical Chemistry, Faculty of Science, Palacky laboratory we focus on the development of dihedral angle University, tr. 17 listopadu 12, Olomouc 771 46, Czech Republic; b modifications capable of modeling non-canonical DNA and Institute of Biophysics of the Czech Academy of Sciences, c RNA forms. The key distinguishing feature of our potentials Kralovopolska 135, Brno 612 65, Czech Republic; Scuola Internazionale Superiore di Studi Avanzati, SISSA, via Bonomea is the inclusion of conformation-dependent solvation effects 265, Trieste 34136, Italy; dLaboratory of Chemical Physics, National that were neglected in previous dihedral parameterization Institute of Diabetes and Digestive and Kidney Diseases, National efforts. Several modifications now available in the OL15 pack- Institutes of Health, Bethesda, MD 20892-0520, USA age (Zgarbova et al., 2015, OL stands for the city of [email protected] Olomouc, Czech Republic) have shown improvements in modeling of Z-DNA, guanine quadruplexes, non-canonical Molecular dynamics (MD) simulations became a leading tool RNAs as well as canonical A-RNA and B-DNA duplexes. for investigation of structural dynamics of nucleic acids. However, current nucleic acid force fields are still far from Despite recent efforts to improve the empirical potentials perfect and many new problems are emerging. One example (force fields, ffs), RNA ffs have persisting deficiencies, which is the A/B equilibrium in DNA, which is not reproduced by hamper their utilization in quantitatively accurate simula- any of the current AMBER force fields (Zgarbova et al., 2018). tions. Previous studies have shown that at least two salient Another is excessive destabilization of some important natur- problems contribute to difficulties in description of free- ally occurring a/c backbone substates in RNA (Zgarbova, energy landscapes of small RNA motifs: (i) excessive stabiliza- Jurecka, Sponer, & Otyepka, 2017). Although some of these tion of the unfolded single-stranded RNA ensemble by intra- problems may be solvable by further dihedral angle refine- molecular base-phosphate and sugar-phosphate interactions ment, it becomes increasingly clear that balance between (Kuhrova et al., 2016) and (ii) destabilization of the native hydrogen bonding, stacking and interaction with water will folded state by underestimation of stability of base pairing have to be tuned to fully explore accuracy potential of the (Sponer et al., 2018). Here, we introduce a general ff term current non-polarizable force fields. (gHBfix) that can selectively fine-tune non-bonding interaction terms in RNA ffs, in particular the H-bonds. gHBfix potential affects the pair-wise interactions between all pos- Funding sible pairs of the specific atom types, while all other interactions remain intact, i.e., it is not a structure-based model. In The authors gratefully acknowledge the support by the order to probe the ability of the gHBfix potential to refine Czech Science Foundation, no. 17-16107S and by the the ff non-bonded terms, we performed an extensive set of Operational Programme Research, Development and folding simulations of RNA tetranucleotides and tetraloops. Education—European Regional Development Fund, project Based on these data we propose particular gHBfix parame- no. CZ.02.1.01/0.0/0.0/16_019/0000754. ters to modify the AMBER RNA ff. The suggested paramet- rization significantly improves the agreement between References experimental data and the simulation conformational ensembles, although our current ff version still remains far from Zgarbova, M., Sponer, J., Otyepka, M., Cheatham, T. E. I. I., Galindo- being flawless. While attempts to tune the RNA ffs by con- Murillo, R., & Jurecka, P. (2015). Refinement of the sugar phosphate À backbone torsion beta for AMBER force fields improves the descrip- ventional reparametrizations of dihedral potentials or non- tion of Z- and B-DNA. Journal of Chemical Theory and Computation, bonded terms can lead to major undesired side effects as we 11, 5723–5736. demonstrate for some recently published ffs, gHBfix has a JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS VOL. 37, SUPPLEMENT, 2019 51 clear promising potential to improve the ff performance Because of this, all organisms have a proteostasis network: while avoiding introduction of major new imbalances. a collection of chaperones and proteases that shepherd protein to the native state or recover or dispose of protein that has misfolded or aggregated. We have used computa- Funding tional models of in vivo protein folding in the presence of The authors gratefully acknowledge the support by the the proteostasis network to deepen our understanding of Ministry of Education, Youth and Sports of the Czech how organisms maintain proteostasis. Here, we will Republic and Czech Science Foundation no. 18-25349S and describe the insights that these models have provided into by the Operational Programme Research, Development and how protein folding energetics read through to proteosta- Education—European Regional Development Fund, project sis and also how these models can, in a sense, be no. CZ.02.1.01/0.0/0.0/16_019/0000754. inverted, to use perturbations in proteostasis to learn about protein folding energetics. References

Kuhrova, P., Best, R. B., Bottaro, S., Bussi, G., Sponer, J., Otyepka, M., & Funding Banas, P. (2016). Computer folding of RNA tetraloops: Identification of This research has been supported by the NIH (grant key force field deficiencies. Journal of Chemical Theory and Computation, 12(9), 4534–4548. no. GM101644). Sponer, J., Bussi, G., Krepl, M., Banas, P., Bottaro, S., Cunha, R. A., … Otyepka, M. (2018). RNA structural dynamics as captured by molecular simulations: A comprehensive overview. Chemical Reviews, 118(8), 4177–4338. 83. Simulations of biomolecules in cellular crowded environments

81. Protein folding and dynamics Yuji Sugitaa,b,c, Kento Kasaharab, Hiraku Oshimab, Isseki Yud, Grzegorz Nawrockie, Suyong Rea and in and out of the cytoplasm Michael Feigb,e aRIKEN Center for Pioneering Research, 2-1 Hirosawa, Wako-shi, Martin Gruebele Saitama, 351-0198, Japan; bRIKEN Center for Biosystems Dynamics Dept of Chemistry, Univ of Illinois, 600 South Mathews Avenue, Research, 2-1 Hirosawa, Wako-shi, Saitama, 351-0198, Japan; Urbana, IL, 61801, USA [email protected] cRIKEN Center for Computational Science, 2-1 Hirosawa, Wako-shi, Saitama, 351-0198, Japan; dMaebashi Institute of Technology, 2-1 e Martin Gruebele, University of Illinois, will describe how the Hirosawa, Wako-shi, Saitama, 351-0198, Japan; Michigan State quinary interactions, sticking and crowding affect protein University, 2-1 Hirosawa, Wako-shi, Saitama, 351-0198, Japan; [email protected] folding and protein–protein interactions in the cytoplasm. Trends from conflicting pressures of sticking and crowding The inside of cell is highly crowded with proteins, nucleic will be unraveled by comparing in-cell with in vitro experi- acids, ribosomes, metabolites and so on. The high concentra- ments, and new techniques to examine protein–protein tion of proteins realizes macromolecular crowding environ- interaction dynamics and thermodynamics inside cells reveal ments, which can affect protein behaviors in cells. The effect important differences to in vitro assays. Systems range from of macromolecular crowding was mainly interpreted via the metabolic enzymes, to loose protein clusters, to the excluded volume effect, which favors the compact and spliceosome. globular structures of proteins in the crowded environment. However, recent in-cell NMR spectroscopy and atomistic molecular dynamics (MD) simulations in explicit solvent 82. Protein folding energetics and (Harada et al. 2012, 2013) have shown the importance of proteostasis: a two-way connection weak protein–protein interactions on protein stability and dynamics. In the talk, we discuss the effect of macromolecu- Evan T. Powers lar crowding on protein-metabolite or protein–ligand interactions. In the simulations of the all-atom model of Department of Chemistry, Scripps Research, La Jolla, CA, 92037, USA [email protected] Mycoplasma Genitalium (Yu et al., 2016), we observed that not only hydrophobic but also hydrophilic metabolites also Proteostasis is the condition of a cell or organism having stay on the surfaces of proteins longer than in the bulk solu- enough natively folded protein to carry out essential bio- tion. Nonspecific and weak protein–metabolite interaction is logical functions while suppressing protein misfolding and likely important for the metabolite distributions. We also aggregation to levels below those that would cause tox- investigated kinase-inhibitor binding processes in dilute solu- icity. Protein folding to the native state can occur spontan- tion and protein crowded environment by all-atom MD simu- eously, but often does not, with protein instead becoming lations and observed different binding processes in those trapped in nonfunctional misfolded or aggregated states. two conditions (Figure 1). 52 BOOK OF ABSTRACTS. ALBANY 2019: THE 20TH CONVERSATION

Figure 1. All-atom MD simulation of the cytoplasm of Mycoplasma genitalium (This figure is taken from Yu et al., 2016).

References National Institutes of Health (R01GM127291). GJP thanks the K.C. Wong Education Foundation for travel support. Harada, R., Sugita, Y., & Feig, M. (2012). Protein crowding affects hydration structure and dynamics. Journal of the American Chemical Society, 134(10), 4842–4849. References Harada, R., Tochio, N., Kigawa, T., Sugita, Y., & Feig, M. (2013). Reduced native state stability in crowded cellular environment due to protein–- Guseman, A. J., Perez Goncalves, G. M., Speer, S. L., Young, G. B., & protein interactions. Journal of the American Chemical Society, 134, Pielak, G. J. (2018). Protein shape modulates crowding effects. 3696–3701. Proceedings of the National Academy of Sciences of the United States of Yu, I., Mori, T., Ando, T., Harada, R., Jung, J., Sugita, Y., & Feig, M. (2016). America, 115(43), 10965–10970. Biomolecular interactions modulate macromolecular structure and Guseman, A. J., Speer, S. L., Perez Goncalves, G. M., & Pielak, G. J. (2018). dynamics in atomistic model of a bacterial cytoplasm. eLife, 5, Surface charge modulates protein–protein interactions in physiologic- e19274. ally relevant environments. Biochemistry, 57(11), 1681–1684. Smith, A. E., Zhou, L. Z., Gorensek, A. H., Senske, M., & Pielak, G. J. (2016). In-cell thermodynamics and a new role for protein surfaces. Proceedings of the National Academy of Sciences of the United States of 84. Understanding protein behavior America, 113(7), 1725–1730. in cells

Gary J. Pielak 85. Effect of insulin on DNA- Department of Chemistry, University of North Carolina, Chapel Internucleosomal fragmentation and Hill, NC 27514, USA [email protected] poly(ADP-ribose)polymerase 1 activity The crowded and complex environment in cells is predicted in rat liver nuclei to affect protein behavior compared to dilute buffer. Some predictions may not be correct. We have examined crowding Anush Asatryan, Karine Matinyan, Irina Artsruni and effects on the stability of proteins and their complexes. I will Emil Gevorgyan focus on equilibrium data acquired in concentrated solutions Department of Biophysics, Yerevan State University, Yerevan, of cosolutes and in living Escherichia coli cells. The cosolutes 0025, Armenia [email protected] include synthetic polymers and their monomers, other proteins and lyophilized cytosol. The results show that crowding Apoptosis is the main type of physiological cell death which affects stability and binding, but not always as predicted by ensures tissue homeostasis by a dynamic balance between simple theory. The differences point to opportunities for the- cell proliferation and death. The final and ‘point of no return’ oretical efforts and simulations. phase of apoptosis is DNA internucleosomal fragmentation resulting in effective degradation of the nuclear DNA and its further elimination by phagocytes (Elmore, 2007). Peptide Funding hormones along with various intra- and extracellular signals Our research is supported by the National Science tightly regulate the onset of apoptosis by modulating tissue Foundation (MCB 1410854 and CHE 1607359) and the and cell-specific responses. Liver is a target organ where JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS VOL. 37, SUPPLEMENT, 2019 53 insulin displays antiapoptotic properties. One of the key fac- simulations revealed alternative binding modes for the linker tors which mediates the cross-talk of various death pathways histone to nucleosomes. From Brownian and molecular is poly(ADP-ribose)polymerase 1 (PARP 1), the activity of dynamics simulations, we proposed that there is an ensem- which is precisely regulated by different exogenous and ble of chromatosome structures. The LH isoform, nucleosome endogenous cell signals. The activation of PARP 1 by DNA dynamics and DNA sequence are all factors that influence strand breaks and inhibition of the enzyme by ATP, estab- the LH binding mode to nucleosomes. Whereas LH proteins lishes PARP 1 as a molecular link between DNA damage, recognize nucleosomes in a sequence independent manner, energy status and chromatin modification in cells [Kim, the pioneer transcription factors (TFs) are a subclass of TFs Zhang, & Kraus, 2005; Hassa & Hottiger, 2008). PARP 1 plays that are able to recognize sequence specific binding sites on a prominent role in switching different cell death programs DNA wrapped in nucleosomes to regulate gene expression. by distinct mechanisms: depleting intracellular NAD and Pioneer TFs were proposed to contribute to chromatin open- þ ATP, controlling chromatin accessibility and the activities of ing. Many of these factors are involved in cell identity transi- various apoptotic endonucleases, which are required for nor- tions, and therefore they are of special importance for mal nuclear degradation. In present study we examined regenerative therapies. For example, the master regulators of whether insulin can modulate the key process of execution stem cell pluripotency, Oct4 and Sox2 were proposed to be phase of apoptosis-internucleosomal DNA fragmentation, in pioneer TFs. The structural basis for the pionner factor- isolated rat liver nuclei after 4 and 24 h during hormone nucleosome binding remains unknown. Moreover, it is still administration to rats. The activity of PARP 1 was concomi- not understood if the binding of TFs to nucleosomes has a tantly investigated. Our results revealed that insulin sup- direct impact on nucleosome dynamics. To characterize the pressed the intensity of DNA fragmentation twofold after 4 h structural basis for Oct4-nucleosome recognition, we first of injection. Nevertheless, during 24 h insulin markedly stimu- performed molecular dynamics simulations of 3 nucleo- lated DNA internucleosomal fragmentation. However, insulin somes: one with an artificial DNA sequence optimized for had no appreciable effect on PARP 1 activity in rat liver nucleosome stability and 2 with native DNA sequences con- nuclei in all examined periods of hormone action. Taking taining 1 or multiple Oct4 binding sites, respectively. We into consideration, that the activity of PARP 1 depends on found that the nucleosome with multiple Oct4 binding sites ability of the enzyme molecules to bind DNA, we suppose was the most mobile. Interestingly, the amplitude of breath- that hydrocortisone’s nuclear receptor can modulate PARP 1 ing and twisting motions in the DNA which were observed activity by competing with the enzyme for binding sites in all nucleosomes, was increased in the nucleosome with on DNA. multiple Oct4 binding sites. Using an approach that com- bines structural modeling, molecular dynamics simulations with experimental approaches, we built different models of References Oct4-nucleosome complexes and show that alternative but Elmore, S. (2007). Apoptosis: A review of programmed cell death. not all possible configurations are stable and compatible Toxicologic Pathology, 35(4), 495–516. with the DNA curvature and DNA-histone interactions. Hassa, P. O., & Hottiger, M. O. (2008). The diverse biological roles of mammalian PARPs, a small but powerful family of poly-ADP-ribose polymerases. Frontiers in Bioscience, 13(13), 3046–3082. Acknowledgments Kim, M. Y., Zhang, T., & Kraus, W. L. (2005). Poly(ADP-ribosyl)ation by The research presented was performed in collaboration with Mehmet PARP-1: ‘PAR-laying’ NADþ into a nuclear signal. Genes & Ozt€ urk,€ Rebecca Wade, Jan Huertas, Caitlin MacCarthy, and Hans R. Scholer.€ Development, 19, 1951–1967. References 86. How do DNA binding proteins Huertas, J., MacCarthy, C., & Scholer,€ H. R. (2019). Do transcription factors interpret nucleosome dynamics? In preparation interpret and modulate Ozt€ urk,€ M. A., Cojocaru, V., & Wade, R. C. (2018a). Towards an ensemble nucleosome dynamics? view of the linker histone - nucleosome complex structure: A paradigm shift from one to many. Structure, 28(8), 1050–1057. Ozt€ urk,€ M. A., Cojocaru, V., & Wade, R. C. (2018b). Dependence of chro- Habil. Vlad Cojocaru matosome structure on linker histone sequence and post-translational In Silico Biolmolecular Structure and Dynamics Group, The modifications. Biophysical Journal, 114(10), 2363–2375. Hubrecht Institute for Developmental Biology and Stem Cell Ozt€ urk,€ M., Pachov, G., Wade, R. C., & Cojocaru, V. (2016). Conformational Research, Uppsalalaan 8, Utrecht, CT, 3584, The Netherlands selection and dynamic adaptation upon linker histone binding to the nucleosome. Nucleic Acids Research, 44(14), 6599–6613. doi:10.1093/ I will present our recent efforts to explore how linker histo- nar/gkw514 nes and pioneer transcription factors interpret and modulate nucleosome dynamics to bind DNA wrapped around histo- 87. Hybrid methods to characterize nes. Linker histones (LH) bind to nucleosomes in a 1:1 stoichiometry to form chromatosomes and to compact nucleosome structure and dynamics chromatin fibers. The geometry of the chromatosome with high precision remains debated. Different structures of the LH-nucleosome complex solved experimentally or proposed from computer Anna Panchenko 54 BOOK OF ABSTRACTS. ALBANY 2019: THE 20TH CONVERSATION

Figure 1.

Shaytan, A. K., Xiao, H., Armeev, G. A., Gaykalova, D. A., Komarova, G. A., National Center for Biotechnology Information, National Library of Wu, C., … Panchenko, A. R. (2018). Structural interpretation of Medicine, National Institutes of Health, Bethesda, MD, 20894, USA DNA–protein hydroxyl-radical footprinting experiments with high [email protected] resolution using HYDROID. Nature Protocols, 13(11), 2535–2556. Shaytan, A. K., Xiao, H., Armeev, G. A., Wu, C., Landsman, D., & Nucleosomes are basic units of chromatin compaction and Panchenko, A. R. (2017). Hydroxyl-radical footprinting combined with hubs in epigenetic signaling pathways. Nucleosomes experi- molecular modeling identifies unique features of DNA conformation ence a broad repertoire of alterations that affect their and nucleosome positioning. Nucleic Acids Research, 45(16), 9229–9243. dynamics and interactions with various binding partners. To Xiao, H., Wang, F., Wisniewski, J., Shaytan, A. K., Ghirlando, R., FitzGerald, gain insights into intrinsic dynamics of the full nucleosome, P. C., … Landsman, D., et al (2017). Molecular basis of CENP-C associ- we performed all-atom microsecond molecular dynamics ation with the CENP-A nucleosome at yeast centromeres. Genes & simulations of nucleosomes including linker DNA segments Development, 31(19), 1958–1972. and full-length histones (Shaytan et al., 2016). Next, we developed several hybrid approaches by combining experimental data with molecular modeling and molecular dynamics simulations. 88. Investigating the structural First approach, HYDROID, allows interpretation of DNA–protein interactions by quantifying Hydroxyl Radical dynamics of DNA in nucleosome Footprinting data and integrating it with atomistic structural core particles models (Shaytan et al., 2018). We applied HYDROID to characterize Saccharomyces cerevisiae centromeric nucleosome Lois Pollack of unknown structure and identified the precise positioning School of Applied and Engineering Physics, Cornell University, of centromeric DNA sequence (Shaytan et al., 2017). In Ithaca, NY 14853, USA [email protected] another study we used HYDROID to pinpoint the footprint of interaction between the inner kinetochore protein Mif2/ DNA is tightly packaged around nucleosome core particles CENP-C and centromeric DNA which plays an important role (NCPs) for efficient storage, yet must remain readily access- for Mif2 recruitment (Xiao et al., 2017). ible for processing. Our interest is in developing experimen- Our second hybrid approach was applied to describe his- tal tools that reveal the global structure(s) of DNA in NCPs, tone octamer distortion preceding DNA entry into nucleo- to understand the molecular mechanisms by which release is somes and processive movement of the ATPase motor of affected by factors including histone or DNA sequence varia- ISW2 on nucleosomal DNA. We performed accelerated tions or the action of partner molecules, such as remodelers molecular dynamics simulations guided by experimental or chaperones. To accomplish this goal, we must be able to chemical crosslinking data to gain an understanding of the detect the global structure(s) of the DNA in the presence of protein partners. Contrast variation small angle X-ray scatter- potential perturbations occurring in the nucleosome struc- ing is an ideal probe of the conformation of the nucleic acid ture and investigate how nucleosomal DNA can adjust to component of a protein-nucleic acid complex (Tokuda, Pabit, perturbations in the histone octamer structure (Figure 1). & Pollack, 2016). This method can be implemented in either static (Chen et al., 2014) or time-resolved studies (Chen et al., References 2017). As a proof of principle, we studied the salt dependent conformations of DNA in NCPs, in both equilibrium and time Shaytan, A. K., Armeev, G. A., Goncearenco, A., Zhurkin, V. B., Landsman, D., & Panchenko, A. R. (2016). Coupling between histone conforma- resolved studies. Data were analyzed using an ensemble tions and DNA geometry in nucleosomes on a microsecond timescale: optimization method that yields plausible structural ensem- Atomistic insights into nucleosome functions. Journal of Molecular bles present under each different solution condition or at dif- Biology, 428(1), 221–237. ferent time during a dynamic release experiment. Initially, we JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS VOL. 37, SUPPLEMENT, 2019 55 employed the tightly positioning Widom 601 DNA sequence mechanisms of DNA access regulation by chromatin dynam- and canonical histones (Chen et al., 2014, 2017); we recently ics are however still poorly understood. expanded our studies to account for DNA sequence using We dissect chromatin signaling on the single-molecules sequence specific models to create ensembles (Mauney scale, combining chemical biology approaches and mechan- et al., 2018), histone variants, and finally the addition of istic biophysics. We recently developed a single-molecule chaperones such as the Chd-1 remodeler (Tokuda et al., FRET (smFRET) approach to directly observe the dynamic 2018). architecture of chemically defined chromatin fibers. We find that local chromatin organization is based on tetranucleo- some units, which undergo structural fluctuations on the micro- to millisecond timescale. These dynamics are regulated by histone PTMs and linked to function. Indeed, internal chromatin dynamics are exploited by transcription factors (TF) to invade chromatin structure. Employing single- molecule fluorescence imaging we could observe how a yeast pioneer TF, Rap1, binds its target promoter in compact chromatin. Importantly, we show that Rap1 binding opens Funding chromatin fiber structure by inhibiting nucleosome-nucleo- LP’s research on NCPs is supported by NIH under some contacts. Finally, we reveal that Rap1 collaborates with R35GM122514. the chromatin remodeler RSC to destabilize promoter nucleosomes, paving the way to form long-lived bound states on now exposed DNA. References In summary, our results provide a mechanistic view of how a pioneer TF gains access and opens chromatin, thereby Chen, Y., Tokuda, J. M., Topping, T., Meisburger, S. P., Pabit, S. A., Gloss, L. M., & Pollack, L. (2017). Asymmetric unwrapping of nucleosomal establishing an active promoter architecture and controlling DNA propagates asymmetric opening and dissociation of the histone gene expression. core. Proceedings of the National Academy of Sciences of the United States of America, 114(2), 334–339. Chen, Y., Tokuda, J. M., Topping, T., Sutton, J. L., Meisburger, S. P., Pabit, S. A., … Pollack, L. et al (2014). Revealing transient structures of nucleosomes as DNA unwinds. Nucleic Acids Research, 42(13), 90. Modeling and informatics of DNA, 8767–8776. Mauney, A. W., Tokuda, J. M., Gloss, L. M., Gonzalez, O., & Pollack, L. nucleosomes and chromatin from (2018). Local DNA sequence controls asymmetry of DNA Unwrapping base pairs to genomes from nucleosome core particles. Biophysical Journal, 115(5), 773–781. Tokuda, J. M., Pabit, S. A., & Pollack, L. (2016). Protein-DNA and ion-DNA interactions revealed through contrast variation SAXS. Biophysical Ran Sun, Zilong Li and Thomas C. Bishop Reviews,1–11. College of Engineering and Science, Louisiana Tech University, Tokuda, J. M., Ren, R., Levendosky, R. F., Tay, R. J., Yan, M., Pollack, L., & Ruston, LA, 71272, USA [email protected] Bowman, G. D. (2018). The ATPase motor of the Chd1 chromatin remodeler stimulates DNA unwrapping from the nucleosome. Nucleic Genomics is a sequence-based informatics science and a Acids Research, 46(10), 4978–4990. structure based molecular science. Nucleosomes are the fundamental building blocks of chromatin, the biomaterial that houses the genome in all higher organisms. There are approximately 160 atomic resolution structures of the 89. Revealing dynamic chromatin nucleosome available from the protein data bank. Collectively they explore histone mutations, species varia- invasion by pioneer transcription tions, binding of drugs and ionic effects but only a few factors on the single-molecule scale sequences of DNA. Given a four-letter code (A, C, G, T) there are on the order of 4147–1088 possible sequences of DNA Beat Fierz that can form a nucleosome. Exhaustive studies are not pos- Ecole Polytechnique Federale de Lausanne, SB ISIC LCBM CH B3 sible. Here, we introduce G-Dash, a web based genome dash- 485, Station 6, Lausanne CH-1015, Switzerland board, specifically designed to integrate informatics and 3 D [email protected] material studies of DNA, nucleosomes and chromatin, and TMB-iBIOMES, a database containing over 20 microseconds The dynamic organization of the eukaryotic genome into of all atom molecular dynamics simulations representing chromatin is integral to genome regulation. Chromatin struc- over 500 different realizations of the nucleosome. G-Dash ture and dynamics, modulated by histone post-translational unites Interactive Chromatin Modeling (ICM), the Biodalliance modifications (PTMs) as well as architectural proteins, dictate genome browser and the JSmol molecular viewer to fold any DNA access for transcription factors and the gene expression DNA sequence into atomic or coarse-grained models of DNA, machinery. While of key importance, the detailed nucleosomes or chromatin. The exchange of data between 56 BOOK OF ABSTRACTS. ALBANY 2019: THE 20TH CONVERSATION

