Wednesday, March 6, 2019 451a
Symposium: Mapping the Cell by four ankyrin repeats, the linker helical domain (LHD1-9), and the pre-S1 elbow. The N-terminus is followed by six membrane-spanning helices (S1-S6) 2230-Symp and a pore helix connecting to a re-entrant loop that forms the selectivity filter. Simultaneous Cross-Evaluation of Heterogeneous E. coli Datasets via The TRP helix is immediately adjacent to S6, and the C-terminus consists of two Mechanistic Simulation alpha-helices connected by a small linker. TRPC3 transmembrane domain re- Markus Covert. sembles those of other TRP channels, whereas the CPD features major differ- Dept Bioengineering, Stanford Univ, Stanford, CA, USA. ences. A C-terminal domain swap occurs at the center of the CPD, where The extensive heterogeneity of biological data poses challenges to analysis and horizontal helices (HH) transition into a coiled-coil bundle, highlighting a interpretation. Construction of a large-scale mechanistic model of Escherichia unique fold for the TRPC subfamily. Comparison of TRPC3 structures reveals coli enabled us to integrate and cross-evaluate a massive, heterogeneous dataset that the HH can reside in two distinct positions. Electrophysiological analyses based on measurements reported by various labs over decades. We identified in- demonstrate that TRPC3 activity increases by shortening the length of the C-ter- consistencies with functional consequences across the data, including: that the minal loop connecting the HH with the TRP helices; likewise elongating the data describing total output of the ribosomes and RNA polymerases is not suffi- length of the loop has the opposite effect. Our findings demonstrate that the C- cient for a cell to reproduce measured doubling times; that measured metabolic terminal loop impacts channel gating by altering the allosteric coupling between parameters are neither fully compatible with each other nor with overall growth; the cytoplasmic and transmembrane domains. Future functional and structural that essential proteins are absent during the cell cycle - and the cell is robust to experiments are underway to validate whether the upward movement of the this absence. Finally, considering these data as a whole leads to successful pre- HHs corresponds to a positive modulation of the TRPC3 gating cycle. dictions of new experimental outcomes, in this case protein half-lives. 2234-Plat 2231-Symp Structural Insights into Ligand Modulation of the TRPV2 Channel Towards a Model of the Human Pancreatic Beta Cell Ruth Pumroy1, Amrita Samanta2, Yuhang Liu3, Franklin Pozo2, Raymond C. Stevens. Taylor Hughes1, George R. Dubyak2, Seungil Han3, David T. Lodowski2, Bridge Institute, University of Southern California, Los Angeles, CA, USA. Vera Moiseenkova-Bell1. A key scientific challenge in biology and chemistry is the integration of data 1Department of Pharmacology, University of Pennsylvania, Philadelphia, across the different scales – molecular, cellular, and whole body. In a collabo- PA, USA, 2Case Western Reserve University, Cleveland, OH, USA, 3Pfizer rative open source effort with several different groups across California and Inc, Groton, CT, USA. Shanghai, we are workings towards a multi-scale model of the human pancre- The transient receptor potential (TRP) family of ion channels permits permeation atic beta cell at atomic resolution. Such a model will be useful for advancing the of essential ions, including Naþ and Ca2þ, through the plasma membrane. Mem- field of structure based drug design from the protein scale to the cellular scale bers of the Vanilloid subset of this family (TRPV) can be activated or inhibited by and provide us with better feedback in the understanding and design of new a variety of stimuli, including heat, pH, mechanosensation, and a variety of small medicines to treat type II diabetes. We invite all groups interested in this effort molecules. Agonist activation of TRPV2 plays a role in the inhibition of a variety to join called the Pancreatic Beta Cell Consortium https://dornsife.usc.edu/ of cancers, including glioblastoma multiforme cell proliferation. The mechanism bridge-institute/pancreatic-beta-cell-consortium/. of TRPV2 activity in these cases seems to be related to increasing the uptake of some anticancer therapeutics, like temozolomide and doxorubicin. Here we pre- Symposium: RNA sent structures of TRPV2 structures in apo and ligand-bound forms resolved by cryo-electron microscopy. In this work we have found a novel ligand binding 2232-Symp pocket for