Figure 1 (Left) TMB-iBIOMES is an iBIOMES-lite database with over 20 ms of trajectory and analysis data for over 500 unique realizations of the nucleosome. Middle: A collection of Molecular Views representing the interactive capabilities of G-Dash. Right: G-dash integrates the informatics capabilities of a Genome Browser (top) with atomic and coarse-grained compute engines via a Control Panel (bottom).

informatics and structure is bi-directional so any informatics Department of Biophysics, Yerevan State University, Yerevan 0025, track can inform a molecular structure (e.g., fold by MNase Armenia [email protected] activity) and structure features can be displayed as informatics tracks in a genome browser (e.g. Roll, Slide, or Twist). Our PAR polymer metabolism play significant role in basal bio- TMB-iBIOMES database serves as a reference for comparative, logical processes involved in cell death and survival path- on-demand simulations of nucleosomes. Every simulation ways. PAR polymer turnover is maintained by coordinated deposited in TMB-iBIOMES contains Amber formatted top- interplay of enzymes responsible for PAR synthesis and deg- ology and coordinate input files, NAMD formatted output, radation, poly(ADP- ribose)polymerase (PARP 1) and log and trajectory files, and RMSD and DNA helical param- poly(ADP-ribose) glycohydrolase (PARG) correspondinglly. eter data. Closely related simulations in TMB-iBIOMES are Nowadays, PARP 1 inhibitors entered into clinical treatment grouped together and a summary analysis is provided. All of cancer patients (Blenn, Wyrsch, & Althaus, 2011) and the data can be navigated in a file browser or iBIOMES-Lite web search of less toxic PARP 1 inhibitors is in progress. It was browser format or downloaded with command line tools. It documented, that natural product-tannic acid (TA) modulates demonstrates that the workflow and simulation protocols poly(ADP-ribose) polymer (PAR) catabolism in the in vitro developed are robust, that DNA sequence can affect the experimental settings (Czeh, Fabian, Sumegi,€ & Scorrano, structure and dynamics of nucleosomal DNA at some loca- 2017). However, little is known about the impact of TA on tions but not others, and that sequence effects on nucleo- PARP1 activity and chromatin condensation in in vivo experi- some structure and dynamics can be observed in 10s of mental settings. Herein, we examine whether intraperitoneal nanoseconds. TMB-iBIOMES and G-Dash work together to (i.p.) injection of TA to rats could influence PARP 1 activity in provide a novel means for investigating structure–function rat liver and thymocytes nuclei. The second goal of the study relationships for regions of the genome ranging from base was investigation of the impact of TA on chromatin accessi- pairs to chromosomes (Figure 1). 2 2 bility to Caþ /Mgþ -dependent endonuclease, coming from the knowledge that PARP 1 determines chromatin condensa- Funding tion. Our data come to show, that i.p. injection of TA to rats suppressed PARP 1 activity both in thymocyte and liver This effort was supported by NIH IDEA grants 5 P20 nuclei and led to augmentation of liver chromatin resistance GM103424-15 and 3 P20 GM103424-15S1 and the NSF OIA- to DNA internucleosomal fragmentation. This data indicated 1541079 and the Louisiana Board of Regents. on chromatin condensation in liver nuclei after administration of TA to rats. The in vivo treatment of rats with TA References affects PARP 1 activity in thymocyte and liver nuclei in dose-

TMB-iBIOMES is available at http://dna.engr.latech.edu/ ibiomes/ and organ-specific manner. TA-induced condensation of liver G-Dash is available at http://dna.engr.latech.edu/ gdash/ nuclei chromatin could provide useful information about the type of cell death involved in TA-induced hepatotoxicity. Funding 91. The effect of tannic acid on PARP This work was supported by the RA MES State Committee of 1 activity and chromatin Science, in the frames of the research project no. 18T-1F011. condensation in rat thymocyte and liver nuclei References Blenn, C., Wyrsch, P., & Althaus, F. R. (2011). The ups and downs of tan- Irina G. Artsruni, Anush L. Asatryan, Karine S. nins as inhibitors of Poly(ADP-Ribose)glycohydrolase. Molecules, 16(2), Matinyan and Emil S. Gevorgyan 1854–1877. doi:10.3390/molecules16021854. JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS VOL. 37, SUPPLEMENT, 2019 57

Czeh, A. M., Fabian, Z., Sumegi,€ B., & Scorrano, L. (2017). Poly(adenosine Widlak, P., Peng, L., Wang, X., & Garrard, W. T. (2000). Cleavage preferen- diphosphate-ribose)polymerase as therapeutic target: Lessons learned ces of the apoptotic endonuclease DFF40 (Caspase-activated DNase from inhibitors. Oncotarget 8(30). 50221–50239. www.impactjournals. or Nuclease) on naked DNA and chromatin substrates. Journal of com/oncotarget Biological Chemistry, 275(11), 8226–8232.

92. The influence of hydrocortizone 93. Topological polymorphism and cisplatin co-administration to rats of chromatin fibers on chromatin olygonucleosomal fragmentation in liver nuclei Davood Norouzi and Victor B. Zhurkin Laboratory of Cell Biology, CCR, National Cancer Institute, NIH, Bethesda, MD 20892, USA [email protected] Anush Asatryan, Irina Artsruni, Karine Matinyan and Emil Gevorgyan Using computer simulations, we found two topologically dis- Department of Biophysics, Yerevan State University, Yerevan, tinct families of the chromatin fiber conformations distin- 0025, Armenia [email protected] guished by the linker length, L. The fibers with L {10n} and ¼ {10n 5} bp have DNA linking numbers per nucleosome þ Accumulation of cisplatin in normal tissues in the course of Lk –1.5 and –1.0, respectively (Norouzi and Zhurkin, 2015). treatment of cancer patients is responsible for severe adverse The fibers with Lk –1.5 (T2) were observed earlier, while effects. Thus, curative effect of cisplatin is attenuated due to the topoisomer with Lk –1.0 (T1) is novel. These predic- dose-limiting toxicity and growing resistance of cancer cells tions were confirmed for circular nucleosome arrays with pre- to the drug. To prevent cisplatin-induced side effects, for cisely positioned nucleosomes (Nikitina, Norouzi, Grigoryev, & example inflammation, nausea and vomiting, hydrocortisone Zhurkin, 2017). We suggest that topological polymorphism of is commonly co-administrated with cisplatin. Cells that chromatin fibers may play a role in the process of transcrip- undergo cytotoxic insults committed life or die decisions tion, i. e., the {10n 5} DNA linkers are likely to produce þ depending on genetic programs, which could be triggered transcriptionally competent chromatin structures. This by modulation of chromatin condensation. From the other hypothesis is consistent with available data for several eukar- hand, it is widely recognized that biological activities of yotes, from yeast to mouse (Norouzi, Katebi, Cui, & Zhurkin, hydrocortisone are mediated by hormone receptors, which 2015; Norouzi and Zhurkin, 2018). Here, we analyzed the bind and locally modify chromatin structure. However, little data from a recent genome-wide radio-probing study of is known about possible effect of co-treatment with cisplatin DNA folding in human cells. The technique uses ionizing and hydrocortisone on chromatin structure. To study Radiation-Induced spatially Correlated Cleavage of DNA whether DNA-binding of hydrocortisone receptors can inter- with sequencing (RICC-seq) to identify the DNA-DNA con- fere with DNA-cisplatin adducts and alter chromatin struc- tacts that are spatially proximal (Risca, Denny, Straight, & ture, we employed chromatin structure-dependent assay, Greenleaf, 2017). We show that the novel topoisomer with 2 using artificially activated intra-nuclear apoptotic Mgþ and Lk –1.0 has to be taken into account to interpret the 2 2 Caþ /Mgþ -dependent endonucleases (Widlak, Peng, Wang, experimental data, especially for the transcriptionally active & Garrard, 2000; Matassov, Kagan, Leblanc, Sikorska, & regions (Figure 1). This is yet another evidence for occur- Zakeri, 2004) and DNase 1 accessibility test. Our data rence of two distinct fiber topoisomers (Norouzi and come to show, that co-administration of cisplatin and Zhurkin, 2018). Potentially, our findings may reflect a gen- hydrocortisone to rats modulated liver chromatin DNase 1 eral tendency of chromosomal domains with different lev- accessibility in time-dependent manner. Elevated resistance els of transcription to retain topologically distinct higher- to DNase 1 in 4 h after treatment of rats with hydrocorti- order fiber conformations, T1 and T2. sone and cisplatin coincided with the rise of liver DNA Tm, and was accompanied by descending intensity of DNA internucleosomal fragmentation. This data prompt that hydrocortisone–cisplatin interaction could prevent loosening References of chromatin in internucleosomal chromatin regions Nikitina, T., Norouzi, D., Grigoryev, S. A., & Zhurkin, V. B. (2017). DNA top- induced by cisplatin treatment and, thus, might modulate ology in chromatin is defined by nucleosome spacing. Science its pharmacological outcomes. Advances, 3(10), e1700957 Norouzi, D., & Zhurkin, V. B. (2015). Topological polymorphism of the two-start chromatin fiber. Biophysical Journal, 108(10), 2591–2600. Norouzi, D., & Zhurkin, V. B. (2018). Polymorphic 30-nm chromatin fiber References and linking number paradox. Evidence for the Occurrence of Two Distinct Topoisomers, bioRxiv 478396; doi: http://dx.doi.org/10.1101/ Matassov, D., Kagan, T., Leblanc, J., Sikorska, M., & Zakeri, Z. (2004). 478396. Measurement of apoptosis by DNA fragmentation. Methods in Norouzi, D., Katebi, A., Cui, F., & Zhurkin, V. B. (2015). Topological diver- Molecular Biology (Clifton, N.J.), 282,1–17. sity of chromatin fibers: Interplay between nucleosome repeat length, 58 BOOK OF ABSTRACTS. ALBANY 2019: THE 20TH CONVERSATION

Figure 1 (Left) Comparison of the experimental RICC-seq data (Risca et al., 2017) with theoretical predictions (Norouzi and Zhurkin, 2018). The experimental genome-wide Fragment Length Distribution (FLD) profiles calculated for the transcriptionally active (H3K27ac, top red curve) and repressed (H3K9me3, top blue curve) regions in human genome correspond to the topoisomer T1 (bottom red curve) and T2 (bottom blue curve), respectively. (Center) Optimal folding of T1 and T2 are shown. (Right) The entry and the exit halves of nucleosomes (gyres) in the left stack are colored differently to emphasize distinct spatial organization of DNA in the T1 and T2 topoisomers, which results in different fragmentation patterns.

DNA linking number and the level of transcription. AIMS Biophysics, sequential imaging to enable RNA imaging at the tran- 2(4), 613. scriptomic scale in individual cells. By allowing single-cell Risca, V. I., Denny, S. K., Straight, A. F., & Greenleaf, W. J. (2017). Variable transcriptomic analysis in the native context of cells and chromatin structure revealed by in situ spatially correlated DNA cleavage mapping. Nature, 541(7636), 237–241. tissues, MERFISH facilitates the delineation of gene regulatory networks, the mapping of RNA distributions inside cells, and the mapping of distinct cell types in complex tissues. In this presentation, I will also talk about our tech- 94. Imaging the 3D organization of nology development of MERFISH and its application to cell the chromosome and transcriptome atlas mapping. in single cells and the cell atlas of tissues 95. The ‘self-stirred’ genome: bulk Xiaowei Zhuang and surface dynamics of the Howard Hughes Medical Institute, Harvard University, 12 Oxford Street, Cambridge, MA 02138, USA chromatin globule [email protected] Alexandra Zidovska The spatial organization of genome plays an important role Center for Soft Matter Research, Department of Physics, New York in many essential genome functions from gene regulation to University, New York City, USA genome replication. However, many gaps remain in our understanding of the three-dimensional (3D) organization of Chromatin structure and dynamics control all aspects of DNA chromatin in the nucleus, partly because of the lack of biology yet are poorly understood. In interphase, time proper imaging tools to directly visualize this organization. between two cell divisions, chromatin fills the cell nucleus in We have developed super-resolution imaging and multiplex its minimally condensed polymeric state. Chromatin serves as fluorescent in situ hybridization (FISH) methods that allow substrate to a number of biological processes, e.g., gene tracing of the 3 D conformation of chromatin in cell nucleus expression and DNA replication, which require it to become with high resolution. In this talk, I will present these methods locally restructured. These are energy-consuming processes and their applications to the studies of chromatin domain giving rise to nonequilibrium dynamics. Chromatin dynamics and compartment organization in individual cells. has been traditionally studied by imaging of fluorescently I will also present a single-cell transcriptome imaging labeled nuclear proteins and single DNA-sites, thus focusing method, multiplexed error-robust FISH (MERFISH). MERFISH only on a small number of tracer particles. Recently, we uses error-robust barcoding, combinatorial labeling and developed an approach, displacement correlation JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS VOL. 37, SUPPLEMENT, 2019 59 spectroscopy (DCS) based on time-resolved image correlation analysis, to map chromatin dynamics simultaneously across 97. Applications of modification the whole nucleus in cultured human cells (Zidovska, Weitz, detection in nanopore sequencing & Mitchison, 2013). DCS revealed that chromatin movement was coherent across large regions (4 and 5 lm) for several Winston Timp seconds. Regions of coherent motion extended beyond the Biomedical Engineering, Johns Hopkins University, Baltimore, MD, boundaries of single-chromosome territories, suggesting elas- USA [email protected] tic coupling of motion over length scales much larger than those of genes (Zidovska, Weitz, & Mitchison, 2013). These Nanopore sequencing is a single molecule characterization large-scale, coupled motions were ATP-dependent and uni- method, allowing direct sequencing of DNA or RNA directional for several seconds. Following these observations, sequencing with read lengths ranging from kilobases to we developed a hydrodynamic theory (Bruinsma, Grosberg, even megabases. Unlike traditional sequencing-by-synthesis Rabin, & Zidovska, 2014) and a microscopic model (Saintillan, methods, it can distinguish covalently modified nucleotides Shelley, & Zidovska, 2018) of active chromatin dynamics. directly through their modulation of the electrolytic cur- Here, we investigate chromatin interactions with nuclear rent. And the long reads allow for straightforward detec- envelope and compare the surface dynamics of the chroma- tion of structural variations, large insertions, deletions or tin globule with its bulk dynamics (Chu, Haley, & Zidovska, transpositions that are often difficult to detect with short- 2017), which we also explore using naturally present cellular read sequencing. probes (Caragine, Haley, & Zidovska, 2018). We demonstrate the power of this technique, as applied with DNA, combined with exogenous labeling, to perform References an integrative, single-molecule characterization of the epige- nome. We used M.CviPI, a GpC methyltransferase, to label accessible chromatin in cancer and normal cell lines. This Bruinsma, R., Grosberg, A. Y., Rabin, Y., & Zidovska, A. (2014). Chromatin allowed us to simultaneously correlate nucleosome position- hydrodynamics. Biophysical Journal, 106(9), 1871–1881. ing and native CpG methylation along long ( 10 kb) single Caragine, C. M., Haley, S. C., & Zidovska, A. (2018). Surface fluctuations and coalescence of nucleolar droplets in the human cell nucleus. molecules. We investigated methods of targeted sequencing Physical Review Letters, 121(14), 148101 for deeper nanopore sequencing at specific genomic loci. Chu, F., Haley, S. C., & Zidovska, A. (2017). On the origin of shape fluctu- These methods resulted in focussed coverage of long native ations of the cell nucleus. Proceedings of the National Academy of nanopore sequencing reads, measuring single-molecule Sciences of the United States of America, 114(39), 10338–10343. methylation patterns, SVs and SNVs. Finally, we have Saintillan, D., Shelley, M. J., & Zidovska, A. (2018). Extensile motor activity drives coherent motions in a model of interphase chromatin. applied native RNA sequencing to interrogate poly(A) tail Proceedings of the National Academy of Sciences of the United States of lengths and modifications to RNA which may be involved America, 115(45), 11442–11447. in posttranscriptional regulation. RNA base modifications are Zidovska, A., Weitz, D. A., & Mitchison, T. J. (2013). Micron-scale coher- detectable via modulation of the nanopore current; our ini- ence in interphase chromatin dynamics. Proceedings of the National tial focus is on the METTL3 motif (GGm6ACU). Poly-A tail Academy of Sciences of the United States of America, 110(39), 15555–15560. lengths inform mRNA lifetime; we can measure these lengths from how long the molecule takes to transit the pore. With these methods, we have measured gene specific and even isoform specific poly(A) tail lengths and modifica- 96. Understanding the human tion signals. genome in 3D

Leonid Mirney 98. Stoichiometric constraints in Institute for Medical Engineering & Science, Massachusetts Institute of Technology, 77 Massachusetts Ave E25-406, protein sequences Cambridge, MA 02139, USA [email protected] Aditya Mittal Leonid Mirny is co-director of the Center for 3D Structure Kusuma School of Biological Sciences, IIT Delhi, Hauz Khas, New and Physics of the Genome at UMass Medical School and Delhi 110016, India [email protected] MIT, funded by the National Institutes of Health’s 4D Nucleome Program. He is working to understand the human About a decade ago, rigorous analyses of structural data of genome in 3D with his team at MIT in collaboration with the thousands of naturally occurring folded proteins yielded a Dekker Lab at UMass Medical School. Using new data uncov- surprising ‘margin of life’ for stoichiometric composition, i.e., ered via Chromosome Conformation Capture (Hi-C) technol- percentage occurrence of individual amino acids, of protein ogy and computer simulations, the collaborators explore sequences (Mittal et al., 2010; Mittal & Jayaram, 2011a, how the genome is organized inside a cell. 2011b). This ‘margin of life’ refers to the lower than expected 60 BOOK OF ABSTRACTS. ALBANY 2019: THE 20TH CONVERSATION variances in percentage occurrence of individual amino acids Day by day, people are dying of one or the other type of in protein sequences (Mezei, 2011). The concept of ‘margin cancer worldwide; oral cancer being the third most common of life’ is about a decade old now and has been confirmed cancer in India and sixth in the world. To treat cancer, the over a large sequence space for almost all known protein roles of different factors involved in triggering cancer needs sequences (even in the absence of structural data for a large to be exploited which needs the exploration of mechanisms number of sequences). While the earlier work was based on of the factors involved and targeting their interactions with the largest structural dataset at that time, in this work further co-operating factors (Takiar et al., 2010; van der Wall, 2013). explorations on compositional constraints for known protein The protein product with accession number BAH14511.1, iso- sequences are presented on the largest dataset analyzed till lated from tongue tumor tissue of Homo sapiens was found date. The relationships between peptide-bonded pairs and to be an isoform b of cGMP-dependent protein kinase II triplets of amino acids, in about half-a-million protein (PKGII) as shown by the results of nonredundant BLAST. sequences, with their various physico-chemical properties PKGII plays an important role in the regulation of signaling and individual proportions (of the peptide-bonded pairs and pathways associated with cancer, and hence prediction of triplets) are explored. While having profound evolutionary the 3 D structure of the isoform would be important for implications towards our insights into occurrence of naturally the prediction of lead molecules that can be further used occurring protein sequences, the results discussed also prom- for designing a drug for the regulation of PKGIIb. In this ise to serve as guide for creating stable and structurally con- study, various tools (PROTPARAM, SMART, TMHMM, SignalP, trolled and/or ‘disordered’ designer proteins. SecretomeP, NetChop and NetPhos) were used for functional annotation. The 3 D structure of the protein was predicted by Homology Modeling approach (Blom, References Gammeltoft, & Brunak, 1999; Yu, Chen, Lu, & Hwang, Mezei, M. (2011). Discriminatory power of stoichiometry-driven protein 2006). The 3 D structure was validated by Verify3D and folding? Journal of Biomolecular Structure and Dynamics, 28(4), WhatIf. The binding site in the 3 D structure of PKGIIb was 625–626. predicted by using SiteMap which gave 5 sites as result Mittal, A., Jayaram, B., Shenoy, S. R., & Bawa, T. S. (2010). A stoichiometry from which the site having highest site score of 1.114 and driven universal spatial organization of backbones of folded proteins: volume 348.145 was further used for docking and virtual Are there Chargaff’s rules for protein folding? Journal of Biomolecular screening. The predicted 3 D structure was used to screen Structure and Dynamics, 28(2), 133–142. Mittal, A., & Jayaram, B. (2011a). Backbones of folded proteins reveal the NCI database for finding potential binders. The struc- novel invariant amino acid neighborhoods. Journal of Biomolecular tures from the NCI database were docked with the 3 D Structure and Dynamics, 28(4), 443–454. structure of the protein and the best docking score of Mittal, A., & Jayaram, B. (2011b). The newest view on protein folding: 12.547 was obtained. All the compounds binding with À Stoichiometric and spatial unity in structural and functional diversity. the protein followed Lipinski’s Rule. The top structures Journal of Biomolecular Structure and Dynamics, 28(4), 669–674. from the NCI database have been reported as potential binders of the protein. The binding affinity of the top binder NSC1972 was found to be 63.296 as predicted by 99. Tongue tumor protein À PRIME MM-GBSA and Induced-fit docking was done to val- BAH14511.1 sequence analysis and idate docking results (Figure 1). homology modeling to find out interacting binder network through References molecular docking Blom, N., Gammeltoft, S., & Brunak, S. (1999). Sequence and structure- based prediction of eukaryotic protein phosphorylation sites. Journal Snigdha Singh and Ramesh Chandra of Molecular Biology, 294(5), 1351–1362. Takiar, R., Nadayil, D., & Nandakumar, A. (2010). Projections of number of Drug Discovery & Development Laboratory, Department of cancer cases in India (2010-2020) by cancer groups. Asian Pacific Chemistry, University of Delhi, Delhi 110007, India Journal of Cancer Prevention, 11(4), 1045–1049. [email protected]

Figure 1. JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS VOL. 37, SUPPLEMENT, 2019 61 van der Waal, I. (2013). Are we able to reduce the mortality and morbid- incomplete, suggesting replication fork impediments; (2) it ity of oral cancer; Some considerations. Medicina Oral, Patologia Oral is inhibition of transcription by pyrimidine dimers (PDs) in y Cirugia Bucal, 18(1), e33–e37. DNA, rather than inhibition of translation by PDs in mRNA, Yu, C. S., Chen, Y. C., Lu, C. H., & Hwang, J. K. (2006). Prediction of protein subcellular localization. Proteins: Structure, Function, and that is responsible for the sensitivity; (3) promotion/stabil- Bioinformatics, 64(3), 643–651. ization of R-loops exacerbates the sensitivity, while their destabilization reduces the sensitivity; (4) PDs are rapidly removed from the bulk DNA, yet 10% of PDs in the chromosome are removed slowly; (5) RNA:DNA hybrids 100. Can R-loops themselves be the accumulate massively in genomic DNA after UV. We are mysterious R-lesions? currently testing whether the chromosomal regions containing UV-induced RNA:DNA hybrids are enriched for slowly- Elena A. Kouzminova and Andrei Kuzminov removable PDs. If yes, then UV-induced R-lesions could be University of Illinois at Urbana-Champaign, B103 CLSL, 601 South RNA polymerases stalled at PDs in template DNA, while Goodwin Avenue, Urbana, IL 61801, USA anchored on their 5’-side by R-loops, an arrangement that [email protected] should block PD removal.

All free-living organisms have two kinds of RNase H enzymes (activities that remove the RNA nucleotides from Funding the RNA:DNA hybrid duplexes): ‘type I’ attacks R-loops and This research is supported by grant GM 073115 from the 4nt long R-tracts, whereas ‘type II’ attacks 5 RNA-DNA3’ 0 National Institutes of Health. junctions, mostly at single RNA nucleotides in DNA. Type II mutants (rnhB in E. coli) behave like WT, suggesting that single rNs in DNA are innocuous, while type I mutants References (rnhA in E. coli) are inhibited, suggesting that not only R- Kouzminova, E. A., Kadyrov, F. F., & Kuzminov, A. (2017). RNase HII saves loops form frequently in the chromosomal DNA, but that rnhA mutant Escherichia coli from R-loop-associated chromosomal they are also quite stable and, unless actively hydrolyzed, fragmentation. Journal of Molecular Biology, 429(19), 2873–2894. interfere with replication and transcription. Surprisingly, the PMID: 28821455 combined type I/II RNase H defect reveals synergy: the double rnhAB mutant E. coli barely grows and forms long fila- mentous cells that have problems with nucleoid 101. Characterization of R loops segregation. We have argued that the chromosomal prob- in class switch recombination lems of the double rnhAB mutant reflect formation of R- lesions: RNA-containing DNA lesions that interfere with Jacqueline Barlow and Hongchang Zhao chromosomal replication and segregation. Department of Microbiology and Molecular Genetics, University of Since the only common substrate for the two bacterial California Davis, Davis, CA 955616, USA RNase H types is R-tract, we propose that the double rnhAB mutants accumulate R-tracts in their DNA that eventually Recurrent DNA translocations characterize blood cell cancers, lead to irreparable double-strand breaks. This idea is con- and are frequently products of aberrant repair of pro- sistent with the following phenotypes of the double rnhAB grammed DNA double-strand breaks (DSBs). Defining the mutants over the single rnhA or rnhB mutants: (1) increased precise molecular mechanisms governing DSB generation SOS response; (2) dependence on recombinational repair; and repair is critical to revealing how lymphoid cancers arise. (3) formation of double-strand breaks in the chromosomal The majority of cancers involving antibody-producing B cells DNA; (4) sensitivity to inhibition of translation (should arise during class switch recombination (CSR), a programmed induce R-loops); (5) sensitivity to inhibition of chromosomal DNA repair event at the immunoglobulin heavy chain (IgH) replication (should preserve R-loops); (6) RNase-treatment- locus. CSR is initiated by DSBs generated by the enzyme AID, induced fragmentation of the chromosomal DNA. The whose recruitment to IgH requires transcription. AID-depend- emerging mechanism envisions that in the absence of ent DSB formation strongly correlates with the appearance RNase H, a fraction of stabilized R-loops is transformed into of R loops—three-stranded nucleotide structures where R-tracts, that are converted into R-gaps by replication, the newly transcribed RNA re-anneals to template DNA. Though latter being attacked by cellular RNases, yielding double- R loops were observed at the IgH locus over 20 years ago, strand DNA breaks. their role in CSR remains undefined. To investigate the role R However, studying one peculiar phenotype of the rnhAB loops play in CSR, we generated mice deficient for two mutants shows how an R-loop could itself be an integral enzymes promoting R loop dissolution, the helicase part of an R-lesion. The rnhAB mutants are extremely UV- Senataxin (SETX-/-) and the nuclease RNase H2 (RNH2Bf/f). We sensitive, but at the same time are not generally sensitive find that B cells from SETX-/- RNH2Bf/f mice have increased R to other forms of DNA damage. We show that in UV-ed loop formation, and over 10% of cells accumulate unrepaired rnhAB mutants: (1) while no additional double-strand breaks DNA breaks and translocations at IgH. In contrast, WT and are formed, post-UV replication recovery is slow and single mutants show modest IgH instability and limited 62 BOOK OF ABSTRACTS. ALBANY 2019: THE 20TH CONVERSATION increases in R loop formation. All 4 genotypes are proficient repeat contractions back to the wild-type level (Su & for CSR, thus DSB repair is largely intact. These results show Freudenreich, 2017). These findings provide evidence for a that Setnataxin and RNase H2 act independently to promote novel mechanism for CAG repeat fragility mediated by cyto- genome stability and suppress aberrant DNA repair during sine deamination of DNA engaged in R-loops. In addition, a CSR. Sequence analysis revealed an increased frequency of second mechanism of R-loop-mediated fragility at CAG insertions and deletions at class switch junctions in double- repeats was identified to be dependent on the Mlh1-Mlh3 deficient cells, a mutational signature associated with nuclease. This result could explain the previously demon- increased repair via alternative end-joining. We propose that strated role of Mlh1-Mlh3 in causing CAG repeat expansions Senataxin and Rnase H2 promote classical NHEJ by removing in a mouse model (Pinto et al., 2013). Thus, R-loop-medi- R loops during CSR. ated cleavage could be an important determinant of instability at unstable and expandable repeats in the human genome. Funding

This research has been supported by an NCI Career References Transition Award, K22CA188106 Pinto, R. M., Dragileva, E., Kirby, A., Lloret, A., Lopez, E., St Claire, J., … Wheeler, V. C. (2013). Mismatch repair genes Mlh1 and Mlh3 modify 102. Mechanisms of R-loop-induced CAG instability in Huntington’s disease mice: Genome-wide and candidate approaches. PLoS Genetics, 9(10), e1003930 chromosome fragility at structure- Su, X. A., & Freudenreich, C. H. (2017). Cytosine deamination and base forming repeats excision repair cause R-loop-induced CAG repeat fragility and instability in Saccharomyces cerevisiae. Proceedings of the National Academy of Sciences of the United States of America, 114(40), Xiaofeng Allen Su, Simran Kaushal, Ruby Ye and E8392–E8401. Catherine H. Freudenreich Department of Biology, Tufts University, Medford, MA 02155, USA [email protected]

CAG repeats are structure-forming repetitive DNA sequences, 103. Understanding and targeting and expansion beyond a threshold of 35 CAG repeats is R loops in cancer cells the cause of many human diseases including Huntington’s disease and myotonic dystrophy. Expanded CAG repeats are Lee Zoua,b prone to breakage, and repair of the breaks can cause repeat aMassachusetts General Hospital Cancer Center, Boston, MA, USA; contractions and expansions. We utilized an expanded CAG- bDepartment of Pathology, Harvard Medical School, Boston, MA, 70 repeat inserted into a yeast artificial chromosome (YAC) USA [email protected] system to evaluate mechanisms of repeat fragility and instability (contractions and expansions). Upon deletion of R loops arising during transcription-induced genomic yeast RNase H genes, RNH1 and RNH201, we found an ele- instability, but how cells respond to the R loop-associated vated level of RNA:DNA hybrids at CAG-70 repeats in vivo by genomic stress is still poorly understood. Our recent studies using chromatin immunoprecipitation (ChIP). Through CAG have suggested that ATR is involved in the sensing of R repeat fragility analysis, we discovered that CAG-70 repeats loops. Here, we show that cells harboring high levels of R show a dramatic and significant increase in fragility in the loops rely on the ATR kinase for survival. In response to absence of RNase H genes compared to wild-type, indicating aberrant R loop accumulation, the ATR-Chk1 pathway is higher levels of breakage events at the expanded repeats activated by R loop-induced reversed replication forks. ATR during R-loop formation (Su & Freudenreich, 2017). In con- protects the genome from R loops by suppressing transcrip- trast, fragility of another structure-forming repeat composed tion-replication collisions, promoting replication fork recov- of an AT dinucleotide was not affected by RNase deletion or ery, and enforcing a G2/M cell-cycle arrest. Furthermore, overexpression. Significant increases in CAG-70 repeat ATR prevents excessive cleavage of reversed forks by instability were also observed in the RNase H deletion back- MUS81. These results suggest that ATR is a key sensor and ground that were dependent on the base excision repair suppressor of R loop-induced genomic instability, uncover- pathway (Su & Freudenreich, 2017). Since disease-causing ing a signaling circuitry that safeguards the genome against CAG repeat expansions occur in transcribed regions shown R loops. to harbor R-loops, our findings indicate that R-loop-mediated We also find that ATR is important for survival of cancer fragility is a mechanism that could cause DNA damage and cells harboring high levels of R loops. Heterozygous somatic repeat-length changes in human cells. mutations in spliceosome genes (U2AF1, SF3B1, ZRSR2 or In a search for the cause of R-loop-mediated fragility, we SRSF2) occur in >50% of myelodysplastic syndrome (MDS) found that a yeast cytosine deaminase, Fcy1, was enriched at and AML patients. We show that RNA splicing perturbation the expanded CAG repeats in rnh1Drnh201D cells. Deletion by expression of the U2AF1(S34F) mutant causes accumula- of Fcy1 significantly decreases CAG-70 repeat fragility in the tion of R loops and elicits an ATR response. ATR inhibitors rnh1Drnh201D background, and reduces the elevated CAG (ATRi)-induced DNA damage and cell death in U2AF1S34F- JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS VOL. 37, SUPPLEMENT, 2019 63 expressing cells, and these effects of ATRi were enhanced by the mechanism through which Dna2 promotes gen- splicing modulating compounds. Our results suggest that ome stability. ATR may represent a novel therapeutic target in MDS patients carrying the U2AF1(S34F) mutation and potentially References other malignancies harboring spliceosome mutations. Bartels, P. L., Zhou, A., Arnold, A. R., Nunez,~ N. N., Crespilho, F. N., David, S. S., & Barton, J. K. (2017). Electrochemistry of the [4Fe4S] cluster in base excision repair proteins: Tuning the redox potential with DNA. Langmuir: The ACS Journal of Surfaces and Colloids, 33(10), 104. The role of the redox active 2523–2530. Budd, M. E., & Campbell, J. L. (1995). A yeast gene required for DNA rep- 4Fe–4S cluster of yeast Dna2 lication encodes a protein with homology to DNA helicases. Proceedings of the National Academy of Sciences of the United States of America, 92(17), 7642–7646. Siobhan Gaustad MacArdle and Jacqueline Barton Lee, S. H., Kim, Y. R., Yoo, N. J., & Lee, S. H. (2010). Mutation and expres- Department of Chemistry and Chemical Engineering, California sion of DNA2 gene in gastric and colorectal Carcinomas. Korean Institute of Technology, 1200 E. California Blvd., MC 127-72, Journal of Pathology, 44(4), 354–359. Pasadena, CA, 91126, USA [email protected] Pinto, C., Kasaciunaite, K., Seidel, R., & Cejka, P. (2016). Human DNA2 possesses a cryptic DNA unwinding activity that functionally integra- Dna2 is an essential nuclease-helicase conserved throughout tes with BLM or WRN helicases. eLife, 5 Strauss, C. (2014). The DNA2 nuclease/helicase is an estrogen-dependent eukaryotic organisms with an impressive repertoire of DNA gene mutated in breast and ovarian cancers. Oncotarget, 5, maintenance activity (Budd & Campbell, 1995). Due to its 9396–9409. major roles in Okazaki fragment processing during DNA replication, double strand break repair, telomere maintenance and mitochondrial DNA maintenance, it is not surprising that both Dna2 upregulation and mutation have been observed in various types of cancer (Lee, Kim, Yoo, & Lee, 2010; 105. Primer handoff between DNA Strauss, 2014). Despite the fact that both the helicase and primase and DNA polymerase a nuclease domains are equally conserved in evolution, the nuclease function of Dna2 dominates on most substrates Aoshu Zhonga, Lauren E. Salayb, Walter J. Chazinb and the functional switch between helicase and nuclease and Jacqueline K. Bartona activity is regulated by an unclear mechanism, which may aDivision of Chemistry and Chemical Engineering, California involve the 4Fe–4S cluster (Pinto, Kasaciunaite, Seidel, & Institute of Technology, Pasadena, CA 91125, USA; bDepartments Cejka, 2016). The goal of this work is to elucidate the effect of Biochemistry and Chemistry, Center for Structural Biology, of the redox properties of the 4Fe–4S cluster on the regula- Vanderbilt University, Nashville, TN 37240, USA [email protected] tion of helicase and nuclease activity in S. cerevisiae Dna2. We have overexpressed wild type S. cerevisiae Dna2 and DNA replication is a fundamental process. In eukaryotes, the nuclease-dead Dna2 E675A in E. coli and purified the recom- first step in daughter strand synthesis is the generation of a binant enzymes with sufficient yield and purity. Using multi- short ( 10 nt) RNA primer by DNA primase, followed by plexed DNA-modified Au electrodes, we have characterized transfer of the primer to DNA polymerase a (Pol a) for exten- the DNA-bound electron transfer activity of isolated Dna2. sion by 20 nts (O’Brien et al., 2017; Burgers & Kunkel, 2017; Cyclic voltammetry on DNA-modified electrodes showed Pellegrini, 2012;O’Brien et al., 2018;O’Brien, Holt, Salay, reversible DNA-bound redox activity centered at 90 mV vs. Chazin, & Barton, 2018). Although the catalytic activity and NHE. This redox potential is similar to that of other DNA- structures of primase and Pol a have been extensively binding 4Fe–4S enzymes that we have studied and supports studied (Burgers & Kunkel, 2017; Pellegrini, 2012;O’Brien a DNA-mediated redox signaling role for the 4Fe–4S cofactor et al., 2018;O’Brien, Holt, Salay, Chazin, & Barton, 2018), the in regulating Dna2 activity. The redox potential of Dna2 molecular mechanism of primer handoff between primase unbound to DNA has been determined by protein film elec- and Pol a remains largely unknown. We have proposed that trochemistry to be 150 mV vs. NHE, 60 mV greater than redox switching of the [4Fe4S] cluster in primase is import- that of the DNA-bound protein. This positive shift in redox ant for primer truncation and handoff to Pol a (O’Brien et al., potential suggests that DNA binding activates the 4Fe–4S 2017;O’Brien et al., 2018). Eukaryotic Pol a is composed of a cluster towards oxidation and is consistent with what has catalytic subunit (p180) and a regulatory subunit (p68). The been observed for other DNA-binding proteins containing a p180 subunit contains a [4Fe4S] cluster. Thus, we hypothe- 4Fe–4S cluster (Bartels et al., 2017). Bulk oxidation and size that the redox partner of primase is Pol a and that the reduction of Dna2 on DNA-modified electrodes has been two [4Fe4S] clusters orchestrate the primer handoff between performed revealing an increase in signal after oxidation the two [4Fe4S] proteins using DNA-mediated charge trans- and decrease upon reduction, consistent with an increase port chemistry. Here, we present results using anaerobically in DNA-binding affinity upon oxidation of the 4Fe–4S clus- purified human [4Fe4S] (C-terminal domain of p180 (p180C) ter to the 3 state. This study illuminates the biochemical in complex with p68. To the best of our knowledge, this is þ role played by the 4Fe–4S cluster and provides insight into the first time that p180C-p68 is isolated with an [4Fe4S] 64 BOOK OF ABSTRACTS. ALBANY 2019: THE 20TH CONVERSATION cluster bound. The cluster loading can be improved to nearly Huge disparity exists in terms of number of sequenced pro- 100% after initial purification using a reconstitution protocol. teins, experimentally available protein structures and func- The protein exhibits electrochemical properties similar to the tionally characterized proteins. With the advent of proteomic p58C [4Fe4S] domain of primase. Using multiplexed DNA- techniques large number of structures are solved for modified Au electrodes, we observe a large cathodic peak ‘proteins of unknown functions’ (PUFs). More than 20% of between –170 and –180 mV versus NHE by cyclic voltamme- the known protein domains are characterized as ‘domains of try only during the initial scan to negative potentials after unknown function’ (DUFs). Bacteria and eukaryotes share oxidation by bulk electrolysis. As with p58C, the data are near about 1000 DUFs. Evolutionary conservation suggests consistent with a model where p180C-p68 is more tightly that DUFs are essential in both eukaryotes and bacterial associated with DNA in the oxidized state compared to the pathogens. However, the function of those proteins and reduced state. We also present results from primer elong- involvement of important amino acids in protein functions ation assays, where truncated products are formed to a remain elusive. Characterization of functions even for a few greater extent in the presence of the primase-p180C-p68 proteins or domains is experimentally laborious and time complex versus primase alone. When electrochemically oxi- consuming. Here, we present annotation of cysteine amino dized p180C-p68 was assayed together with primase, the acid functions based on protein microenvironments in length of the products is similar to the products produced known protein dataset and apply those annotations to with primase alone. Together, these data are consistent with unknown proteins. Protein microenvironment is described by our hypothesis that the [4Fe4S] clusters in p180C-p68 and two parameters, buried fraction (BF) and rHpy (a quantitative primase regulate the primer handoff from primase to Pol . a property descriptor to describe the hydrophobicity of local environment around an amino acid) (Bandyopadhyay & References Mehler, 2008). Cysteine functions were curated from literature and structures were obtained PDB database with reso- Burgers, P. M. J., & Kunkel, T. A. (2017). Eukaryotic DNA replication fork. lution greater than 1.5 Å. Statistically meaningful Annual Review of Biochemistry, 86, 417–438. O’Brien, E., Holt, M. E., Thompson, M. K., Salay, L. E., Ehlinger, A. C., characterization was done for four cysteine modifications, Chazin, W. J., & Barton, J. K. (2017). The [4Fe4S] cluster of human namely disulfide formation (Bhatnagar, Apostol, & DNA primase functions as a redox switch using DNA charge transport. Bandyopadhyay, 2016), thioether formation, sulfenylation Science, 355, eaag1789. and metal binding (Bhatnagar & Bandyopadhyay, 2018). O’Brien, E., Salay, L. E., Epum, E. A., Friedman, K. L., Chazin, W. J., & Barton, J. K. (2018). Yeast require redox switching in DNA primase. Distinct contours (within the space of BF and rHpy) were Proceedings of the National Academy of Sciences of the United States of identified for different cysteine modifications. Training sets America, 115(52), 13186–13191. on four cysteine functions were validated on test sets of O’Brien, E., Holt, M. E., Salay, L. E., Chazin, W. J., & Barton, J. K. (2018). cysteines obtained from protein structures in PDB database Substrate binding regulates redox signaling in human DNA primase. with resolution lower than 1.5 Å. These contours will be Journal of the American Chemical Society, 140, 17153–17162. Pellegrini, L. (2012). The Pol a-primase complex. Sub-Cellular exploited to annotate cysteine functions in PUFs and DUFs Biochemistry, 62, 157–169. (Figure 1).

106. Annotation of cysteine functions Funding in unknown proteins—a new This research is supported by Funding from Department of approach based on protein Science and Technology (DST-SERB), Govt. of India (File No. EMR/2017/002953) microenvironments

Debashree Bandyopadhyay, Akshay Bhatnagar and References Shubham Paliwal Bandyopadhyay, D., & Mehler, E. L. (2008). Quantitative expression of Birla Institute of Technology and Science, Pilani, Hyderabad protein heterogeneity: Response of amino acid side chains to their Campus, Hyderabad 500078, India local environment. Proteins: Structure, Function and Bioinformatics, [email protected] 72(2), 646–659.

Figure 1. JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS VOL. 37, SUPPLEMENT, 2019 65

Bhatnagar, A., Apostol, M. I., & Bandyopadhyay, D. (2016). Amino acid function relates to its embedded protein microenvironment: A study on disulphide-bridged cystine. Proteins: Structure, Function, and Bioinformatics, 84(11), 1576–1589. Bhatnagar, A., & Bandyopadhyay, D. (2018). Characterization of cysteine thiol modifications based on protein microenvironments and local secondary structures. Proteins: Structure, Function, and Bioinformatics, 86(2), 192–209.