TRPV2 which could lead to future drug development. Untangling Messenger RNA Structure with Dead-Box RNA Helicases Elizabeth Tran. 2235-Plat Biochemistry, Purdue University, West Lafayette, IN, USA. Multimerization of Human TRPA1 Ion Channel Cytoplasmic Domains DEAD-box proteins constitute the largest RNA helicase family in eukaryotes, Gilbert Q. Martinez, Sharona E. Gordon. functioning in all aspects of gene expression. However, the mechanism of ac- Dept Physiol/Biophys, Univ Washington, Seattle, WA, USA. tion in cells for these helicases remains elusive. Over the last several years, we The transient receptor potential Ankyrin-1 (TRPA1) ion channel is modu- have used genetics and classic biochemical techniques to provide insight into lated by myriad noxious stimuli that interact with multiple regions of the the role of the DEAD-box RNA helicase Dbp2 in S. cerevisiae, revealing con- channel, including the cytoplasmic N-terminal Ankyrin repeat domains nections between RNA duplex unwinding and nuclear messenger RNA matu- (ARDs) via covalent modification. The way in which TRPA1 cytoplasmic ration steps. However, the precise enzymatic targets of Dbp2 and the vast domain modification is transmitted across large spatial distances to open majority of the 25-40 DEAD-box RNA helicases in eukaryotes. Using a com- thechannelporehasyettobeelucidated.Thecryo-EMstructureof bination of genome-wide techniques including iCLIP-seq, Structure-seq, and TRPA1 revealed a tetrameric C-terminal coiled-coil (CC) surrounded by RNA seq, we have now characterized the enzymatic targets of Dbp2. This re- the ARDs, an architecture shared with the canonical transient receptor po- veals a role for dynamic changes in RNA structure during transcriptional termi- tential (TRPC) ion channel family. Similarly, structures of the TRP mela- nation. Moreover, we find evidence for regulation of Dbp2 and, consequently, statin (TRPM) ion channel family also showed a C-terminal coiled-coil Dbp2-dependent mRNAs, in response to glucose availability. These findings enclosed by N-terminal cytoplasmic domains. This conserved architecture correlate with changes in metabolic gene expression and provide support for may indicate a common gating mechanism in which modification of cyto- widespread regulation of RNA structure as a mechanism of gene regulation. plasmic domains can transduce conformational changes to open the ion- conducting pore. We developed an in vitro system in which N-terminal ARDs and C-terminal CC domains can be expressed in bacteria and main- Platform: TRP Channels tain the ability to interact. We tested whether temperature, the polyphos- phate compound IP6, and the covalent modifier allyl isothiocyanate alters 2233-Plat N- and C-terminal interactions. We found that none of the modifications Structural and Functional Analyses of TRPC3 Reveal Allosteric Gating tested altered ARD-CC interactions. We found that CCs tetramerize in a Modulation by the Cytoplasmic Domain concentration dependent manner, with monomers and trimers observed at Francisco J. Sierra Valdez1, Caleigh M. Azumaya2, Luis O. Romero1, lower concentrations. Although we did not observe ligand- or Terunaga Nakagawa2, Julio F. Cordero-Morales1. temperature-dependent disruption of ARD-CC interactions, our system pro- 1Dept Physiology, Univ Tennessee, Memphis, TN, USA, 2Vanderbilt vides a method for examining the mechanism of oligomerization of TRPA1 University, Nashville, TN, USA. cytoplasmic domains. TRPC3 is associated with neurodegenerative diseases, memory loss, and hyper- tension. In blood vessels, TRPC3 is an essential component of the cellular mech- 2236-Plat anisms by which vasoconstrictors regulate blood pressure. Vasoconstrictors, Approaching to the Molecular Mechanism of the Fast Inactivation of such as angiotensin II, bind to phospholipase C (PLC)-coupled receptors, Calcium Selective TRP Channels increasing the intracellular levels of DAG, which in turn activates TRPC3. Lisandra Flores Aldama1, Kattina Zavala2, Daniel Bustos3, Upon activation, TRPC3 channels induce depolarization increases intracellular Wendy Gonzalez3, Juan Opazo2, Sebastian E. Brauchi1. þ Ca2 and promotes vasoconstriction. Here, we show the full-length and cyto- 1Physiology, Univ Austral de Chile, Valdivia, Chile, 2Faculty of Science, plasmic domain (CPD) cryoEM structures for human TRPC3 in the apo state Universidad Austral de Chile, Valdivia, Chile, 3Universidad de Talca, Talca, at 5.8 and 4.0 A˚ resolution. A TRPC3 subunit consists of an N-terminus formed Chile.