107. Change in the yeast Figure 1 Effect of point mutations in C-terminal end of the Pma1 ATPase on Pma1 Hþ-ATPase regulation causes the distribution of PolyP fractions during glucose-dependent activation. redistribution of polyphosphates (PolyP) fold, while mutant enzymes exhibited already elevated basal Alexandr A. Tomashevsky and Valery V. Petrov activity by 1.6- to 10.2-fold. S911D ATPase was constitutively activated by ca. 10-fold regardless the presence of glucose, Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, 142290, whilst S911A and T912D exposed ability to be additionally Russia [email protected] activated by 1.7-5.0-fold upon addition of glucose. During

glucose fermentation, orthophosphate (Pi) content dropped Plasma membrane Pma1 Hþ-ﬀase is a key enzyme of 3 to 4 times in all strains as compared to starvation mode yeast metabolism. The enzyme functioning is regulated by pointing to its use for phosphorylation; however, S911D glucose, which fermentation leads to producing ATP being mutant with changed type of phosphorylation had signifi- used for phosphorylation of proteins and other substrates cantly elevated Pi content in both the absence and presence and maintaining energy metabolism. Glucose-induced activa- of glucose (Figure 1). This points either to inability of altered tion of Pma1 is structurally accompanied by the enzyme acylphosphate site at D911 to be phosphorylated or, more multiple and reversible phosphorylation during intracellular likely, to irreversible phosphorylation at this site, which traffic via secretory pathway on route to plasma membrane. should disturb cascade of reactions ignited by glucose fer- There is evidence that the Pma1 activation is linked to Ser/ mentation. PolyP content in the wild-type strain did not dif- Thr phosphorylation within the regulatory C-terminus and it fer much in the presence and absence of glucose; in mutant leads to essential conformational changes; S911 and T912 strains, there was a difference seen with the most pro- were identified as two major phosphosites, which phosphor- nounced effect for the shortest PolyP1 fraction, which ylation goes tandemly (Lecchi et al., 2007). The location of decreased in S911A and T912D and completely disappeared all sites of phosphorylation is still unclear; however, it seems in S911D in the absence of glucose; glucose addition logical that phosphorylation of Pma1 goes step by step and brought PolyP1 amount up to control level and higher in such sites could be located both in the enzyme intra- and the case of S911D. This strain had also the most noticeable extracellular parts. Both ATP and PolyP can phosphorylate changes in PolyP2-5 fractions, which were higher comparing Ser and Thr like in the case of Lys (Azevedo et al., 2015); we to the wild type. This points to involvement of PolyP directly have earlier shown that Ala replacement of phosphorylable or indirectly—through P pool—in phosphorylation. residues in the extracellular L9-10 loop, which is close to the i Presented data allow to suggest interaction between ATPase C-tail, has affected PolyP distribution (Tomashevsky & Petrov, functioning and PolyP metabolism; removal of phosphosite 2011, 2013). Other extracellular phosphorylable residues at S911 or altering its type causes changing in P content were found not to be important for the enzyme structure- i and redistribution between PolyP fractions, which may point function relationship (Petrov, 2011, 2015). We have also to preventing or altering P usage for phosphorylation both shown that mutations at S911 and T912 affected PolyP i of the Pma1 and other proteins and substances at different metabolism and phosphate distribution among PolyP1-5 steps. Further study is needed to get helpful insights into fractions (Tomashevski & Petrov, 2015). Results described here extend our study towards interaction of PolyP metabol- fine mechanisms of functioning and regulating of the ism and the enzyme regulation at S911 and T912 phosphor- Pma1 ATPase. ylation sites. Replacement by Ala led to removing phosphosite of phosphoester type and by Asp, to altering References site to acylphosphate type. Yeast cells carrying wild type (NY13) and mutant ATPases (S911A, S911D and T912D) were Azevedo, C., Livermore, T., & Saiardi, A. (2015). Protein polyphosphoryla- tion of lysine residues by inorganic polyphosphate. Molecular Cell, grown until mid-log phase, harvested and incubated with or 58(1), 71–82. without 2% glucose. Then PolyP were extracted and plasma Lecchi, S., Nelson, C. J., Allen, K. E., Swaney, D. L., Thompson, K. L., Coon, membranes were obtained to assay ATPase activity. Wild- J. J., … Slayman, C. W. (2007). Tandem phosphorylation of Ser-911 type ATPase underwent glucose-dependent activation by 9- and Thr-912 at the C terminus of yeast plasma membrane Hþ-ATPase 66 BOOK OF ABSTRACTS. ALBANY 2019: THE 20TH CONVERSATION

leads to glucose-dependent activation. Journal of Biological Chemistry, HL-60 leukemia cells. So, hGMPR-II attracts an excellent 282(49), 35471–35481. potential target for design of isoform-specific antileukemic Petrov, V. V. (2011). Role of M5-M6 loop in the biogenesis and function agent. The catalytic domain of hGMPR is a homo-tetramer of the yeast Pma1 Hþ-ATPase. Journal of Biomolecular Structure and Dynamics, 28, 1024–1025. with 37-kDa subunits and monomer of this enzyme has a 8 Petrov, V. V. (2015). Point mutations in the extracytosolic loop between (a/b) structure of TIM barrel. The polypeptide chain of this transmembrane segments M5 and M6 of the yeast Pma1 Hþ-ATPase: domain has folded to form two separate nucleotide binding Alanine-scanning mutagenesis. Journal of Biomolecular Structure and pockets (Figure 1a). One of them accommodates cofactor Dynamics, 33(1), 70–84. NADP (di-nucleotide) and the other binds to substrate GMP Tomashevsky, A. A., & Petrov, V. V. (2011). Point mutation in M9-M10 loop of the yeast Pma1 Hþ-ATPase affects both ATPase functioning (mono-nucleotide). Few crystal structures of hGMPR are and polyphosphate distribution. Journal of Biomolecular Structure and observed in different conformations. The analysis of crystal Dynamics, 28, 1025–1026. structures explores that active site residue or Cys186 attacks Tomashevski, A. A., & Petrov, V. V. (2013). Point mutations in the yeast C2 of substrate (GMP) to create an intermediate Pma1 Hþ-ATPase affects polyphosphate (PolyP) distribution. Journal of (Cys186SG—C2GMP) whereas ammonia leaving group is acti- Biomolecular Structure and Dynamics, 31, 124–125. Tomashevski, A. A., & Petrov, V. V. (2015). Mutations in the yeast vated by proton relay through Thr188 and Glu289 dyad. So, Pma1 Hþ-ATPase regulatory domain affects polyphosphate metabol- interaction between Cys186 and Thr188/Glu289 is Cys186— ism. Journal of Biomolecular Structure and Dynamics, 33(Suppl), GMP—Glu289/Thr188 in ligand bound state (Figure 1d). 105–106. Consequently, cofactor or NADPH is stabilized by Lys78, Thr105, Ser270 and Arg286/Tyr285 (Figure 1b). In spite of this distinction, no studies have been carried out on catalytic 108. Conformational transition of domain to identify the new conformation of native state of hGMPR enzyme. To investigate the dynamic behavior of catalytic domain in hGMPR enzyme native conformation of catalytic domain, molecular dynamics from native to ligand bound state: simulation was carried out using CHARMM27 force field with a MD simulation study Nanoscale Molecular Dynamics (v.2.9) program In fact, MD result suggest that 22 water molecules are observed to Hridoy R. Bairagya occupy at position of GMP and NADPH binding region (in Department of Biophysics, All India Institute of Medical Sciences, unliganded form). The Cys186, Thr188, Glu289 of substrate New Delhi 110029, India [email protected] and Lys78, Glu289, Ser270, Arg286/Tyr285 of cofactor binding residues move away from their respective binding pocket

The human Guanosine-50-Monophosphate Reductase during MD simulation and stabilized by conserved water (hGMPR) enzyme involves in inosine biosynthesis pathway by mediated interactions. Four water molecules (Cys186—W1— converting GMP to IMP and ammonia with concomitant oxi- W2—W3—W4—Thr188/Glu289) at substrate binding region dation of NADPH and plays a vital role in re-utilization of (Figure 1e) and eighteen water molecules (Lys78—W5— free intracellular bases. Type I hGMPR is expressed in all cells W6—W7(—Thr105)—W8—W9—W10—W11—W12—W13— whereas isoform-II can promote monocytic differentiation of OB(Arg286)—W14—W15—W16—W17—W18—W19—OG

Figure 1. (a) GMP with NADPH bound conformation. (b, d) Residues of NADPH and IMP binding pocket in X-ray structure. (c, d) Water-mediated H-bonding interaction of NADPH and IMP binding pocket in native conformation of MD structures. JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS VOL. 37, SUPPLEMENT, 2019 67

(Ser270)—W20—W21—W22—Lys78) at cofactor binding by increased activities of hexokinase, glucose-6-phosphate pocket (Figure 1c) are observed to stabilize in unliganded dehydrogenase, fructose bisphosphate aldolase, pyruvate kin- state of catalytic domain, but they move and vacate those ase, citrate synthase and aconitate hydratase; oxaloacetate in positions when enzyme (hGMPR)-substrate cofactor-complex this case is presumably resynthesized by means of pyruvate is formed. Our computational study highlights structural carboxylase reaction. and functional importance of water molecules in catalytic Similar effect of a thiamine deficiency has been previously domain of hGMPR enzyme and suggests a rethink of con- demonstrated for Blastobotrys adeninivorans grown on glu- ventional definition of chemical geometry of inhibitor bind- cose (Kamzolova & Morgunov, 2016b) and Y. lipolytica VKM ing site. Y-2378 grown on glycerol-containing waste of biodiesel production (Kamzolova & Morgunov, 2018), an active superpro- ducers of -keto acids. Therefore, it can be suggested that Funding a this phenomenon is a common regularity associated with This research has been supported by C.S.I.R. for Senior the limitation of the activity (or synthesis) of enzymes Research Associateship (Pool Scientist scheme). involved in carbohydrate utilization and a-keto acids production.

109. Deficiency of thiamine shifts References metabolism toward the a-keto acids Kamzolova, S. V., & Morgunov, I. G. (2015). Inhibition of isocitrate lyase oversynthesis in Yarrowia shifts metabolism toward the isocitrate overproduction in yeast Yarrowia lipolytica. Journal of Biomolecular Structure and Dynamics, lipolytica yeast 33(Supp1), 107–108. doi:10.1080/07391102.2015.1032802 Kamzolova, S. V., Allayarov, R. K., Lunina, J. N., & Morgunov, I. G. (2016). Igor G. Morgunov and Svetlana V. Kamzolova The effect of oxalic and itaconic acids on threo-Ds-isocitric acid production from rapeseed oil by Yarrowia lipolytica. Bioresource G.K. Skryabin Institute of Biochemistry and Physiology of Technology, 206, 128–133. doi: 10.1016/j.biortech.2016.01.092. Microorganisms RAS, Pushchino 142290, Moscow region, Russia Kamzolova, S. V., & Morgunov, I. G. (2016). Biosynthesis of pyruvic [email protected] acid from glucose by Blastobotrys adeninivorans. Applied Microbiology and Biotechnology, 100(17), 7689–7697. doi: 10.1007/ Earlier, we found that the inhibition of isocitrate lyase by s00253-016-7618-1. oxalic and itaconic acids shifts yeast metabolism towards the Kamzolova, S. V., & Morgunov, I. G. (2018). Biosynthesis of pyruvic acid threo-Ds-isocitric acid synthesis, a biologically active com- from glycerol-containing substrates and its regulation in the yeast Yarrowia lipolytica. Bioresource Technology , 266, 125–133. doi: pound, an intermediate of Krebs cycle (Kamzolova & 10.1016/j.biortech.2018.06.071. Morgunov, 2015; Kamzolova, Allayarov, Lunina, & Morgunov, 2016). The purpose of this work was to study the effect of limited growth of Yarrowia lipolytica yeast by a thiamine, which 110. Genome-wide survey of protein is essential for multiple biochemical pathways involved in structural domains and analysis of glucose catabolism. It was shown that the growth curve of Y. lipolytica VKM Y- domain architectures 2378 had three phases: exponential phase (up to 12 h), retardation phase (from 12 to 24 h) caused by exhaustion of Meenakshi S. Iyer, Adwait G. Joshi and thiamine, and stationary phase. The production of a-keto R. Sowdhamini acids (pyruvate and a-ketoglutarate) began in the growth National Centre for Biological Sciences, TIFR, GKVK, Bellary Road, retardation phase and continued in the stationary phase. At Bangalore 560065, India [email protected] the end of cultivation, cells synthesized 41 g/L pyruvate and 8 g/L a-ketoglutarate. The thiamine deficiency disturbed the In the past few years, the increase in genome and transcrip- metabolic flow through the thiamine-dependent enzymes of tome studies has resulted in a plethora of protein sequences glucose catabolism. The inhibition of pyruvate dehydrogen- being deposited in the sequence databases. But, there exists ase complex leads to excretion of pyruvate from the yeast a huge deficit between the number of structures and cells. As the inhibition of pyruvate dehydrogenase is not sequences available. In this work, we have tried to bridge complete, the formation of acetyl-CoA continues, providing this gap using SCOP superfamily domain sequences to iden- for the synthesis of a-ketoglutarate in Krebs cycle. tify homologues in nonredundant database (from NCBI) (Iyer, a-Ketoglutarate is not oxidized in Krebs cycle since thiamine Joshi, & Sowdhamini, 2018). The sequences were validated deficiency limits a-ketoglutarate dehydrogenase, and hence using structure-based sequence alignment HMMs derived it is excreted from the yeast cells. The deficiency of energy from SCOP (v1.75) superfamily members in PASS2.4 database. at the level of thiamine-dependent enzymes affected the Domain architectures were computed for the validated hits respiratory chain of cells. To maintain the with induced at structure level (SCOP) and sequence level (Pfam). The a-keto acids production, the cells activated the initial reac- associated domains in a domain architecture were found to tions of glucose catabolism and Krebs cycle. It is confirmed be involved in the same biological process. A 68 BOOK OF ABSTRACTS. ALBANY 2019: THE 20TH CONVERSATION

Figure 1. correspondence of Pfam and SCOP superfamilies was obtained It is known that exposure to electromagnetic fields (EMF) of for each superfamily covering about 61% of Pfam families. The millimeter range or extremely high frequencies (EHF) has structural census (Chothia, 1992; Caetano-Anolles, Wang, beneficial effects on seed germination and plant growth Caetano-Anolles, & Mittenthal, 2009) was revisited and distri- (Naueiene et al., 2017). This application in contrast to chem- bution of homologues across superfamilies, folds and classes ical methods of seeds pre-germination treatment is noninva- were analyzed. About 27% of NR database and 41% of the tax- sive and environmentally appropriate technology what is onomy database (from NCBI) were covered in the study. The very important for farming industry. results from the above analysis have been presented in the The study aimed to investigate the effects of pre-sowing form of a database named GenDiS (Pugalenthi, 2005; Iyer, seed treatment with physical factor-EHF EMF (50.3 GHz fre- þ 2018). Profiles derived from the alignments of superfamily quency, 3–10 min) on seed germination and seedling per- homologues can be used in sequence searches and for assign- formance. For this, seeds of wheat (Triticum aestivum L. of ing structural domains to sequences (Figure 1). ‘Bezostaya’ variety) were imbibed in water for 12 hour then treated once with EMF with 50.3 GHz frequency, for 3, 5 and 10 min., then left to germinate on wet filter paper in Funding Petri dishes at 23 C in the dark for 8 days. The irradiation This research has been supported by NCBS, TIFR. was performed using the generator G4-141 type (State Scientific-Production Enterprise ‘Istok’, Russia) with working interval of 37.50–53.57 GHz and power flux density 64 mWt/ References cm2. The germination rate, seedling length and fresh Caetano-Anolles, G., Wang, M., Caetano-Anolles, D., & Mittenthal, J. E. weight were determined at the third and seventh day after (2009). The origin, evolution and structure of the protein world. seeds sowing. Our findings show that the most positive Biochemical Journal, 417(3), 621–637. germination effects were in short-time EMI treatments (3 Chothia, C. (1992). Proteins. One thousand families for the molecular and 5 min, respectively) groups. The germination tests biologist. Nature, 357(6379), 543–544. Iyer, M. S., Joshi, A. G., & Sowdhamini, R. (2018). Genome-wide survey of revealed that EMI-treatments induced increase in germin- remote homologues for protein domain superfamilies of known struc- ation rate in seeds as compared to control. Thus, the ger- ture reveals unequal distribution across structural classes. Molecular mination rate at the third day after sowing was significantly Omics, 14(4), 266–280. (p < 0.05) higher (by 12% and by 9%) for EMF-exposed Iyer, M. S. (2018). GenDiS : improved sequence search and validation and þ seeds, while on the seventh day these indexes did not sig- update of features for homologues of Structural Superfamily members. (Manuscript under review) nificantly differ from control. On the other hand, the longer Pugalenthi, G. (2005). GenDiS: Genomic distribution of protein structural time EMI –treatment (10 min) did not change germination domain superfamilies. Nucleic Acids Research, 33, D252–D255. rate for both studied days after sowing. Data also show that seedlings grown from seeds EMI-treated (5 and 10 min) groups had increased seedling weight (up to 12%) and 111. Germination response of Triticum length (up to 8%) compared to control. aestivum seeds to different exposure times of low-intensity EMI treatment References

Naueiene, Z., & Zukiene, R. (2017). Transgenerational stress memory in E. Gayane H. Poghosyan and Zhanna H. Mukhaelyan purpurea: Seed treatment with cold plasma and electromagnetic field Department of Biophysics, Yerevan State University, Yerevan, induces changes in germination, Abstract Book, 1st Inter. Conf. “Smart Armenia [email protected] Bio”, Kaunas, p.159. JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS VOL. 37, SUPPLEMENT, 2019 69

irreversible rise of entropy. It is proposed the next step of 112. Intermediate stage of the experiments on the origin of life: various types of prebiotic transition of prebiotic microsystems models should be explored under multilevel and high-fre- to life quency oscillations of physico-chemical parameters in the chamber. Vladimir N. Kompanichenko Institute for Complex Analysis RAS in Birobidzhan, 4, Sholom References Aleyhem Str, Birobidzhan 679016, Russia [email protected] Kompanichenko, V. N. (2017). Thermodynamic Inversion: Origin of Living Systems. Cham (Switzerland): Springer International Publishing. Following the author’s inversion concept, the primary forms https://link.springer.com/book/10.1007/978-3-319-53512-8. of life arise through thermodynamic inversion in non-living prebiotic microsystems, when the contributions of free energy (F) and information (I) become prevalent over the contribution of entropy (S) (Kompanichenko, 2017). In this 113. MD simulation based study to way the ‘thermodynamic corridor’ for any chemical scenario understand the role of pre-hydrolysis of life origin is substantiated: F I < S (prebiotic microsys- þ tem) F I S (intermediate stage, inversion moment) network in the comparative dynamics ! þ ! F I > S (primary form of life). Prebiotic organic microsys- of A59G-H-Ras and wild-type H-Ras þ tems can achieve the intermediate stage only under far from equilibrium conditions, including mandatory high-frequency Neeru Sharma, Uddhavesh B. Sonavane and oscillations of physico-chemical parameters (pressure, tem- Rajendra Joshi perature and chemistry) in hydrothermal environments. Such HPC-M&BA Group, Centre for Development of Advanced oscillations are considered as the fourth required condition Computing, Panchawati Road, Pune, 411008, India for life origin, in addition to the three well-known ones: [email protected] availability of organic matter, aqueous medium and a source of energy. A prebiotic organic microsystem at the intermedi- The Ras family of proteins plays an important role in cell-pro- ate stage is characterized by internal oscillations around the liferation, differentiation, apoptosis, membrane localization point of bifurcation and relative efficiency of chemical reac- and ultimately signal transduction (Downward, 2003). The tions producing free energy and entropy (Figure 1). A main protein Ras has three isoforms namely H-Ras, K-Ras and N- idea for discussion is that self-maintained life processes were Ras and belongs to a class of G-proteins. In normal condi- launched through continuous response of prebiotic microsys- tions, wild-type Ras regularly cycles between active GTP tems to incessant physic-chemical oscillations in the outside bound and inactive GDP bound conformations. Also being a world (stress), in connection with the Le Chatelier principle. GTPase, Ras also possess an intrinsic capability to hydrolyze No substantial fluctuations in a medium (for instance, in GTP to GDP and maintain a highly controlled regulation quiet ocean), no incessant recombination of (macro)mole- between active and inactive states (Lu et al., 2016). In gain- cules within containing organic microsystems and no per- of-function mutations of Ras, the intrinsic hydrolysis activity spective for their transformation into living units. The as well GAP-assisted GTP hydrolysis activity is hindered and oscillating microsystem at the intermediate stage is unstable: hence the protein tends to stuck in the permanently active further it can either be transformed into a primary living oncogenic state. The oncoprotein Ras is found to be mutated unit through thermodynamic inversion, or degrade due to in more than 30% of human cancers (Adjei, 2001). The

Figure 1. Intermediate state between ‘non-life and life’. 70 BOOK OF ABSTRACTS. ALBANY 2019: THE 20TH CONVERSATION present work aims to explore the dynamics of the wild-type Glomalin-related soil protein (GRSP), is an insoluble glyco- and mutant A59G-HRas using long-time scale MD simula- protein, produced in copious amounts on hyphae of arbus- tions, followed by advance MD analytics techniques like cular mycorrhizal fungi (AMF). It provides unique properties MSM based analysis. The in-depth insight into the crucial to the soil such as mechanical stability, amelioration of salin- interactions of wild-type and mutant, especially for the pre- ity and aggregate water stability. Due to high sequence simi- hydrolysis network is reported in the study. Conventional as larity of GRSP sequence identified from Rhizophagus well feature-based PCA analyses have also been performed irregularis, a mycorrhizal fungus, with heat shock proteins 60 to differentiate the wild-type and mutant conformations. The (Hsp60) of various other species, this protein is said to be a study focuses on the relation between disruption of the cru- putative homolog of Hsp60. There is a lack of evidence at cial pre-hydrolysis network and the oncogenic conformation both the experimental and theoretical level with respect to in the mutant A59G-HRas ensemble. Pivotal roles of residues the structural information of GRSP and its interaction with from SwI, SwII and P-loop have been reported. humic acid, one of the components of soil organic matter. Thus, the present study has focussed on evolutionary studies of Hsp60, followed by homology based 3D modeling and molecular docking of GRSP with humic acid. Based on the phylogenetic studies, it is evident that Hsp60 protein from R. irregularis from Glomeromycota shares a very close evolutionary relationship with fungi belonging to Ascomycota and Basidiomycota, with classical a-Proteobacteria as the common ancestor. Comprehensive analysis of mitochondrial hsp60 from selected fungal taxa from the evolutionary point of view explains the possibility of gene duplication and or horizontal gene transfer of this gene across various fungal species. Molecular clock analysis carried out by BEAST programme for the 14 common protein cod- ing genes (inclusive of hsp60),identifiedfrom21mitochondrial genomes belonging to fungal divisions Ascomycota, Basidiomycota and Glomeromycota estimates their time for diversification to be around 1425 Mya. Population genetics study cred- ibly explains high genetic variability associated with fungal hsp60 presumably brought by random genetic recombination events. The results also confirm a high level of genetic differentiation of hsp60 among all the three fungal populations analyzed. PsiBlast search and Swiss-Model server identified Hsp60 from Chlorobaculum tepidum (5DA8_A) as the potential template for generating 3 D model of GRSP. Accuracy of the 3 D model was References subsequently validated using Ramachandran plot analysis, ERRAT, Adjei, A. A. (2001). Blocking oncogenic Ras signaling for cancer therapy. ProCheck and Verify 3 D. Among the two identified N-Linked gly- Journal of the National Cancer Institute, 93(14), 1062–1074. cosylation sites, the one that was present towards the C-terminal Downward, J. (2003). Targeting RAS signalling pathways in cancer ther- end was included in the truncated 94 residue fragment of the 3 D apy. Nature Reviews Cancer, 3(1), 11. model. Using Glycam Server, a glycoprotein model of GRSP was Lu, S., Jang, H., Muratcioglu, S., Gursoy, A., Keskin, O., Nussinov, R., & built by attaching the carbohydrate moiety rich in mannose, gal- Zhang, J. (2016). Ras conformational ensembles, allostery, and signal- 94 ing. Chemical Reviews, 116(11), 6607–6665. actose and N-acetyl galactosamine to the asp residue of the truncated model. AutoDock software was successful in docking the standard 3 D model of leonardite humic acid (chain L and U), onto the GRSP- carbohydrate complex. This was followed by docking the ‘GRSP glycoprotein—humic acid’ complex with 50 114. Molecular modelling and water molecules. These findings open up new avenues for explor- docking studies of glomalin related ing the interactions of glomalin related soil protein with the soil organic matter, and its influence in aggregating the water mole- soil protein from Rhizophagus cules in improving soil productivity. irregularis References

Dipti Mothay and K. V. Ramesh Gadkar, V., & Rillig, M. C. (2006). The arbuscular mycorrhizal fungal pro- Department of Biotechnology, Jain University, School of Sciences, tein glomalin is a putative homolog of heat shock protein 60. FEMS #18/3, 9th Main Road, Jayanagar East, Jaya Nagar 3rd Block, Microbiology Letters, 263(1), 93–101. Bengaluru 560041, Karnataka, India Petrov, D., Tunega, D., Gerzabek, M. H., & Oostenbrink, C. (2017). [email protected] Molecular dynamics simulations of the standard leonardite humic acid: Microscopic analysis of the structure and dynamics. Environmental Science & Technology, 51(10), 5414–5424. JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS VOL. 37, SUPPLEMENT, 2019 71

State Key Laboratory for Conservation and Utilization of Bio- 115. Sex-dependent differences in Resources in Yunnan & School of Life Sciences, Yunnan University, poly(ADP-ribose)polymerase 1 activity Kunming 650091, China [email protected] of rat liver and thymocyte nuclei The molecular mechanisms of enzymatic temperature adaptation are dictated by the delicate balance between the sta- Karine S. Matinyan, Anush L. Asatryan, Irina G. bility, flexibility and activity of the extremophilic enzymes; Artsruni, Arshak A. Karapetyan and therefore, identifying the factors that rule the stability–flexi- Emil S. Gevorgyan bility–activity relationship is essential for both fundamental Department of Biophysics, Faculty of Biology, Yerevan State research and industrial applications (Papaleo, Tiberti, University, Yerevan 0025, Armenia [email protected] Invernizzi, Pasi, & Ranzani, 2011). To investigate the role of electrostatics in temperature adaptation, we have performed Sex-dependent differences among therapeutic effects of a comparative study on three differently temperature- PARP inhibitors were documented in treatment of various adapted subtilisin-like serine proteases, the psychrophilic pathologies. The purpose of this study was to examine VPR, the mesophilic PRK and the thermophilic AQN, using whether PARP 1 activity displays sexual dimorphism in liver multiple-replica molecular dynamics simulations followed by cells and thymocytes of rat (Virag & Szabo, 2002; Waxman & continuum electrostatics calculations and comparison of elec- Holloway, 2009). PARP 1 inhibition by benzamide (Bam) and trostatic surface potentials. Comparison of the salt-bridge ATP in liver and thymocyte nuclei after in vivo administration electrostatic strength at 300 K reveals that they on average of cis-diammine-1,1-cyclobutanedicarboxylate platinum (cis- provide greater stabilization to VPR and PRK than to AQN, DDP) to animals was studied. Our data show that female indicating that salt bridge may not be a crucial factor in thymocyte and liver nuclei, in contrast to male counterparts, determining the overall thermostability of subtilisin-like ser- exhibit organ-specific differences in PARP 1 activities. PARP 1 ine proteases. The most significant difference lies in the dis- activity of female thymocyte nuclei was unaffected by Bam, tribution of different electrostatic surface potentials, while in liver nuclei it was suppressed by 45%. PARP 1 activ- especially across the back surfaces, that is, those of PRK and ity of the male rats liver nuclei was completely suppressed AQN are dominated by the electro-positive potential with by Bam. It was demonstrated that in liver nuclei of male rats sporadic electro-neutral patches interspersed, and VPR’s back treated with cis-DDP PARP 1 activity decreased more than 3 surface is dominated by the electro-negative potential, times, whereas in liver nuclei of females it was suppressed although the catalytic centers (on the front surface) of the only by 45%. In addition, the studies revealed that thymo- three proteases collectively feature the electro-negative cyte nuclei of both sexes were more resistant to in vivo and potential. Because water is a protic solvent, it interacts more ex vivo chemical interventions than liver nuclei. It was shown favorably with the electro-negative surface than with the that in vivo administration of anti-cancer drug cis-DDP is electro-positive and neutral surfaces, and this explains why a capable to modulate PARP 1 inhibition by Bam in liver nuclei protein with more negative surface charge is more soluble in in sex-dependent manner which should be considered for water than its homologue with more positive surface charge. improving therapeutic outcomes in adjuvant cancer chemo- Furthermore, since water molecules around the negatively therapy by PARP 1 inhibitors. charged and polar uncharged surfaces form a high-density, collapsed structure with weak hydrogen-bonding interactions and fast dynamics, they would facilitate conformational fluc- References tuations, hence enhancing the local flexibility of the electro- negative surface regions. On the contrary, the low-density, Virag, L., & Szabo, C. (2002). The Therapeutic Potential of Poly(ADP- expanded structure of hydration shell formed around the Ribose)Polymerase Inhibitors. Pharmacological Reviews, 54(3), 375–418. Waxman, D. J., & Holloway, M. G. (2009). Sex Differences in the positively charged and nonpolar/neutral surfaces would Expression of Hepatic Drug Metabolizing Enzymes. Molecular restrain their fluctuations and hence increase the local rigid-

Pharmacology, 76(2), 215–228. ity. For the mesophilic PRK at 300 K, comparison of the Ca root-mean-square-fluctuation values averaged over the solvent-exposed residues covered by the electro-negative, electro-positive and electro-neutral potentials reveals that the 116. The distribution of electrostatic electro-negative surface is indeed more flexible than the electro-positive and nonpolar/neutral surfaces. For all three surface potentials is an important proteases, the electro-negative surface potential around the factor underlying the temperature active center could provide the active-center flexibility neces- adaptation of subtilisin-like sary for nucleophilic attack and proton transfer. For VPR, the predominant distribution of the electro-negative potential on serine proteases the back surface could ensure sufficient low-temperature solubility and surface flexibility crucial for VPR’s low-tempera- Yuan-Ling Xia, Xiao-Yan Zhang, Yan Tao and ture catalytic activity. For AQN, the predominant distribution Shu-Qun Liu of the electro-positive potential likely contributes to 72 BOOK OF ABSTRACTS. ALBANY 2019: THE 20TH CONVERSATION enhancing the surface rigidity necessary for the mainten- and CD4-bound states, which structural elements determine ance of the structural integrity at high temperatures. these differences, and how gp120 regulates transition Therefore, differently charged surface patches can affect/ between different states, still remain answered. To answer modulate via differential interactions with water molecules these questions, we have performed a series of multiple-rep- the protein solubility and the local structural flexibility/ lica molecular dynamics simulations on the three structural rigidity (Xia et al., 2018) and, therefore, the electrostatic models (i.e., the unliganded gp120, the CD4-free gp120 in surface potential distribution is an important factor under- the CD4-bound state and the gp120-CD4 complex), with the lying the temperature adaptation of subtilisin-like ser- cumulative simulation time length reaching 3 ms. ine proteases. Comparative analyses of their respective concatenated equilibrium trajectories in terms of structural deviation and conformational flexibility reveal that the CD4-free and CD4- Funding complexed gp120s are more structurally mobile and confor- This research has been supported by NSFC of China (nos. mationally flexible than the unliganded gp120. The most 31160181 and 31370715) and Programs for Excellent Young pronounced differences in the local flexibility between the Talents and Donglu Scholar in the Yunnan University. unliganded and CD4-complexed/free gp120s were observed in the V1/V2 region and V3 loop, which exhibit significantly

higher Ca root-mean-square-fluctuation values in the latter References than in the former due to the dissociation between and full exposure of V1/V2 and V3. Analyses of the largest-amplitude Papaleo, E., Tiberti, M., Invernizzi, G., Pasi, M., & Ranzani, V. (2011). Molecular determinants of enzyme cold adaptation: Comparative motion modes indicate that in the CD4-free gp120, the structural and computational studies of cold- and warm-adapted mutual approach between V1/V2 and V3 could lead to the enzymes. Current Protein & Peptide Science, 12, 657–683. conformational transition to the unliganded state. Xia, Y.-L., Sun, J.-H., Ai, S.-M., Li, Y., Du, X., Sang, P., … Liu, S.-Q. Comparison of the constructed free-energy landscapes (2018). Insights into the role of electrostatics in temperature adapta- (FELs) shows that CD4-complexed and CD4-free gp120s tion: A comparative study of psychrophilic, mesophilic, and thermo- have larger conformational entropy, richer conformational philic subtilisin-like serine proteases. RSC Advances, 8(52), 29698–29713. diversity and lower thermal stability than the unliganded gp120. Further comparison of the representative structures extracted from the free energy basins of FELs reveals that the presence of CD4 weakens the reorientation ability of V1/V2 relative to V3 and therefore restricts the conform- 117. The mutual orientation between ational transition from the CD4-bound state to the unli- V1/V2 and V3 is the structural ganded state. Therefore, it can be concluded that the mutual orientation of the V1/V2 region with respective to determinant of HIV-1 gp120 the V3 loop is not only a major marker for distinguishing conformational states and dynamics between the unliganded and CD4-bound states, but determines also the differences in the conformational dynamics Yi Li, Lei Deng, Yan Tao and Shu-Qun Liu and thermodynamics between these two states. Locking State Key Laboratory for Conservation and Utilization of Bio- gp120 conformation via restraining mutual reorientations Resources in Yunnan & School of Life Sciences, Yunnan University, between V1/V2 and V3 with small molecules seems to be Kunming, 650091, China [email protected] a promising strategy to control HIV-1 infection (Li, Deng, Yang, Sang, & Liu, 2019). The trimeric envelope spike (Env), which is the infection machinery of HIV-1 and the sole target of protective neutral- izing antibodies, is composed of three external glycoprotein gp120 subunits associated non-covalently with three trans- Funding membrane glycoprotein gp41 subunits. The entry of HIV-1 is This research has been supported by NSFC of China (nos. initiated by binding of gp120 to the cell-surface receptor 31370715 and 31160181) and Programs for Excellent Young CD4, which induces substantial changes in gp120 conform- Talents and Donglu Scholar in the Yunnan University. ation involving the reorientation of the V1/V2 region relative to the V3 loop, rearrangements of the bridging-sheet elements, and formation and exposure of the coreceptor-bind- References ing site, ultimately leading to the transition of Env from an Guttman, M., Garcia, N. K., Cupo, A., Matsui, T., Julien, J.-P., Sanders, unliganded ‘closed’ state to a CD4-bound ‘open’ conform- R. W., … Lee, K. K. (2014). CD4-Induced activation in a soluble ation (Guttman et al., 2014). Although structural studies have HIV-1 Env trimer. Structure, 22(7), 974–984. provided valuable information on various states of Env/ Li, Y., Deng, L., Yang, L. Q., Sang, P., & Liu, S. Q. (2019). Effects of CD4 gp120, the detailed questions on many dynamic aspects of binding on conformational dynamics, molecular motions, and thermo- gp120, such as what the differences in conformational dynamics of HIV-1 gp120. International Journal of Molecular Sciences, 20(2), 260. dynamics and thermodynamics are between the unliganded JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS VOL. 37, SUPPLEMENT, 2019 73

carrying the dominant-negative E219K polymorphism. Biochemical 118. Understanding the basis for Journal, 446(2), 243–251. Friedman-Levi, Y., Meiner, Z., Canello, T., Frid, K., Kovacs, G. G., Budka, H., protective role of E219K prion variant … Gabizon, R. (2011). Fatal prion disease in a mouse model of gen- using molecular dynamics simulations etic E200K Creutzfeldt-Jakob disease. PLoS Pathogens, 7(11), e1002350. Jahandideh, S., Jamalan, M., & Faridounnia, M. (2015). Molecular dynamics study of the dominant-negative E219K polymorphism in human Vinod Jani, Uddhavesh Sonavane and Rajendra Joshi prion protein. Journal of Biomolecular Structure and Dynamics, 33(6), High Performance Computing-Medical & Bioinformatics 1315–1325. Application Group, Centre for Development of Advanced Computing (C-DAC), C-DAC Innovation Park, Panchavati, Pashan, Pune 411007, India [email protected]@cdac.in

The scrapie form of prion protein is responsible for various 119. Thermodynamic stability and neurodegenerative diseases. Mutation in prion protein is one molecular dimensions of pseudoknots of the factors which leads to pathogenic conversion of normal prion PrPc protein to scrapie form PrPSc (Friedman-Levi et al., Calliste Reiling-Steffensmeier and Luis A. Marky 2011). But, previous studies on the prion protein also suggest Department of Pharmaceutical Sciences, College of Pharmacy, that E219K prion mutant (glutamate to Lysine mutation at resi- University of Nebraska Medical Center, Omaha, NE, USA due position 219) is more stable than the wild type protein [email protected] (Biljan et al., 2012; Jahandideh, Jamalan, & Faridounnia, 2015). Hence, in this study the wild type prion protein, prion mutant Pseudoknots are prevalent RNA structures that play signifi- E200K (glutamate to Lysine mutation at residue position 200) cant roles in the biological function of RNA. In this work, we and E219K, a naturally occurring mutant, which is considered have investigated the unfolding thermodynamics and solu- to protect against Creutzfeldt-Jakob disease (CJD) have been tion molecular dimensions of two pseudoknots with stem studied. In order to understand the structural changes, we loop complementarity to model the frameshift occurrence of have carried out detailed atomistic simulation of three sys- ribosomal pseudoknots. tems. Principal component analysis, free-energy analysis, We used a combination of UV and DSC melting techni- dynamic cross correlation analysis and other analyses have ques and SAXS to determine the thermodynamic stability been carried out. Our analysis shows that the extra stability of and solution molecular dimensions of two pseudoknots

E219K mutant is result of increase in number of native con- with sequences: d(TCTCTT1111AAAAAAAAGAGAT5TTTTTTT) tacts, strong hydrogen bond network and less random (PsK11-DNA) and r(UCUCU-U1111AAAAAAAAGAGAU5 motions. The pathogenicity of E200K may be the result of loss UUUUUUU) (PsK11-RNA), ‘T11’ and ‘U1111’ are the loop of some crucial salt–bridge interaction and anti-correlated sequences complementary to one stem. We used three buf- motion between helix 2(H2) and helix 3 (H3) and increase in fer conditions: 10 mM Sodium Phosphate (NaPi), 10 mM solvent accessible surface area of hydrophobic residues. Thus, Sodium Cacodylate (NaCac) and 10 mM Sodium Cacodylate 2 this study shows that native contacts, salt–bridge interaction with 10 mM MgCl2 (NaCac & Mg þ). and hydrogen bonds between H2 and H3 play an important Both pseudoknots form intramolecularly with PsK11-DNA role in the maintaining stability/pathogenicity of the prion pro- in the ‘B’ conformation and PsK11-RNA in the ‘A’ conform- tein (Figure 1). ation, determined by their circular dichroism spectra. These pseudoknots were slightly more stable in the NaPi buffer by 2 0.9 kcal/mol. However, the addition of Mg þ to the NaCac buffer significantly increased their stability, by 10.8 (PsK11- References DNA) and 8.7 kcal/mol (PsK11-RNA). Overall, PsK11-RNA is Biljan, I., Giachin, G., Ilc, G., Zhukov, I., Plavec, J., & Legname, G. (2012). more stable by 5 kcal/mol, due to its higher TM and unfold- Structural basis for the protective effect of the human prion protein ing enthalpy. However, this is reduced to 3 kcal/mol in the

Figure 1. 74 BOOK OF ABSTRACTS. ALBANY 2019: THE 20TH CONVERSATION

2 presence of Mg þ. The TM-dependencies on salt yielded simi- indicating that we are indeed able to detect the thermody- lar ion uptakes of 3 mol Naþ/mol in both buffers and namics of a KLI. The two bimolecular kissing complexes also 2 0.1 mol of Naþ/mol in NaCac & Mg þ. The T -dependencies displayed KLI but only at high concentrations. Addition of M on osmolyte (ethylene glycol) yielded lower water uptakes the complementary strand to the base of Kissing Complex for PsK11-DNA by 13 mol H2O/mol, in NaPi and NaCac. abolished the KLI while maintaining integrity of the individ- 2 However, in the presence of Mg þ PsK11-DNA has a greater ual hairpin stems. water uptake by 10 mol H20/mol. The SAXS data indicate PsK11-DNA has a length of 80 Å Funding and width of 32 Å, while PsK11-RNA has a length of 65 Å and width of 30 Å; both molecules have a thickness of This research has been supported by Grant MCB-1122029 about 14 Å. Thus, PsK11-RNA is more compact. from NSF. The main conclusion is that the higher compactness of the RNA pseudoknot is consistent with its greater thermodynamic stability. 121. Accounting for electrostatic polarization in gas-phase simulations Funding of ion mobility spectrometry This research has been supported by grant MCB-1122029 from the National Science Foundation. Christopher A. Myersa and Alan A. Chenb,c aPhysics Department; bChemistry Department; cRNA Institute, University at Albany (SUNY), Albany, 12222, NY [email protected] 120. Unfolding thermodynamics Molecular dynamics (MD) simulations coupled with ion of DNA kissing-loop interactions mobility spectrometry (IMS), a gas-phase extension to mass spectrometry that further filters the analytes by their top- Carolyn E. Carr and Luis A. Marky ology, allows researchers to perform three-dimensional struc- Department of Pharmaceutical Sciences, University of Nebraska tural elucidation of nucleic acids. Recently, combined IMS- Medical Center, Omaha, NE 68198-6025, USA MD studies of short DNA duplexes (Porrini et al., 2017) and [email protected] hairpins (Lippens, Ranganathan, D’Esposito, & Fabris, 2016), however, have struggled with obtaining accurately compact Kissing-loop interactions (KLI) are common biological motifs found in RNA–RNA interactions. While significant computa- structures that agree with the experimentally determined tional work has been done on the thermodynamics and collision cross sections for parts of their work. Because IMS structure of RNA kissing-loop interactions, there are only a experiments are performed on charged molecules, long- few experimental studies on the thermodynamics of kissing range electrostatic interactions, as well as the changes in and none give complete and detailed thermodynamic charge density due to protonated phosphate sites along the reports. As a proof of concept, we modeled a monomolecu- backbone, play a crucial role in understanding how nucleic lar and bimolecular KLI and determined a complete thermo- acids behave in a gaseous environment and can benefit from dynamic description of the folding/unfolding of an a detailed quantum chemical analysis. intramolecular DNA kissing complex (Kissing Complex), two In the work presented here, we explore what a properly bimolecular kissing complexes (KHp1 KHp2, KS1 KS2), a tuned electrostatic force field for nucleic acids might look þ þ non-kissing control complex (No Kiss Com) and their appro- like. Based off of density functional theory calculations of priate control stem-loop motifs (KHp1, KHp2 and TLKHp2). We small oligonucleotides, we examine how one could adjust also tested the ability of the kissing complex to engage in the partial charges in an MD force field to more accurately kissing when sterically strained by adding a complementary replicate Coulomb interactions for charged and protonated strand to the base. The transition temperature of each oligo- nucleic acids. Since the electrostatic interactions used in nucleotide remains constant over a ten-fold range of total most force fields are often based off of single monomer strand concentration, indicating all molecules formed intra- quantum chemistry data and do not account for hydrogen molecularly, with the exception of KHp2, which displayed bonding energies, the work presented here proposes new two transitions, one bimolecular and one monomolecular methods for parameterizing force fields using multiple oligo- which was attributed to the formation of a duplex. Kissing nucleotide geometries and, more generally, how polarization Complex unfolded triphasically, with the last two peaks corre- is accounted for throughout an MD simulation. sponding to the two hairpins that make up the kissing complex and the first peak corresponding to the KLI (DH cal ¼ 23.7 kcal/mol). No Kiss Com unfolded biphasically, with two Funding peaks corresponding to the two individual hairpins that make up the complex, but no peak relating to a KLI, Alan A. Chen is supported by NSF award #MCB1651877. JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS VOL. 37, SUPPLEMENT, 2019 75

References Value of the enthalpy change (DH 2.2 ccal/mol) is À about three-fold less, than in the case of intercalation bind- Lippens, J. L., Ranganathan, S. V., D’Esposito, R. J., & Fabris, D. (2016). ing of EtBr to double-stranded NA and the binding constant Modular calibrant sets for the structural analysis of nucleic acids by ion mobility spectrometry mass spectrometry. The Analyst, 141(13), value K is about an order less, than the analogous value cor- 4084–4099. responding to EtBr intercalation into the plane of ds-NA base Porrini, M., Rosu, F., Rabin, C., Darre, L., Gomez, H., Orozco, M., & pairs. Most probably, it is the fact that at the mentioned con-

Gabelica, V. (2017). Compaction of duplex nucleic acids upon native ditions EtBr binds to poly(G)4 by semi-intercalation mode. It electrospray mass spectrometry. ACS Central Science, 3(5), 454–461. is indicated by the fact that the values of DG and DS are in Rueda, M., Kalko, S. G., Luque, F. J., & Orozco, M. (2003). The structure correspondence to those obtained for EtBr complexes with and dynamics of DNA in the gas phase. Journal of the American single- or double-stranded DNA at the binding semi-intercal- Chemical Society, 125 (26), 8007–8014. ation mode. The obtained data indicate that EtBr binding modes to nucleic acids depend on structural state of the latters. Particularly, in the case of NA structure dense packaging, 122. Binding peculiarities of ethidium EtBr intercalation becomes difficult and the main binding bromide to polyguanine acid mode remains semi-intercalation, as it was revealed for this ligand complexes with poly(G)4. Consequently, by investigating interactions of synthetic four-stranded polyguanylic acid Marine A. Parsadanyana, Ara P. Antonyana, Mikayel V. b c with EtBr, we can understand the structural differences of Minasyants and Armen T. Karapetian studied synthetic and right-handed B- or A-conformational a Department of Biophysics, Yerevan State University, Yerevan, types of nucleic acids, at the same external conditions. Armenia; bUWC Dilijan College, Dilijan, Armenia; cDepartment of Physics and Electrotechnics, Natioanl University of Architecture and Construction of Armenia, Yerevan, Armenia References [email protected] Bochman, M. L., Paeschke, K., & Zakian, V. A. (2012). DNA secondary structures: Stability and function of G-quadruplex structures. Nature Ethidium bromide (EtBr) binding to poly(G)4 has been Reviews Genetics, 13(11), 770–780. studied using spectroscopic methods at the solution ionic strengths 0.11, 0.31 and 0.51 M. Spectral characteristics (changes of absorption and fluorescence spectra) of EtBr- poly(G)4 complexes are analogous to those obtained for EtBr complexes with DNA or poly(rA)-poly(rU) (Bochman, 123. Clustering of stacked nucleobases Paeschke, & Zakian, 2012). The studies were carried out at in a stretched DNA conformation the temperatures 293, 298 and 300 K to determine the binding thermodynamic parameters. Based on the absorption enhances facilitated diffusion of DNA and fluorescence spectra, the binding curves in Scatchard’s binding proteins as revealed by coordinates were constructed at the mentioned ionic molecular dynamics simulations strengths of the solution and temperatures (EtBr binding curves to poly(G)4 in Scatchard’s coordinates are similar to Anupam Mondal and Arnab Bhattacherjee those obtained for this ligand complexes with DNA), and the School of Computational and Integrative Sciences, Jawaharlal values of binding constant K and binding site size n, DG, DH Nehru University, New Delhi, 110067, India and DS were determined by standard formulas. [email protected]

Figure 1. 76 BOOK OF ABSTRACTS. ALBANY 2019: THE 20TH CONVERSATION

DNA damage and repair refers to the creation and correction (PGOs). Guanidine residue can be introduced into desired posi- of DNA lesions that threat genome integrity. Homologous tions of an oligonucleotide by the standard automatic phos- recombination is the common mechanism to prevent the phitamide protocol of DNA synthesis (Stetsenko, Kupryushkin, DNA from the deleterious effects caused by mutations due & Pyshnyi, 2014; Kuprushkin, Pyshnyi, & Stetsenko, 2014). to the error in replication or induced by environmental In the present work, studied the absolute configuration of changes. Experiments (Chen, Yang, & Pavletich, 2008; Chen phosphate atom in PGO. First, we analyzed monomodified et al., 2017) show that during the process, RecA protein oligomer d(TpCpÃA) (Ã - shows N,N,N0,N0-tetramethylguani- binds with genomic DNA, which is unusually elongated dine moiety). Reverse-phase HPLC analysis allowed to isolate (approximately 1.5 times to its contour length in B-DNA) and two individual diastereomers of monomodified oligomer its nucleobases are clustered together in a specific manner. (fast and slow). Both diastereomers of d(TpCpÃA) were not How these structural changes in DNA affect its interaction digested with a snake venom phosphodiesterase after 7 days, with the protein? What are the implications of such structural whereas native analog was fully degraded after 30 min. diversity in identifying damaged DNA sites? Aided by exten- Ultraviolet and circular dichroism spectroscopy methods, as sive Langevin dynamics simulations we probe these issues, well as 1 D and 2 D NMR spectroscopy showed high similar- where we study the diffusion of a protein on a force-induced ity of the diastereomers. Only detailed computational ana- stretched DNA. Upon pulling systematically at the 30 termini lysis of restraint penalty energies via restrained molecular of a 100 bp DNA by constant forces, the DNA exhibits a con- dynamics simulations allowed us to conclude that most formational transition from B-DNA to ladder-like S-DNA con- likely, the fast isomer is the Sp- and the slow one is the Rp- formation via -DNA (Figure 1) intermediate. Our analysis diastereomer. suggests that the resulting stretched DNA features non-uni- To study PGO properties more deeply we designed DNA P form clusters of nucleotide bases such as doublets, triplets, duplex (d(50-CAGCGGCGÃTG-30)/d(CACGCCGCTG)) with single quadruplets and so on separated by large rise gap. We found N,N0-dimethyl-N,N0-ethylguanidine modification. Using RP- that relative population of these clusters govern the rugged- HPLC we isolated individual diastereomers of PGO. 1 D (1H, ness of the protein–DNA binding energy landscape and 32P) and 2 D (1H-1H NOESY, 1H-1H COSY, 1H-1H TOCSY, 1H-31P thereby the ability of a protein to locate its target DNA sites. COSY, 1H-13C HSQC, 1H-13C HMBC) NMR spectra were By analyzing different force regimes, we delineate the under- recorded. After assignment of all non-exchangeable protons lying translocation mechanism of a DNA binding protein and in NOESY spectra, using the restrained molecular dynamics underscore the significance of triplex formation in regulating method, we determined that the fast diastereomer is Rp and the protein diffusion on DNA. In my presentation, I shall the slow-Sp. The structural analysis showed that the intro- describe our recent findings and discuss them in the light of duction of the PG group into the oligomer does not disturb recent experimental observations. the B-form of the double DNA helix. Computer analysis of the Taq DNA polymerase complexes with PGO duplexes and experimental 10-mer PGO primer References elongation were carried out. The significant difference in the Chen, Z., Yang, H., & Pavletich, N. P. (2008). Mechanism of homologous processing of the Rp and Sp isomers was observed. The recombination from the RecA-ssDNA/dsDNA structures. Nature, detailed comparative analysis of the data obtained allowed 453(7194), 489–494. us to determine the origin of the different processing of the Chen, J., Tang, Q., Guo, S., Lu, C., Le, S., & Yan, J. (2017). Parallel triplex diastereomers by the enzyme. structure formed between stretched single-stranded DNA and homologous duplex DNA. Nucleic Acids Research, 45(17), 10032–10041. The data obtained show that the phosphorylguanidine oligonucleotides are promising for a number of applications such as PCR analysis, biosensors (Dmitrienko et al., 2016) and 124. Comparative physico chemical therapeutics. and biological studies of phosphorylguanidine oligonucleotide Funding diasteriomers This research has been supported by the Russian Science Foundation [grant No. 18-14-00357]. Alexander A. Lomzov, Valeria S. Apukhtina, Andrey V. Shernyukov, Maxim S. Kupryushkin, References Georgy Yu. Shevelev and Dmitrii V. Pyshnyi Institute of Chemical Biology and Fundamental Medicine SB RAS, Dmitrienko, E., Naumova, O., Fomin, B., Kupryushkin, M., Volkova, A., Novosibirsk State University, Novosibirsk, 630090, Russia Amirkhanov, N., … Pyshnyi, D. (2016). Surface modification of SOI- [email protected] FET sensors for label-free and specific detection of short RNA analyte. Nanomedicine, 11(16), 2073–2082. Synthetic oligonucleotide analogs are widely used in various Kupryushkin, M. S., Pyshnyi, D. V., & Stetsenko, D. A. (2014). Phosphoryl guanidines: A new type of nucleic Acid analogues. Acta Naturae, 6 (4), fields of molecular biology, nanobiotechnology and medicine. 116–118. Recently, we developed a new type of phosphate-modified Stetsenko, D. A., Kupryushkin, M. S., & Pyshnyi, D. V. 2014. International nucleic acids: phosphorylguanidine (PG) oligonucleotides patent application WO2016/028187 A1, August 22. JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS VOL. 37, SUPPLEMENT, 2019 77

Vardevanyan, P. O., Antonyan, A. P., Parsadanyan, M. A., Shahinyan, 125. Comparative study of binding M. A., & Sahakyan, V. G. (2017). Peculiarities of interaction of synthetic polyribonucleotide poly(rA)-poly(rU) with some intercalators. Journal of some intercalating compounds of Biomolecular Structure and Dynamics, 35,1–7. doi: 10.1080/ with DNA 07391102.2017.1402708.

Poghos O. Vardevanyana, Ara P. Antonyana, Marine A. Parsadanyana, Mariam A. Shahinyana and 126. Concentrational dependence Victoria G. Sahakyanb a of melting temperature: possible Department of Biophysics, Yerevan State University, Yerevan, Armenia; bDepartment of Clinical Pathology, Yerevan State explanation of non- Medical University after M.Heratsi, Yerevan, Armenia [email protected] monotonic behavior

The comparative study of DNA-acridine orange (AO), DNA- A. Asatryan, Sh. Tonoyan, Y. Mamasakhlisov and ethidium bromide (EtBr) and DNA-methylene blue (MB) com- V. Morozov plexes has been carried out using UV melting method at the Department of Molecular Physics, Yerevan State University, solution ionic strength 0.02 M, in the interval of ligand/DNA Yerevan 0025, 1 Alex Manoogian, Republic of Armenia [email protected] concentration ratio change—0

References References

Asatryan, A., Tonoyan, S., Mirtavoosi, S., Mamasakhlisov, Y., & Morozov, V. Case, B. C., Hartley, S., Osuga, M., Jeruzalmi, D., & Hingorani, M. M. (2015). 192 The helix-coil transition in two-component solvent in the (2019). The ATPase mechanism of UvrA2 reveals the distinct roles of frames of GMPC. Ligands effects on the characteristics of the transi- proximal and distal ATPase sites in nucleotide excision repair. Nucleic tion. Journal of Biomolecular Structure and Dynamics, 33(supp1), Acids Research, under revision. 126–127. Jaciuk, M., Nowak, E., Skowronek, K., Tanska, A., & Nowotny, M. (2011). Badasyan, A., Tonoyan, S., Giacometti, A., Podgornik, R., Parsegian, V., Structure of UvrA nucleotide excision repair protein in complex Mamasakhlisov, Y., & Morozov, V. (2014). Unified description of solv- with modified DNA. Nature Structural & Molecular Biology, 18(2), ent effects in the helix-coil transition. Physical Review E, 89(2), 022723. 191–197. De Costa, N., & Heemstra, J. (2013). Evaluating the effect of ionic Kisker, C., Kuper, J., & van Houten, B. (2013). Prokaryotic nucleotide strength on duplex stability for PNA having negatively or positively excision repair. Cold Spring Harbor Perspectives in Biology, 5(3), charged side chains. PLoS One, 8(3), e58670 a012591. Vardevanyan, P., Antonyan, A., Parsadanyan, M., Torosyan, M., & Pakotiprapha, D., Samuels, M., Shen, K., Hu, J. H., & Jeruzalmi, D. (2012). Karapetian, A. (2016). Joint interaction of ethidium bromide and Structure and mechanism of the UvrA-UvrB DNA damage sensor. methylene blue with DNA. The effect of ionic strength on binding Nature Structural & Molecular Biology, 19(3), 291–298. thermodynamic parameters. Journal of Biomolecular Structure and Dynamics, 34(7), 1377–1382.

128. Dynamics changes of CRISPR- 127. Coordinated actions of UvrA2 during initiation of nucleotide Cas9 system induced by high excision repair fidelity mutations

Brandon C. Case and Manju M. Hingorani Liangzhen Zheng and Yuguang Mu Department of Molecular Biology & Biochemistry, Wesleyan School of Biological Sciences, Nanyang Technological University, University, Middletown, CT 06459, USA 637551, Singapore [email protected] [email protected] CRISPR-Cas9 as a powerful genome editing tool has widely Nucleotide excision repair (NER) protects genomic DNA from been applied in biological fields. Ever since the discovery of a large number of chemically diverse lesions, including CRISPR-Cas9 as an adaptive immune system, it has been nucleotide adducts and pyrimidine dimers. In bacteria, NER is gradually modified to perform precise genome editing in eukaryotic cells by creating double-strand breaks. Though initiated by UvrA2 or UvrA2(B2) complex that scans DNA for changes in structure/dynamics. Once the complex encoun- being robust and efficient, current CRISPR-Cas9 system faces a major flaw: the off-target effect, which has not been well ters a potential damage site, UvrA2 dissociates, leaving UvrB to confirm lesion recognition and signal UvrC to nick the understood. Several Cas9 mutants show significant improve- damaged strand for removal (Kisker, Kuper, & van Houten, ment, with very low off-target effect, however, relatively lower cleavage efficiency for on-target sequences as well. In 2013). Each monomer in the UvrA2 dimer has two ATPase sites (proximal and distal), both of which are required for NER and this study, the dynamics of the wild-type Cas9 from S. pyo- are implicated in lesion recognition and loading of UvrB onto genes and the high fidelity (HF) Cas9 mutant has been the DNA (Jaciuk, Nowak, Skowronek, Tanska, & Nowotny, 2011; explored using molecular dynamics simulations. It turns out Pakotiprapha, Samuels, Shen, Hu, & Jeruzalmi, 2012). Our that the mutations cause reduction of electrostatic interactions between Cas9 and R-loop. Consequently, the flexibility recent study of the UvrA2 ATPase kinetic mechanism revealed that the two sites exhibit asymmetric ATP binding and hydroly- of the tDNA/sgRNA heteroduplex is reduced, which may sis, which is modulated differentially by undamaged and dam- explain the reason of less tolerance of mismatches in the aged DNA. The findings suggest that key steps in the ATPase heteroduplex region. The mutations also affect the protein mechanism may be coupled to changes in UvrA2 interactions dynamics and the correlation networks among Cas9 domains. with DNA, ultimately leading to lesion recognition and UvrB In mutant Cas9, weakened communications between two loading (Case, Hartley, Osuga, Jeruzalmi, & Hingorani, 2019). catalytic domains, as well as the mutations induced slight We are now investigating the mechanochemical coupling opening of the conformation, may account for the lower on- between the ATPase reaction and other UvrA2 activities to target cleavage efficiency, and probably lower off-target as determine how the nucleotide-bound state of UvrA2 influences well. These findings would facilitate more precise Cas9 its conformation and interactions with DNA. Preliminary results engineering in future. indicate the UvrA2-lesion complex adopts a distinct conformation in the presence of ATP that is not observed with ADP or Funding without any nucleotide. Additionally, analysis of Walker A and B mutants to prevent ATP binding or hydrolysis, respectively, is This research has been supported by MOE (Tier 1 RG146/17). JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS VOL. 37, SUPPLEMENT, 2019 79

beginning with concentration increasing of ligands in the 129. Effect of concentration of ligands solution DNA-biosensor output signal dispersion increases, on DNA-biosensor average output then passing through the maximum it decreases and tends signal and dispersion to zero at the further enhancement of ligand concentration. The obtained results can be used in the practice at preparation of micro DNA-biosensors and analysis of the measure- Valeri B. Arakelyana, Marine A. Parsadanyanb and ment data. Poghos O. Vardevanyanb aDepartment of Molecular Physics; bDepartment of Biophysics, Yerevan State University, Yerevan, Armenia References [email protected] Arakelyan, V. B., Babayan, S. Y., Tairyan, V. I., Arakelyan, A. V., Parsadanyan, M. A., & Vardevanyan, P. O. (2006). Kinetics of ligand Nowadays, DNA-biosensors are intensively applied for reso- binding to nucleic acids. Journal of Biomolecular Structure and lution of actual bio-analytical and medical problems, such as Dynamics, 23(4), 479–484. identification of biological material by primary sequence of Kavita, V. (2017). DNA Biosensors – A Review. Journal of Bioengineering nucleotides, improvement of early diagnostic and treatment and Biomedical Science, 7, 222. doi:10.4172/2155-9538.1000222 methods of oncological diseases, determination of pharmacological preparations of anti-cancerous action (Kavita, 2017). Application of DNA-biosensors significantly decreases both analysis cost and time of its carrying out as compared to 130. Expanding the structural traditional medico-biological methods. It is connected to the fact that DNA-biosensors have small sizes and demand rele- diversity of DNA beyond the vantly less amounts of the studying samples. Meanwhile, the double helix decrease of DNA-biosensor sizes inevitably results in both falling down of its output signal value and increasing of level Betty Chu and Paul J. Paukstelis of the signal fluctuations. It should be mentioned that DNA- Department of Chemistry and Biochemistry, Center for biosensor output signal fluctuations may be conditioned by Biomolecular Structure and Organization, University of Maryland, arbitrary character of the binding of small molecules (ligands) College Park, MD 20742, USA [email protected] in the solution with DNA duplexes on the biosensor as well. By this reason, the studies of the effect of ligands on average The iconic model of DNA is the Watson-Crick double helix, value of DNA-biosensor output signal and its fluctuations but it can form other types of structures. The extent of the become actual and important. In this work in the framework structural diversity of DNA is not well understood. We are of relativistic approach the dependences of DNA-biosensor interested in discovering new types of DNA structure output signal average value and its dispersion on ligand con- through the screening of a library of short DNA sequences centration in the solution were theoretically calculated. The and subsequent structure determination for those that crys- characteristic peculiarities of DNA-biosensor output signal tallize. From the screen, we aim to create a library of 3 D were studied. It was shown that the output signal of DNA- DNA crystal structures and identify previously unknown DNA biosensors monotonously increases with ligand concentration motifs. We report two new crystal structures of single oligo- enhancement in the solution (Arakelyan et al., 2006). It was nucleotides that interact via noncanonical base pairing. also shown that the output signal relaxation time decreases d(CGTAAGGCG) forms a non-G-quadruplex fold-back struc- along with ligand concentration increasing in the solution. ture through both Watson-Crick and noncanonical interac- Calculations showed that the dispersion dependence of tions. The tetrameric assembly encloses a central cation DNA-biosensor output signal on the ligand concentration in binding pocket and features a hexad base pairing arrange- the solution has a form of a curve with maxima. From the ment through two C-G-G base triples. This is the first fold-

Figure 1. 80 BOOK OF ABSTRACTS. ALBANY 2019: THE 20TH CONVERSATION back structure that forms a tetramer and is specific for diva- some preference to DNA (to B-form). Our obtained data also lent cations. We have also determined three variant sequen- indicate that MTX binds to DNA by analogous mechanism with ces that form the same structure, suggesting that there is a methylene blue (MB):—at relatively high ionic strengths and large number of potential fold-back sequences in genomes low concentrations it semi-intercalates into ds-structure of (Chu, Zhang, Hwang, & Paukstelis, 2018). Fold-back struc- nucleic acid (NA), at high concentrations of the ligands—binds tures are biologically relevant since they have been to helix electrostatically from the outer side (Vardevanyan, observed in promoter regions of development genes (Mir Antonyan, Parsadanyan, Torosyan, & Karapetian, 2016). It was 2 et al., 2017). d(CCAGGCTGCAA) features a Ba þ-stabilized also revealed that at the joint binding of both ligands to NA G-quadruplex, which is flanked on either side by a base and their low concentrations a competition between bound triple formed through noncanonical interactions and an EtBr molecules and MTX does not emerge, meanwhile their i-motif. This tetramer is the first structure of a hybrid DNA general effect on stabilization of DNA ds-structures is less, than G-quadruplex/i-motif and demonstrates the possibility of at separate impacts. At high concentrations of both ligands in the coexistence of G-quadruplexes and i-motifs in a single the case of ds-DNA a competition between them emerges, strand of DNA in genomes. The fold-back quadruplex and though, the EtBr effect becomes prevailing. At the same time hybrid G-quadruplex/i-motif highlight the growing struc- in the case of the joint interaction of both ligands with tural diversity of DNA and suggest greater biological roles poly(rA)-poly(rU) the main part of stabilization of ds-structures for non-duplex structures. These two structures demon- corresponds to EtBr, which may be the result of MTX weak strate that DNA assemblies beyond the traditional double affinity to ds-poly(rA)-poly(rU). helix exist and suggest that DNA can form even more Thus, the experimental data indicate that despite the ver- diverse structures (Figure 1). satility of the main (semi-intercalation) binding mode, MTX interacts with RNA much weaker, than with DNA. From this point of view, in the case of EtBr it was not revealed a pref- Funding erence to DNA or RNA. It was shown that the EtBr binding This research has been supported by the NSF (Career modes to the NA does not depend on the absence or the Award 1149665). presence of MTX.

References Reference

Chu, B., Zhang, D., Hwang, W., & Paukstelis, P. J. (2018). Crystal structure Vardevanyan, P. O., Antonyan, A. P., Parsadanyan, M. A., Torosyan, M. A., of a tetrameric DNA fold-back quadruplex. Journal of the American & Karapetian, A. T. (2016). Joint interaction of ethidium bromide and Chemical Society, 140(47), 16291–16298. methylene blue with DNA. The effect of ionic strength on binding Mir, B., Serrano, I., Buitrago, D., Orozco, M., Escaja, N., & Gonzalez, C. thermodynamic parameters. Journal of Biomolecular Structure and (2017). Prevalent sequences in the human genome can form mini i- Dynamics, 34(7), 1377–1382. DOI: http://dx.doi.org/10.1080/07391102. motif structures at physiological pH. Journal of the American Chemical 2015.1079557. Society, 139(40), 13985–13988.

132. Mismatched base pairs locally 131. Interaction of ethidium bromide distort DNA structure, leading to and mitoxantrone with double- increased DNA-binding by stranded poly(rA)-poly(rU) transcription factor proteins

a a Armen T. Karapetyan , Margarita A. Torosyan and a,b c d b Ariel Afek , Honglue Shi , Atul Rangadurai , Mariam A. Shahinyan Hashim M Al-Hashimic,d and Raluca M Gordana,b a Department of Physics and Electrotechnics, Natioanl University of aCenter for Genomic and Computational Biology, Duke University, Architecture and Construction of Armenia, Yerevan, 0009, b b Durham, NC 27708, USA; Department of Biostatistics and Armenia; Department of Biophysics, Yerevan State University, Bioinformatics, Duke University, Durham, NC 27708, USA; Yerevan, 0025, Armenia [email protected] cDepartment of Chemistry, Duke University, Durham, NC 27708, USA; dDepartment of Biochemistry, Duke University, Durham, NC The study of binding of the ligands ethidium bromide (EtBr) 27708, USA [email protected] and mitoxantrone (MTX) with double-stranded (ds-) DNA (B- form) and ds-poly(rA)-poly(rU) (ds-RNA, A-form) has been car- The local structure of genomic DNA can vary drastically from ried out. The obtained results reveal that EtBr forms several the ideal B-form double helix, and one cause for structural types of complexes with both ds-DNA and ds-poly(rA)- deformations is the pairing of non-complementary bases (i.e., poly(rU) structures: by intercalation, semi-intercalation and mis-paired bases, or mismatches). DNA mismatches are fre- electrostatic modes simultaneously. Though, it was revealed quently formed by spontaneous chemical reactions (such as that EtBr shows practically the same affinity to ds-DNA and nucleotide deamination), replication errors, and genetic ds-poly(rA)-poly(rU). MTX binds to ds-DNA and ds-RNA by recombination. Mismatches alter the local DNA structure semi-intercalation and electrostatic modes and performs (Figure 1a), which can affect interactions with DNA-binding JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS VOL. 37, SUPPLEMENT, 2019 81

Figure 1. (a) DNA mismatches have non-canonical base-pair geometries and induce large structural. (b) An example of protein-induced DNA structural deformation that could be mimicked by specific mismatches. proteins, including regulatory transcription factors (TFs). Quantitative and Computational Biology, Department of Biological Currently, very little is known about the effects of mis- Sciences, University of Southern California, Los Angeles, CA, USA matches on TF binding. [email protected] We present Saturation Mismatch Binding Assay (SaMBA), the first assay to characterize the effects of mismatches on Transcription factor (TF)-DNA binding is a fundamental compo- TF-DNA binding in high throughput. For genomic sequences nent of gene regulatory processes, but how these proteins recog- of interest, SaMBA generates DNA duplexes containing all nize their target sites in the genome is still not completely possible single-base mismatches, and quantitatively assesses understood. TFs can recognize their binding sites by having a sur- the effects of the mismatches on TF-DNA interactions. face physicochemically complementary to the physicochemical We applied SaMBA to measure binding of 21 TFs (cover- signature of DNA, forming a series of contacts between amino ing 14 structural families) to thousands of mismatched acids and nucleotides. These contacts include direct hydrogen sequences, and mapped the impact of mismatches on these bonds, water-mediated hydrogen bonds, and hydrophobic con- TFs. Remarkably, for all 21 TFs examined, introduction of mis- tacts. In the last decade, several high-throughput technologies matches at certain positions resulted in significantly have been developed for a better understanding of the TF-DNA increased binding, with some mismatches creating high-affin- binding mechanisms by quantitatively measuring the binding ity binding sites in nonspecific DNA and some converting affinities of a TF against thousands or even millions of different known binding sites into ‘super-sites’ stronger than any DNA sequences in vitro. Relevant methods include high-through- canonical Watson-Crick site. put sequencing technologies, such as SELEX-seq, HT-SELEX or Structural analyses of mismatches that increase TF binding SMiLE-seq. These methods provide an alternative path to infer TF- revealed that these mismatches are oftentimes distorting the DNA binding mechanisms without requiring time-consuming naked DNA such that its structure becomes similar to that of structural biology experiments. Here, we present DeepRec (Deep bound DNA sites (Figure 1(b)), thus explaining the increased Recognition for TF-DNA binding), a multi-module deep learning binding measured in our assay. Our results reveal that the framework capable of building a precise predictive model for TF- intrinsic energy cost of deforming the DNA structure is an DNA binding based on large-scale in vitro experimental data. The important, widespread layer of control in protein-DNA recog- method integrates a forward perturbation-based interpretation nition. Furthermore, since several recent studies have shown approach to highlight the important patterns for deciphering the that bound TFs at damaged DNA are likely to be a significant binding mechanisms. We demonstrate here applications of our cause for mutations (Reijns et al. 2015), characterizing the method to SELEX-seq data for human helix-loop-helix (bHLH) pro- binding preferences of TFs to mismatched DNA is an import- tein MAX, the human myocyte enhancer factor-2B (MEF2B), and ant step toward understanding the mutational landscape in the human tumor suppressor protein p53. We accurately pre- the genome. dicted DNA binding specificities and were able to unravel important insights into the binding mechanisms. References

Reijns, M. A. M., Kemp, H., Ding, J., Marion de Proce, S., Jackson, A. P., & 134. Modeling physical analogs Taylor, M. S. (2015). Lagging-strand replication shapes the mutational of DNA landscape of the genome. Nature, 518(7540), 502–506. Rohs, R., Jin, X., West, S. M., Joshi, R., Honig, B., & Mann, R. S. (2010). Valentina N. Balashovaa, Ludmila V Yakushevichb and Origins of specificity in protein-DNA recognition. Annual Review of a Biochemistry, 79, 233–269. Farit K. Zakiryanov aBashkir State University, Ufa, 450076, Russian Federation; bInstitute of Cell Biophysics RAS, Pushchino, 142290, Russian 133. Modeling of protein–DNA Federation [email protected] binding with a multi-module deep The creation of mechanical, electronic and other physical learning framework analogs of living systems, including whole organisms, individual organs, cells, and even biological molecules, helps to Tsu-Pei Chiu, Satyanarayan Rao, Ana Carolina Dantas deepen our knowledge of the nature of these systems. In Machado and Remo Rohs this paper, we consider a possibility to create the mechanical 82 BOOK OF ABSTRACTS. ALBANY 2019: THE 20TH CONVERSATION and electronic analogs of the DNA molecule (Figure 1), as Federal Research Center Pushchino Center for Biological Research well as mathematical methods that allow to calculate the of the Russian Academy of Sciences, G.K. Skryabin Institute of parameters of these analogs. The goal of this research is to Biochemistry and Physiology of Microorganisms RAS, Pushchino, find the coefficients of the transformation that relates the Moscow region, 142290, Russia [email protected] structural and dynamic parameters of DNA with similar parameters of the mechanical and electronic analogs of the The study of oxidative and constructive metabolism under molecule. The basis of the proposed approach is the ideas of conditions of limitation of growth of microbial cultures is Scott (1969) and of Englander, Kallenbach, Heeger, important from the point of view of the directional produc- Krumhansl, and Litwin, (1980), supplemented by recently tion of practically valuable metabolites, enzymes, biomass proposed mathematical model of the DNA torsion dynamics with the special composition. (Grinevich, Ryasik, & Yakushevich, 2015). They allowed us to The object of the study was the yeast Yarrowia lipolytica, find (1) the transformation required, (2) the values of the the uniqueness of which lies in the ability to synthesize signifi- parameters of the mechanical analog (pendulums mass m, cant quantities of organic acids, lipids and enzymes from a pendulums length r, stiffness of springs K, the distances wide range of carbon sources (Lazar, Liu, & Stephanopoulos, between neighboring pendulums a), (3) the values of the 2018; Morgunov & Kamzolova, 2015; Zinjarde, 2014). Ethanol electronic circuit parameters (L, C, I0, d) and (4) the relation- was used as carbon source. Ethanol as a substrate for growth ship between the parameters of the mechanical and elec- of producers of practically valuable compounds has several tronic analogues and the parameters of DNA (M, K b, R). The advantages over other substrates. It does not contain harmful obtained results show that the creation of the mechanical impurities, it is well assimilated by yeast and dissolves in water and electronic analogs of the DNA molecule can be realized in any proportions. Since ethanol is used in the human diet, and used to study various dynamic regimes that occur in the the products derived from it do not require additional purifica- molecule, in particular, the modes associated with the emer- tion from the residual substrate. As reported by Weusthuis, gence and spread of the transcription bubbles that play an Aarts, and Sanders (2011) the several companies in the USA important role in the functioning of the DNA molecule. and Switzerland created the food products based on microbial biomass produced from ethanol. References In the course of the research, it was established that under conditions of unlimited (exponential) growth of Y. lipo- Scott, A. C. (1969). A nonlinear Klein-Gordon equation. American Journal lytica VKM Y-2373 the efficiency of ethanol oxidation attained of Physics, 37(1), 52–61. Englander, S. W., Kallenbach, N. R., Heeger, A. J., Krumhansl, J. A., & a maximum. Under those conditions the cells contained the Litwin, S. (1980). Nature of the open state in long polynucleotide dou- maximum amount of protein, and the excretion of metabo- ble helices: Possibility of soliton excitations. Proceedings of the lites (citric, isocitric, a-ketoglutaric and other acids) did not National Academy of Sciences of the United States of America, 77(12), occur (Table 1). In other words, the metabolism of Y. lipoly- 7222–7226. Grinevich, A. A., Ryasik, A. A., & Yakushevich, L. V. (2015). Trajectories of tica was relatively balanced. DNA bubles. Chaos, Solitons & Fractals, 75, 62–75. On the contrary, under conditions of Y. lipolytica VKM Y- 2373 growth limitation by nitrogen or thiamine, the complex of functional changes takes place (Table 1), of which the 135. Modifications in the oxidative most significant modifications are following: and constructive metabolism of yeast Yarrowia lipolytica caused by 1. the orientation of biosynthetic processes shifts into the direction of a sharp decline in protein synthesis; limitation the growth by nitrogen 2. the intensive excretion of cellular metabolites became or thiamine active; the composition and quantity of the excreted organic acids depend on the nature of the growth-limit- Svetlana V. Kamzolova, Julia N. Lunina, Nadezhda N. ing component: the limitation with a nitrogen stimu- Stepanova, Ramil K. Allayarov and Igor G. Morgunov lated the excretion of citric and isocitric acids (6.8 and

Figure 1. JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS VOL. 37, SUPPLEMENT, 2019 83

Table 1. The effect of limiting factors on the growth and product synthesis in Y. lipolytica VKM Y-2373. 136. Structure and hybridization Limitation of growth by: properties of phosphorylguanidine Unlimited Nitrogen Thiamine Parameters growth (63 mg/L) (0.3 mg/L) oligonucleotides Protein (%) 50.75 21.2 27.50 Lipids (%) 10.12 10.30 12.37 Citric acid (g/L) 0 6.8 0.50 Alexander A. Lomzov, Victor M. Golyshev, Isocitric acid (g/L) 0 11.2 0.25 Evgeniya S. Dyudeeva, Maksim S. Kupryushkin and a-Ketoglutaric acid (g/L) 0 0 11.00 Dmitriy V. Pyshnyi Other organic acids (%) 0 3.5 10.0 Enzyme activite (U/mg protein) Institute of Chemical Biology and Fundamental Medicine SB RAS, Alcohol dehydrogenase 0.07 0.06 0.05 Novosibirsk State University, Novosibirsk 630090, Russia Aldehyde dehydrogenase 0.04 0.03 0.02 [email protected] Citrate synthase 1.71 1.80 1.65 Aconitate hydratase 0.39 0.40 0.38 NAD-isocitrate dehydrogenase 0.11 0.020 0.10 Oligonucleotide analogs have been a hot topic for the past NADP-isocitrate dehydrogenase 0.14 0.080 0.090 three decades because of their present and potential thera- Oxoglutarate dehydrogenase 0.11 0.070 0.010 peutic applications. Recently, a new type of oligonucleotide Isocitrate lyase 0.036 0.060 0.060 derivatives with a modified backbone was developed—phosphorylguanidine oligonucleotides (PGO) (Kuprushkin et al., 2014). PGOs are unique due to the possibility of synthesis via 11.2 g/L, respectively), while with a thiamine deficiency standard automated phosphoroamidite protocol using basic cells synthesize a-ketoglutaric acid (11.0 g/L). set of unmodified phosphoroamidites, allowing to introduce 3. the ratio of the enzymes responsible for the metabolism PG modification at any position of the oligomer sequence. of the excreted products changes. Under unlimited The high potential of the PGOs was shown in biosensors, in growth the activity of all enzymes involved in primary biological applications and in therapy (Lebedeva et al., 2015; oxidation of ethanol, Krebs and glyoxylate cycles was Dmitrienko et al., 2016)(Figure 1). sufficiently high. Under conditions of nitrogen limitation, In this work, we studied the solution structure of PGOs a high activities of citrate synthase and aconitate hydra- and their complementary complexes with unmodified DNA tase, necessary for the intensive synthesis of citric and or RNA as well as thermal stability of these complexes under isocitric acids, in distinction from the subsequent conditions of different ionic strength. There were no signifi- enzyme of Krebs cycle, NAD-isocitrate dehydrogenase, cant differences between the CD spectra of unmodified oli- were observed. Hence, these acids formed in Krebs cycle gonucleotides and PGOs as well as between spectra of can be presumably excreted from the yeast cell rather unmodified duplexes and duplexes with one fully modified than metabolized via the cycle. Similar data were previ- strand, indicating that PG modification insignificantly disturbs ously obtained for Y. lipolytica - producers of citric- and the structure of modified oligonucleotides and their duplexes isocitric acids from biodiesel-derived waste (Morgunov & with native DNA or RNA. Kamzolova, 2015). In contrast, in the case of thiamine At high ionic strength (1 M NaCl), the thermal stability of deficiency, the metabolic flux is blocked at the level of PGO containing duplexes decreases with each added PG the thiamine-dependent enzyme, 2-oxoglutarate modification. At low ionic strength (<100 mM NaCl), com- dehydrogenase, and a-ketoglutaric acid is excreted into plexes with one fully modified strand are more stable than the cultural medium rather than metabolized via the the native ones. Moreover, a fully modified PGO formed cycle. The continuous formation of excreted products highly stable duplexes with DNA or RNA even in deionized was ensured by the functioning of the glyoxylate cycle, with is confirmed by the high activities of citrate synthase, aconitate hydratase and isocitrate lyase which operate both in the glyoxylate cycle and in Krebs cycle.

References

Lazar, Z., Liu, N., & Stephanopoulos, G. (2018). Holistic approaches in lipid production by Yarrowia lipolytica. Trends in Biotechnology, 36(11), 1157–1170. doi:10.1016/j.tibtech.2018.06.007 Morgunov, I. G., & Kamzolova, S. V. (2015). 166 Metabolism of biodiesel- derived waste in the yeast Yarrowia lipolytica—producers of citric- and isocitric acids. Journal of Biomolecular Structure and Dynamics, 33(supp1), 108–109. doi:10.1080/07391102.2015.1032803 Weusthuis, R., Aarts, J., & Sanders, J. (2011). From biofuel to bioproduct: Is bioethanol a suitable fermentation feedstock for synthesis of bulk chemicals? Biofuels, Bioproducts and Biorefining, 5(5), 486–494. Zinjarde, S. S. (2014). Food-related applications of Yarrowia lipolytica. Food Chemistry, 152,1–10. doi:10.1016/j.foodchem.2013.11.117 Figure 1. Structure of the phosphorylguanidine modification. 84 BOOK OF ABSTRACTS. ALBANY 2019: THE 20TH CONVERSATION water. PGOs have sequence dependent hybridization proper- a simple sum of such influences. Moreover, in the certain ties and can be used for single mismatch discrimination like degree, the joint effect of two ligands on thermodynamic other native or modified oligonucleotides . characteristics of DNA helix-coil transition is possible to consider as the effect of ‘generalized’ ligand, which shows intrinsic properties for the latter (Nafisi, Saboury, Keramat, Neault, Funding & Tajmir-Riahi, 2007). It is also obvious that the long polymer This research has been supported by the Russian Science chain of DNA acts as one integer, since even at non-relevant Foundation (grant no. 18-14-00357). concentrations of two different ligands, when the regions between these compounds outlying from each other are filled by molecules, it behaves not as in the case of separate References binding at the same concentrations. This fact may elucidate Dmitrienko, E., Naumova, O., Fomin, B., Kupryushkin, M., Volkova, A., some questions, connected to DNA functioning regulation Amirkhanov, N., … Pyshnyi, D. (2016). Surface modification of SOI- (initiation, inhibition, expression of genes) in cell. FET sensors for label-free and specific detection of short RNA analyte. Nonetheless, the obtained data do not put in a claim for Nanomedicine (London), 11(16), 2073–2082. absoluteness, since it cannot be excluded the additive effect Kupryushkin, M. S., Pyshnyi, D. V., & Stetsenko, D. A. (2014). Phosphoryl guanidines: A new type of nucleic acid analogues. Acta Naturae, 6(4), of two different ligands on parameters of DNA conform- 116–118. ational transitions, which may also have a certain biological Lebedeva, N. A., Anarbaev, R. O., Kupryushkin, M. S., Rechkunova, N. I., value for DNA functioning regulation. Pyshnyi, D. V., Stetsenko, D. A., & Lavrik, O. I. (2015). Design of a new fluorescent oligonucleotide-based assay for a highly specific real-time detection of apurinic/apyrimidinic site cleavage by tyrosyl-DNA References phosphodiesterase 1. Bioconjugate Chemistry, 26(10), 2046–2053. Karapetian, A. T., Grigoryan, Z. A., Mamasakhlisov, Y. S., Minasyants, M. V., & Vardevanyan, P. O. (2016). Theoretical treatment of helix-coil transition of complexes DNA with two different ligands having differ- 137. Study of three-component ent binding parameters. Journal of Biomolecular Structure and Dynamics, 34(1), 201–202. doi: 10.1080/07391102.2015.1010584. system, consisted of DNA complexes Nafisi, S., Saboury, A. A., Keramat, N., Neault, J.-F., & Tajmir-Riahi, H.-A. with ligands of different type (2007). Stability and structural features of DNA intercalation with ethidium bromide, acridine orange and methylene blue. Journal of Molecular Structure, 827(1-3), 35–43. Ara P. Antonyan, Marine A. Parsadanyan, Narek H. Shahnazaryan and Poghos O. Vardevanyan Department of Biophysics, Yerevan State University, Yerevan, Armenia [email protected] 138. The effect of porphyrins configuration on the hydrodynamic The joint binding of intercalators EtBr and AO, intercalator EtBr and semi-intercalator MB, intercalator EtBr and groove- behavior of DNA solutions binding compound Hoechst 33258 (H33258) with DNA has been studied using UV melting method at ionic strengths of Vigen G. Barkhudaryan and Gayane V. Ananyan the solution 2 and 20 mM. The values of melting temperature Department of Molecular Physics, Yerevan State University, 1 Alex

(Tm) and melting interval width (DT) of DNA-ligand com- Manoogian St. 0025, Yerevan, Armenia [email protected] plexes were obtained and based on them the values of 2 The present work summarizes possibilities of a viscometry changes of these parameters—(1/Tm) and (DT/Tm ) on ligand concentrations were determined. The obtained results indi- method for studying the conformational changes of DNA at cate that at the joint binding of two different ligands with interaction with porphyrins. For elucidating the influence of DNA, the solution ionic strength has a determining effect of porphyrin structure by varying the metal center, peripheral on their binding modes. Meanwhile, the joint interaction of substituents and their positions in pyridylic ring on binding intercalator and non-intercalator or two intercalators with mode and intensity of interaction with DNA water soluble DNA is not a simple sum of their separate binding. cationic meso-tetra-(3N- and 4N-hydroxyethylpyridyl) Moreover, at their joint interaction with DNA a competition (Barkhudaryan & Ananyan, 2014, 2016), (3N- and 4N-allylpyr- between them emerges. These results also indicate that the idyl) (Barkhudaryan & Ananyan, 2017, 2018) porphyrins and joint interaction of two different multimodal ligands with their metal complexes with Cu and Co were studied via visc- DNA is not a result of binding by at least two modes of one ometry and UV–vis absorption spectroscopy. It was shown type of ligand (Karapetian, Grigoryan, Mamasakhlisov, that planar porphyrins interact with DNA by intercalation Minasyants, & Vardevanyan 2016). mode (Figure 1a, b), excepting CuTAllPyP4 which interacts Results of the experimental studies of DNA complexes with DNA by partial intercalation mod (Barkhudaryan & with two different ligands reveal that the immediate binding Ananyan, 2018). Moreover, the planar porphyrins with allyl- of any type of the ligand with DNA leaves a separate effect pyridyl peripheral substituents interact with DNA consider- on its thermodynamic characteristics, though the joint ably more intensively than with hydroxyethylpyridyl impact of two (and, probably, more) different ligands is not substituents. Comparison of different locations of peripheral JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS VOL. 37, SUPPLEMENT, 2019 85

Figure 1.

substituents on pyridylic rings leads to the conclusion that The study and understanding of the mechanisms of radiation the H2THOEtPyP3 and H2TALPyP3 and their metallocom- causing damage to DNA is one of the important problems in plexes bind to DNA more preferably than H2THOEtPyP4, the development of new cancer therapies and effective radio- H2TALPyP4 and their metallocomplexes. This fact can be sensitizers. The fast electrons directly ionize the DNA molecule, explained by the preferred arrangement of 3N-porphyrins on causing damage. These include single-strand breaks and dou- the DNA helix compared to 4N-porphyrins. The change of ble-strand breaks, DNA–DNA or DNA–protein cross-links. The chemical structure of peripheral radicals and their positions usage of the porphyrin-based Photodynamic Therapy (PDT) in on pyridylic ring of porphyrins does not absolutely affect on conjunction with electron beam radiation therapy can be one the mechanism of interaction of outside binders such as Co- of the effective treatment methods for cancer. Porphyrins have porphyrins (Figure 1c). The viscometry and spectrophotomet- attracted the attention of researchers globally for application ric measurements are in good agreement (Barkhudaryan & as photosensitizing agents in medicine. However, studies of Ananyan, 2014, 2016, 2017, 2018). the radiation damage at the molecular level in the presence of such PDT agents and anticancer drugs like porphyrins are still References necessary. The goal of our investigations was to examine DNA damage in the presence of different amounts of Cu containing Barkhudaryan, V. G., Ananyan, G. V., Dalyan, Y. B., & Haroutiun, S. G. (2014). Development of viscometric methods for studying the inter- water soluble cationic meso-tetra-(4N-oxyethylpyridyl) porphy- action of various porphyrins with DNA. Part I. Journal of Porphyrins rin induced by 3 and 4 MeV electron radiation depending on and Phthalocyanines, 18(07), 594–599. radiation doses in vitro. The samples with different relative Barkhudaryan, V. G., & Ananyan, G. V. (2016). Development of viscomet- concentrations of porphyrins per base pair were irradiated by ric methods for studying the interaction of various porphyrins with DNA. Part II. Journal of Porphyrins and Phthalocyanines, 20(07), the electron beam. After the irradiation of samples, the melting 766–772. curves (the dependence of percentage of denaturized DNA on Barkhudaryan, V. G., & Ananyan, G. V. (2017). Development of viscomet- temperature) of investigated complexes were obtained. As the ric methods for studying the interaction of various porphyrins with melting temperature of DNA is sensitive to double helix stabil- DNA. Part III. Journal of Porphyrins and Phthalocyanines, 21(02), 110–115. ity, it can be used as an indicator of strand breaks of DNA mol- Barkhudaryan, V. G., & Ananyan, G. V. (2018). Development of viscomet- ecules after radiation. Our results indicate that at the radiation ric methods for studying the interaction of various porphyrins with dose 1 Gy when the relative concentration of porphyrins DNA. Part IV. Journal of Porphyrins and Phthalocyanines, 22(11), equals 0.01 and 0.02 per DNA base pair, the radiation effect on 1022–1029. the DNA structure is higher than the stabilizing effect of porphyrins. At the higher value of the relative concentration of porphyrins (0.04 and 0.06) the DNA stabilizing effect is more 139. The influence of CuTOEPyP4 significant than DNA damage produced by an electron beam. porphyrin on DNA damage induced On the contrary at the radiation dose equal to 2 Gy the radiation effect exceeds the porphyrins stabilizing effect on by high energy electron DNA at all examined relative concentrations. beam radiation

L.R. Aloyan, Y.B. Dalyan and S.G Haroutiunian Acknowledgments Yerevan State University, Al. Manoogian 1, Yerevan 0025, Armenia The experimental studies were conducted within the framework of RA [email protected] MES Committee of Science Projects no. 18T-1F055. 86 BOOK OF ABSTRACTS. ALBANY 2019: THE 20TH CONVERSATION

References Studies have demonstrated that viscometry and spectro- photometric measurements are in good agreement. Ananyan, G., Avetisyan, A., Aloyan, L., & Dalyan, Y. (2011). The stability of DNA-porphyrin complexes in the presence of Mn(II) ions. Biophysical Chemistry, 156(1), 96–101. References Dalyan, Y. B., Haroutiunian, S. G., Ananyan, G. V., Vardanyan, V. I., Yu.Lando, D., Madakyan, V. N., … Benight, A. S. (2001). Interaction of Karapetyan, N. H., Torosyan, A. L., Malakyan, M. H., Bajinyan, S. A., & meso-tetra-(4-N-oxyethylpyridyl) porphyrin, its 3-N analog and their Haroutiunian, S. G. (2016). Investigation of irradiated rats DNA in the metallocomplexes with duplex DNA. Journal of Biomolecular Structure presence of Cu(II) chelates of amino acids Schiff bases. Journal of and Dynamics, 18(5), 677–687. [10.1080/07391102.2001.10506698] Biomolecular Structure and Dynamics, 34(1), 177–183. O’Connor, A. E., Gallagher, W. M., & Byrne, A. T. (2009). Porphyrin and nonporphyrin photosensitizers in oncology: Preclinical and clinical advances in photodynamic therapy. Photochemistry and Photobiology, 85(5), 1053 Swiderek, P. (2006). Fundamental processes in radiation damage of DNA. 141. Changes in the stability and Angewandte Chemie International Edition, 45(25), 4056. Vrouenraets, M. B., Visser, G. W., Snow, G. B., & van Dongen, G. A. (2003). conductivity of BLM in the presence Basic principles, applications in oncology and improved selectivity of of zinc oxide nanoparticles photodynamic therapy.. Anticancer Research, 23(1B), 505 Wilson, B. C. (2002). Photodynamic therapy for cancer: Principles. Anahit L. Torosyan, Gayane V. Ananyan and Canadian Journal of Gastroenterology Journal Canadien de ¼ Gastroenterologie, 16(6), 393 Valeri B. Arakelyan Department of Molecular Physics, Yerevan State University, 1 Alex Manoogian St. 0025, Yerevan, Armenia [email protected]

Currently, nanoparticles are intensively implanted in almost 140. Molecular alterations in DNA all areas of scientific and practical human activity, although under UV-irradiation there are fears that they may damage biological systems (Limbach et al, 2005). The effect of nanoparticles beginning with their primary interaction with biological membranes. Gayane V. Ananyan, Nelli H. Karapetyan and However, since biological membranes display a very complex Vigen G. Barkhudaryan composition in terms of lipids and proteins, it seems appro- Department of Molecular Physics, Yerevan State University, 1 Alex priate to investigate the effect of nanoparticles on a bilayer Manoogian St. 0025, Yerevan, Armenia [email protected] lipid membrane (BLM), which mimics well the lipid bilayer of a biological membrane. This work is devoted to the study of The ionizing radiation of matter produces a large number of the effect of zinc oxide nanoparticles (ZnO), with an average ions, radicals, which can subsequently cause both physical size of 50 nm, on the stability and conductivity of BLM. The and chemical modification in the biological media. Moreover experiments were performed on BLM obtained from a mix- the irradiation can induce strand break formation in double- ture of 1,2-dipalmitoyl-sn-glycero-3-[phospho-1-serine](DPPS) stranded supercoiled DNA. and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) of In present work the influence of UV-irradiation on the the company Avanti Polar Lipids in a ratio 1:1. Voltage, in structural transformations of DNA in buffer solutions was the range of 0.20–0.55 V, was applied to the BLM using studied using UV-absorption spectroscopy and viscometry. chlorine-silver electrodes connected to the ADC and con- Detection of DNA structural changes such as single trolled by a computer. Experimental points were obtained stranded and double stranded breaks under UV-irradiation depicting the dependence of the average BLM lifetime on were characterized by their melting curves, in which tem- the potential in the absence and presence of zinc oxide perature-dependent spectral measurements were carried nanoparticles in solution. The parameters characterizing the out in buffered solutions for different exposure time/dose BLM stability were determined from fitting the experimental of irradiation. The changes of melting temperature (Tm)and data with a theoretical curve for the dependence of the aver- interval of helix-coil transition (DT) at different exposure age lifetime of the BLM on the potential (Pastushenko, times indicate damages to the secondary structure of DNA Chizmadzev, & Arakelyan, 1979). This is the BLM tension, the (Karapetyan, Torosyan, Malakyan, Bajinyan, & Haroutiunian, linear tension of the pore edge in the BLM, as well as the 2016). The decreasing of melting temperature testifies to parameter which is the product of the number of pores on DNA destabilization, which could occur due to base modifica- the BLM and the pore diffusion coefficient in radius space. It tions, strand breaks or breaks of hydrogen bonds between was shown that ZnO nanoparticles increase BLM stability in the DNA strands during irradiation. The increases of damaged an electric field. With an increase in the concentration of sites create favorable conditions for helix-coil transition. nanoparticles, the stability of BLM also increases. It was Moreover, an increase in DT (degree of heterogeneity) also shown that ZnO nanoparticles increase the value of the lin- indicates the presence of defective sites. The relative viscosity ear tension coefficient of the pore edge, which is formed in of the UV-irradiated DNA solution decreased with increasing BLM, which also leads to an increase in the stability of BLM irradiation time, which indicates a reduction of macromole- in an electric field. It was also shown that ZnO nanoparticles cules size. lead to a decrease in the conductivity of BLM. JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS VOL. 37, SUPPLEMENT, 2019 87

References Studied indexes may indicate possible health implications of such exposure and should be taken into account. Limbach, L. K., Li, Y., Grass, R. N., Brunner, T. J., Hintermann, M. A., Muller, M. … , Stark, W.J. (2005). Oxide Nanoparticle Uptake in Human Lung Fibroblasts: Effects of Particle Size, Agglomeration, and References Diffusion at Low Concentrations. Environmental Science & Technology, v39(23), 9370–9376. Ramundo-Orlando, A. (2010). Effects of millimeter waves on cell mem- Pastushenko, V. F., Chizmadzev, Y. A., & Arakelyan, V. B. (1979). Electric brane—a brief review. Journal of Infrared, Millimeter, and Terahertz Breakdown of Bilayer Lipid Membranes II. Calculation of the mem- Waves, 31(12), 1400–1411. brane lifetime in the steady-state diffusion approximation. Bioelectrochemistry and Bioenergetics, v. 6 p, 37–52.

143. Nitrogen limitation triggers lipid 142. Influence of low-intensity synthesis of Torulaspora globose on millimeter waves on lipid various carbon substrates peroxidation and antioxidant Nadezhda N. Stepanova, Svetlana V. Kamzolova and enzymes activity in male Wistar rats Igor G. Morgunov Federal Research Center Pushchino Center for Biological Research Gayane H. Poghosyan, Marieta S. Mikaelyan and of the Russian Academy of Sciences, G.K. Skryabin Institute of Anahit V. Nerkararyan Biochemistry and Physiology of Microorganisms RAS, Pushchino, Department of Biophysics, Yerevan State University, Yerevan, Moscow region 142290, Russia [email protected] Armenia [email protected] It is well known that the nature of the growth-limiting com- Electromagnetic waves of millimeter range (MM EMW) corres- ponent has a significant effect on lipid synthesis in microor- pond to the extremely-high-frequency (EHF) electromagnetic ganisms with a typical diphase process. The limitation of irradiation (EMI) band (f 30–300 GHz) always exist in sur- yeast growth by mineral components of nutrition medium ¼ rounding environment and intensively influence living organ- (for example, nitrogen) stimulates the intensive storage of isms. It has been suggested that initial cellular event affected lipids by the lipid-producer yeast, while with a carbon source by exposure to EMI might be the increase of free radical deficiency, microorganisms synthesize a negligible amount of level, which may enhance lipid peroxidation (LPO) and anti- lipids (Dourou et al., 2018; Lazar et al., 2018). oxidant enzymes activity (Ramundo-Orlando, 2010). The The purpose of this work was to study the effect of the investigation concerned with the effect of low-intensity nature of the growth-limiting component on biomass com- MMW on whole body exposure of male Wistar rats. Twenty position and lipid synthesis of yeast Torulaspora globose rats were divided into two groups: sham-exposed (control) VKPMY-953, cultivated on ethanol or glucose. and experimental (10 animals each). Animals were exposed The selected strain T. globose VKPM Y-953 has a unique with 50.3 GHz frequency EMI (power flux density 64 mWt/ feature—the ability to intensively synthesize lipids in parallel cm2) for 1 h/day, for 5 days (day after day). As a source of with the growth of the culture, unlike the classical producers monochromatic EMI EHF generator G4-141 type with work- of lipids, in which intensive synthesis of lipids occurs after ing interval of 37.50–53.57 GHz (State Scientific-Production the completion of active growth. Enterprise Istok’, Russia) was used. The whole body specific As seen from Table 1, in the medium with ethanol as a absorption rate (SAR) was 0.05 W/kg. Exposure took place in carbon source under conditions of limitation of T. globose a ventilated plexiglas cage, where rats could move inside growth by nitrogen, a lipid synthesis was higher (43.8% of given space. After completion of each exposure period, rats the biomass) than with an ethanol deficiency (17.8% of the were sacrificed to analyze malondialdehyde (MDA)-rate and biomass). Conversely, with ethanol deficiency, the biomass enzyme activity (catalase and glutathione peroxidase) in was characterized by a higher protein content (23.4% of bio- brain, liver, heart and skeletal muscle. Our findings indicate a mass) and a biomass yield from the consumed substrate (YX/ significant increase (p < 0.05) in level of MDA and CAT activ- S) (54.4%) than with nitrogen limitation (16.7% of biomass ity in brain of EMI-exposed group of animals as compared to and 31.1%, respectively); under these conditions, the lipid sham-exposed, depending on EMI exposure duration. At the yield (YL/S) was higher by 27% than with a nitrogen defi- same time, we recorded slightly elevated MDA-rate in experi- ciency. It is considered that lipids synthesized by microorgan- mental animals liver in comparison with the control group. isms under deficiency of carbon substrate perform mainly a The MDA values concerning heart and skeletal muscles functional role in metabolism (take part in transport proc- remained at the same levels in the EMI- exposed rats and esses, regulate the activity of enzymatic systems, etc.). The did not differ significantly from control. Data show a signifi- purpose of growth-coupled lipid synthesis in T. globose is still cant decrease of GPX activity in experimental rats brain unknown. It is possible that the studied strain is character- (p < 0.001) and liver (p < 0.05), whereas CAT activity signifi- ized by a higher proportion of functional lipids than other cantly increased in the same organs compared to control. types of yeast. 88 BOOK OF ABSTRACTS. ALBANY 2019: THE 20TH CONVERSATION

Table 1. The effect of the nature of the growth-limiting component on physiological indices of T. globose. 144. Zinc supplementation stimulates Nitrogen Ethanol Parameters limitation limitation lipid production in Torulaspora Lipids (% of biomass) 43.8 27.8 globose yeast Protein ((% of biomass) 16.7 23.4 1 Specific rate of lipid synthesis (hÀ ) 0.04 0.03 a a Specific rate of protein synthesis (h-1) 0.02 0.05 Svetlana V. Kamzolova , Nadezda N. Stepanova , b a Biomass yield by mass (YX/S) (g/g) 31.1 54.4 Grigorii I. Morgunov , Ramil K. Allayarov and Lipid yield by mass (YL/S) (g/g) 13.6 17.3 Igor G. Morgunova Fatty acids (% of lipids) a C14 0.2 0.1 Federal Research Center Pushchino Center for Biological Research C14:1 Trace Trace of the Russian Academy of Sciences, G.K. Skryabin Institute of C16 14.5 15.5 Biochemistry and Physiology of Microorganisms RAS, Pushchino, b C16:1 26.2 29.2 Moscow region, 142290, Russia; Peoples’ Friendship University of C18 2.3 1.3 Russia (RUDN University), Miklukho-Maklaya str. 6, Moscow, P.O. C18:1 51.5 41.5 Box 117198, Russia [email protected] 18:2 5.3 12.4 Unsaturated fatty acids (%) 83.0 83.0 The data represent the means of four measurements. Standard deviation did The practical importance of microbial lipids causes great inter- not exceed 10%. est of researchers to the problem of their synthesis and regulation. The experimental approaches in which the cell growth and the direction of various biosynthetic processes can be con- The specific rates of lipid and protein synthesis were cal- trolled are well known. The limitation of cell growth by nutri- culated per the unit of a lipid-free biomass fraction. The data tion elements is one of the ways to shift metabolism into presented in Table 1 show that the specific rate of lipid syn- directional synthesis of lipids (Athenaki et al., 2018). thesis exceeded the specific rate of protein synthesis by 2 At present, the ethanol is considered to be a promising carbon source in various biotechnological processes, because it times with a nitrogen limitation and it was reduced by 1.7 can be produced from sugarcane, beet, corn, lignocellulose times with an ethanol deficiency. Thus, the lipid accumula- and other renewable materials. It does not contain harmful tion in the cells of T. globose depends on the ratio between impurities; it is well assimilated by yeast and dissolves in water the rates of lipid and protein synthesis. in any proportions. Several companies in the US and As seen from Table 1, the nature of the growth-limiting Switzerland have created food products based on microbial component had no significant effect on the fatty acid (FA) biomass produced from ethanol (Weusthuis et al., 2011). It composition of lipids in T. globose. The predominant fatty should be noted that the large-scale production of lipids from acids included the unsaturated acids —oleic acid (C ) 18:1 ethanol is still limited by the lack of basic knowledge about fer- (41.5–51.5% of total FA), palmitoleic acid (C ) 16:1 mentation conditions conducive to product overproduction. (26.2–29.2% of total FA) and palmitic acid (C ) 16 Analysis of the literature data shows the exceptional role of (14.5–15.5% of total FA). zinc in the regulation of cell metabolism and physiology. Lipogenesis of T. globose cultivated on glucose had the Besides acting as a cofactor for many enzymes (dehydrogen- distinctive features: the lipid accumulation was reduced by ases, aldolases, polymerases and proteases) (Dedyukhina & 2.7 times in comparison with cells, grown on ethanol; the Eroshin, 1991; Tomaszewska et al., 2014; Kamzolova et al., maximum amount of lipids (16% of the biomass) was synthe- 2018) zinc is also required for the structural stability of zinc fin- sized with a nitrogen deficiency, while with a glucose defi- ger proteins, many of which exert important controls on cellu- ciency, the biomass yield from the consumed substrate (Y ) X/S lar metabolic processes (Hamed & Arya, 2016). was maximum (21.8%); the fraction of oleic acid (C ) in the 18:1 The purpose of this work was to study the effect of vari- lipids was increased up to 63% (of the total amount of FA). ous zinc concentrations on growth and lipid synthesis of Thus, nitrogen limitation triggers lipid synthesis of T. glo- yeast Torulaspora globosa VKPMY-953 using a chemostate bose on various carbon substrates. 1 regimen (D 0.05 hÀ ). ¼ As seen from Table 1, the limitation of the growth of T. glo- Funding bosa by zinc (0.001 mg/L) sharply reduced the biomass yield (YX/S) (in 2.7 times), lipid content (in 3 times) and lipid yield (in The reported research was funded by RFBR (project @ 18- 3.6 times) as compare to high Zn concentration (2.8 mg/L). At 38-00794_mol_a). the same time, the protein content was maximum (37.96% from dry biomass) under condition of Zn deficiency. – References As seen from Table 1, the lipids contained 14 18 acids with a prevalence of palmitic (C16:0), palmitoleic (C16:1) and Dourou, M., Aggeli, D., Papanikolaou, S., & Aggelis, G. (2018). Critical oleic (C18:1) acids. steps in carbon metabolism affecting lipid accumulation and their In this study, we focused on the palmitoleic acid (C16:1), regulation in oleaginous microorganisms. Applied Microbiology and which is considered as a novel lipokine (Athenaki et al., 2018). Biotechnology, 102(6), 2509–2523. doi: 10.1007/s00253-018-8813-z. Lazar, Z., Liu, N., & Stephanopoulos, G. (2018). Holistic approaches in As seen from Table 1, a large amount of palmitoleic acid lipid production by Yarrowia lipolytica. Trends in Biotechnology, 36(11), (37.5–41.7% of lipids) was synthesized at zinc concentration in 1157–1170. doi:10.1016/j.tibtech.2018.06.007 the range from 0.1 to 2.8 mg/L. JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS VOL. 37, SUPPLEMENT, 2019 89

Table 1. Effect of zinc amount on biomass yield and the amount of lipids in References T. globosa. Zinc content (mg/L) Athenaki, M., Gardeli, C., Diamantopoulou, P., Tchakouteu, S. S., Sarris, D., Philippoussis, A., & Papanikolaou, S. (2018). Lipids from yeasts and Parameters 0.001 0.1 0.28 2.8 fungi: Physiology, production and analytical considerations. Journal of Biomass yield by mass (YX/S) (g/g) 0.21 0.57 0.57 0.56 Applied Microbiology, 124(2), 336–367. doi: 10.1111/jam.13633. Lipids (% of biomass) 9.2 20.0 26.4 27.8 Dedyukhina, E. G., & Eroshin, V. K. (1991). Essential metal ions in the con- Lipid yield by mass (YL/S) (g/g) 0.048 0.112 0.141 0.172 trol of microbial metabolism. Process Biochemistry, 26(1), 31–37. Protein (% of biomass) 37.96 26.82 20.23 23.03 10.1139/cjm-2018-0050 Fatty acids (% of lipids) Hamed, M. Y., & Arya, G. (2016). Zinc finger protein binding to DNA: An C 0.8 2.1 3.3 2.5 14 energy perspective using molecular dynamics simulation and free C14:1 0.7 0.6 1.0 0.4 energy calculations on mutants of both zinc finger domains and their C16 16.5 18.6 23.8 24.6 C16:1 29.2 41.7 40.7 37.5 specific DNA bases. Journal of Biomolecular Structure and Dynamics, C18 3.3 2.1 1.2 2.5 34(5), 919–934. doi: 10.1080/07391102.2015.1068224. C18:1 42.2 24.9 30.0 32.5 Kamzolova, S. V., Shamin, R. V., Stepanova, N. N., Morgunov, G. I., Lunina, 18:2 7.3 0 0 0 J. N., Allayarov, R. K., … Morgunov, I. G. (2018). Fermentation condi- 16:1/C16 (D-9 desaturase) 1.87 2.24 1.71 1.52 tions and media optimization for isocitric acid production from ethanol by Yarrowia lipolytica. BioMed Research International, 2543210, The activity of D-9 desaturase involved in the conversion 2018, 1. doi:10.1155/2018/2543210 Tomaszewska, L., Rymowicz, W., & Rywinska, A. (2014). Mineral sup- of palmitic into palmitoleic acid was evaluated by measuring plementation increases erythrose reductase activity in erythritol the ratio 16:1/16. In our experiments, the ratio 16:1/16 biosynthesis from glycerol by Yarrowia lipolytica. Applied completely correlated with a change in the palmitoleic acid Biochemistry and Biotechnology, 172(6), 3069–3078. doi: 10.1007/ content of lipids (Table 1). s12010-014-0745-1. Weusthuis, R., Aarts, J., & Sanders, J. (2011). From biofuel to bioproduct: Is bioethanol a suitable fermentation feedstock for synthesis of bulk Funding chemicals? Biofuels, Bioproducts and Biorefining, 5(5), 486–494. Zhao, X. Q., & Bai, F. W. (2012). Zinc and yeast stress tolerance: The reported research was funded by RFBR (project no. 18- Micronutrient plays a big role. Journal of Biotechnology, 158(4), 38-00794_mol_a). 176–183. doi: 10.1016/j.jbiotec.2011.06.038. JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS https://doi.org/10.1080/07391102.2019.1604468

Author Index

Abolghasemi, M. 30 Brehove, M. S. 44 Friesner, R. A. 20 Abrahmsen, L. 39 Buragohain, M. 13 Gamazo, P. A. 18 Afek, A. 80 Bussi, G. 50 Garipova, M. I. 43 Agajanian, S. 24 Caetano-Anolles, G. 46 Ge, P. 2 Alexander, S. P. 34 Calvopina,~ K. 16 Gevorgyan, E. S. 21, 22, 56, 71 Alexandrov, B. 47 Cantero, J. 18 Gevorgyan, E. 52, 57 Al-Hashimi, H. M. 80 Carr, C. E. 74 Ghosh, I. 28 Allayarov, R. K. 82, 88 Case, B. C. 78 Gnanakaran, S. 47 Aloyan, L.R. 85 Chandra, N. 15 Golfetto, O. 44 Alvareda Migliaro, E. M. 18 Chandra, R. 12, 20, 27, 31, 35, 60 Golovenko, D. 37, 38, 39 Ananyan, G. V. 84, 86 Chaudhary, C. 27 Golyshev, V. M. 83 Andrianov, A. M. 22, 23 Chazin, W. J. 63 Gonen, T. 3 Antonyan, A. P. 75, 77, 84 Chen, A. A. 74 Gordan, R. M. 80 Apukhtina, V. S. 76 Chen, R. 6 Grintsevich, E. E. 2 Arakelyan, V. B. 79, 86 Chiranjeevi, P. 34, 40 Gross, M. L. 8 Arjmand, B. 36 Chiu, T.-P. 81 Gruebele, M. 51 Arora, R. 29, 33 Chu, B. 79 Gu, C. 45 Artsruni, I. G. 56, 71 Chugh, H. 35 Gupta, M. K. 42 Artsruni, I. 52, 57 Clark, S. 9 Guttula, P. K. 42 Asatryan, A. L. 56, 71 Coalson, R. D. 45 Hakobyan, N. R. 21 Asatryan, A. 52, 57, 77 Cojocaru, H. V. 53 Hakobyan, N. 22 Astl, L. 24 Colina, R. 18 Haran, T. E. 37, 38 Avagyan, G. A. 21 Coutsias, E. 48 Haroutiunian, S.G 85 Avison, M. B. 16 Dalyan, Y.B. 85 Harvey, S. C. 8 Bairagya, H. R. 66 Das, S. 2 Hema, K. 32, 42 Balashova, V. N. 81 Dantas Machado, A. C. 81 Hengartner, N. 47 Bandyopadhyay, D. 64 DasGupta, D. 17 Hogberg,€ B. 12 Banas, P. 48, 50 Dasgupta, S. 17 Hinchliffe, P. 16 Barbar, E. 9 Davoodi, M. 36 Hingorani, M. M. 78 Barkhudaryan, V. G. 84, 86 Degtjarik, O. 38, 39 Hirvonen, V. H.A. 16 Barlow, J. 61 Deng, L. 72 Hon, J. 6 Barton, J. K. 63 Dey, S. 4 Hong Zhou, Z. 2 Barton, J. 63 Diskin-Posner, Y. 39 Honig, B. 44 Basu, A. 7 Dong, Y. 6 Hosseini, E. 36 Beattie, N. R. 8 Dowson, C. 14 Hovhannisyan, A. G. 21, 22 Belfort, G. 19 Dunker, K. 9 Ignatov, M. 48 Bellini, D. 14 Dyudeeva, E. S. 83 Issar, U. 29, 33 Best, R. B. 50 Fang, R. 5, 6 Iyer, M. S. 67 Beveridge, D. L 6 Farkhutdinov, R. G. 43 Janecek, M. 48 Bhadra, K. 21 Fawzi, N. L. 10 Jani, V. 73 Bhat, R. 17 Fayez Aziz, M. 46 Jara, K. 9 Bhatnagar, A. 64 Fedyaev, V. V. 43 Jarosz, D. F. 10 Bhattacherjee, A. 75 Feig, M. 51 Jayaraj, A. 17 Bischoff, N. 24 Fierz, B. 55 Jayaram, B. 17 Bishop, T. C. 55 Finley, D. 6 Johari, S. 13 Biswas, S. 44 Fogg, J. M. 5 Joshi, A. G. 67 Blacklock, K. 24 Frank, J. 1 Joshi, R. 69, 73 Bondos, S. E. 10 Freudenreich, C. H. 62 Jovanovic-Talisman, T. 44

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Jurecka, P. 49, 50 Mlynsky, V. 50 Safieh, J. 37 Kadirvelraj, R. 8 Mondal, A. 75 Sahakyan, V. G. 77 Kakkar, R. 29, 33 Morgunov, G. I. 88 Salay, L. E. 63 Kamzolova, S. V. 67, 82, 87, 88 Morgunov, I. G. 67, 82, 87, 88 Salimraj, R. 16 Kang, H. 6 Morozov, V. 77 Schaper Bergman, E. T. 8 Karapetian, A. T. 75 Mothay, D. 70 Schiffer, C. 25 Karapetyan, A. A. 71 Mothay, K.V. D. 18 Seeman, N. C. 12 Karapetyan, A. T. 80 Mu, Y. 78 Sengupta, J. 4 Karapetyan, N. H. 86 Mughal, F. 46 Serindag, K. 6 Kasahara, K. 51 Mukhaelyan, Z. H. 68 Shahinyan, M. A. 77, 80 Kaushal, S. 62 Mukherjee, S. 17 Shahnazaryan, N. H. 84 Kaushik, A. C. 8 Mukhopadhyay, B.P. 17 Shakked, Z. 38, 39 Kaushik, R. 17 Mulholland, A. J. 16 Shalev-Benami, M. 2 Ke, Y. 11 Munikumar, M. 42 Shandilya, A. 17 Keul, N. D. 8 Munsamy, G. 37 Sharma, N. 69 Kirschner, M. W. 6 Myers, C. A. 74 Shekhar, S. 17 Klimov, D. K. 25 Naik, V. U. 40 Shekhar, V. 17 Kompanichenko, V. N. 69 Narayanan, S. 18 Shernyukov, A. V. 76 Kornoushenko, Y. V. 22, 23 Nawrocki, G. 51 Shevelev, G. Y. 76 Kouzminova, E. A. 61 Nerkararyan, A. V. 87 Shi, H. 80 Kozakov, D. 48 Newman, H. 14 Shigapova, A. I. 43 Krepl, M. 50 Nikolaev, G. I. 22, 23 Shirbandi, K. 36 Kuhrov€ a, P. 48, 50 Norouzi, D. 57 Singh, A. 12, 17 Kumar, K. S. 29, 34, 40, 42 Olotu, F. A. 37 Singh, S. 60 Kumar, N. 20, 31 Oruganty, K. 8 Smith, A. K. 25 Kupryushkin, M. S. 76, 83 Oshima, H. 51 Soliman, M. E. 37 Kuzminov, A. 61 Otyepka, M. 48, 49, 50 Sonavane, U. B. 69 Langley, D. R. 6 Ouyang, Q. 6 Sonavane, U. 73 Lennon, K. M. 44 Oztug Durer, Z. A. 2 Soni, A. 17 Li, Y. 72 Pakala, S. 29 Sood, D. 27 Li, Z. 55 Paliwal, S. 64 Sotnikova, J. M. 43 Lim, R. Y. H. 46 Panchenko, A. 53 Sowdhamini, R. 67 Liu, S.-Q. 71, 72 Panneerselvan, N. 33 Spencer, J. 16 Lloyd, A. 14 Parsadanyan, M. A. 75, 77, 79, 84 Sponer, J. 48, 49, 50 Lomzov, A. A. 76, 83 Pasala, C. 29 Srinivasan, K. 4 Lopez, C. A 47 Paukstelis, P. J. 79 Stepanova, N. N. 82, 87, 88 Lu, C. 26 Petrov, V. V. 65 Su, X. A. 62 Lu, Y. 5, 6 Phillips, R. S. 8 Subbarao, N. 28 Luan, B. 6 Pielak, G. J. 52 Subramaniam, S. 1 Lunina, J. N. 82 Poghosyan, G. H. 68, 87 Sugita, Y. 51 Ma, W. 6 Pollack, L. 54 Sun, R. 55 MacArdle, S. G. 63 Powers, E. T. 51 Swargam, S. 32, 42 Madendran, R. 33 Pradeep, N. 32, 42 Tao, Y. 71, 72 Madhukar, G. 28 Pyshnyi, D. V. 76, 83 Thayer, K. M. 6 Madhulitha, N. R. 29, 34, 40 Rahim, F. 30, 36 Timp, W. 59 Mamasakhlisov, Y. 77 Ramesh 18 Tomar, V. 31 Mansbach, R. 47 Ramesh, K. V. 70 Tomashevsky, A. A. 65 Mao, Y. 6 Rangadurai, A. 80 Tonoyan, Sh. 77 Marky, L. A. 73, 74 Rangunathan, M. 33 Tooke, C. L. 16 Matinyan, K. S. 56, 71 Rao, S. 81 Torosyan, A. L. 86 Matinyan, K. 52, 57 Re, S. 51 Torosyan, M. A. 80 Maxwell, A. 14 Reardon, P. 9 Tort, F. L. 18 McDonald, W. E. 8 Reiling-Steffensmeier, C. 73 Tuzikov, A. V. 22 Mikaelyan, M. S. 87 Reisler, E. 2 Umamaheswari, A. 29, 32, 34, 40, 42 Minasyants, M. V. 75 Rohs, R. 81 Usanov, S. A. 23 Mirney, L. 59 Rosenberg, S. M. 15 van der Kamp, M. W. 16 Mitchell, J. 48 Rozenberg, H. 38, 39 Vardevanyan, P. O. 77, 79, 84 Mittal, A. 59 Ruggerone, P. 47 Vashishtha, A. 13 92 BOOK OF ABSTRACTS. ALBANY 2019: THE 20TH CONVERSATION

Veisi, A. 30 Yan, H. 11 Zheng, L. 78 Verkhivker, G. M. 24 Yavroyan, Z. V. 21 Zheng, T. 45 Victoria, M. 18 Yavroyan, Z. 22 Zhong, A. 63 Wakefield, D. L. 44 Ye, R. 62 Zhou, X. 26 Wei, D. Q. 8 Yu, I. 51 Zhu, Y. 6 Wilson, B. S 47 Zakiryanov, F. K. 81 Zhuang, X. 58 Wood, Z. A. 8 Zarezade, V. 30 Zhurkin, V. B. 57 Wu, J. 6 Zechiedrich, L. 5 Zidovska, A. 58 Xia, Y.-L. 71 Zgarbova, M. 49, 50 Zilman, A. 45 Xu, S. 26 Zgurskaya, H. I. 16, 47 Zou, L. 62 Xu, Z. 26 Zhang, X.-Y. 71 Zunini, M. P. 18 Yakupova, A. B. 43 Zhang, Z. 26 Yakushevich, L. V. 81 Zhao, H. 61