BPJ 9439_9446 452a Wednesday, March 6, 2019
TRPV5 and TRPV6 are inwardly rectifying calcium selective channels, 2239-Plat considered as gatekeepers of epithelial calcium transport and key elements Gain-Of-Function Mutationsin TRPM4 Activation Gate Cause Skin Dis- for calcium homeostasis. Intracellular calcium exert a negative control over ease PSEK the activity of these channels. In mammals, TRPV6 channels show a charac- Huijun Wang1, Zhe Xu2, Bo Hyun Lee3, Simon Vu3, Linghan Hu1, teristic fast calcium-dependent inactivation phase, that is absent in TRPV5 Mingyang Lee1, Dingfang Bu1, Xu Cao1, Samuel Hwang4, Yong Yang1, channels at physiological conditions. It has been evidenced that the intracel- Jie Zheng3, Zhimiao Lin1. lular loop located between the transmembrane segments (TM) S2-S3 and res- 1Dept Dermatology, Peking University First Hospital, Beijing, China, 2Dept. idues downstream the TM S6 are involved in the mechanism of fast Dermatology, Beijing Children’s Hospital, Beijing, China, 3Dept Physiol inactivation, however the molecular mechanism driving fast inactivation is Memb Biol, Univ Calif Davis, Davis, CA, USA, 4Dept. Dermatology, Univ. not known. In the present study we establish a structural-functional correlation California Davis, Davis, CA, USA. of this process. By means of electrophysiological recordings we identified a Here we report the first cases of mutations in TRPM4 which encodes TRP þ set of conserved residues at the Helix-Loop-Helix (HLH) domain that modu- melastatin 4 (TRPM4), a Ca2 -activated monovalent cation channel, as a lates the inactivation phenotype. Molecular dynamics simulations suggest that cause of an autosomal-dominant form of progressive symmetric erythrokera- the inactivation phenotype can be explained by specific conformational toderma (PSEK). In three separate families with PSEK, we identified two changes induced by calcium coordination to a structural triad formed by the missense mutations (c.3099C>G and c.3119T>C) that produce I1033M and HLH domain, the intracellular connector between TM S2-S3, and the TRP I1040T, both of which are located in the S6 transmembrane domain of helix. TRPM4 protein. The substitutions are expected to directly affect activation gating of TRPM4 according to the cryo-EM structures. Electrophysiological 2237-Plat studies of the mutants showed substantial hyperactivity, as evidenced by pro- Antagonist-Induced Clockwise Rotation in the TRPV1 nounced baseline activity, enhanced sensitivity to intracellular Ca2þ, and an Shoko Fujimura1, Kazuhiro Mio1, Masahiro Kuramochi2, elevated resting membrane potential. In vitro studies revealed enhanced pro- Hiroshi Sekiguchi3, Muneyo Mio1, Tai Kubo1, Yuji C. Sasaki2. 1 liferation in keratinocytes overexpressing either of the mutants. We also de- Operand OIL, National Institute of Advanced Industria, Kashiwa, Japan, tected an upregulationof markers for proliferation and differentiation of 2Graduate School of Frontier Sciences, The Univ. Tokyo, Chiba, Japan, 3 keratinocytes in the affected skin tissues. Our study identified TRPM4as a Japan Synchrotron Radiation Research Institute, Hyogo, Japan. new player in the pathogenesis of skin TRP channelopathies, as well as a po- The TRPV1 is a nonselective cation channel that responds to various signals tential target for treatment of skin hyperkeratotic disorders. including capsaicin, heat, and low pH condition. The Cryo-EM structures of TRPV1 show twisting of transmembrane helices around the pore axis. To un- derstand the intramolecular dynamics of the TRPV1 associating with the 2240-Plat gating event, we adopted the Diffracted X-ray Tracking (DXT) technique. In TRPM8 Regulates Sexual Reward and Satiety DXT, an individual protein was labelled with gold nanocrystals. Trajectories Yelena Nersesyan1, Ekaterina Gribkova2, Padmamalini Baskaran3, of Laue diffraction spots from nanocrystals attached to the immobilized target Daniel Llano2, Baskaran Thyagarajan4, Eleonora Zakharian1. proteins were investigated as the intramolecular movement of the target pro- 1Dept Cancer Bio/Pharm, University Illinois College of Medicine, Peoria, IL, teins in real time. DXT can monitor the rotational motion of the nanocrystal USA, 2University Illinois, Urbana-Champaign, IL, USA, 3University of at several milliradian scales with two rotational axis views, tilting (q) and Wyoming, Laramie, WY, USA, 4Sch Pharmacy, Univ Wyoming, Laramie, twisting (c) motions. The motion of TRPV1 was evaluated by mean square WY, USA. angular displacement (MSD) curves of the Laue diffraction spots from DXT. Testosterone regulates dimorphic sexual behaviors in all vertebrates. However, The slopes of the MSD curves obtained from TRPV1 were shifted upward the molecular mechanism underlying these behaviors remains inconclusive. in response to capsaicin, which reflects DXT successfully extracted the internal Here, we report that a newly identified rapid testosterone signaling receptor, motion of TRPV1. We then applied a velocity dependent filtering method to TRPM8, regulates dimorphic sexual behaviors. We found that TRPM8 / sampling a biased motion. The filtering method clearly distinguished the male mice exhibit a delayed sexual satiety, increased mounting frequency agonist- and competitive antagonist (AMG9810) -induced intramolecular mo- indiscriminate of sexes, and augmented aggression compared to controls, while tion. The motions under the capsaicin and antagonist were 1 mrad (clockwise TRPM8 / females display increased olfaction-exploratory behaviors. Re- from the Au(111) direction) and 0.5 mrad (counterclockwise) after 3 ms, sponses to the acute testosterone application onto the amygdala were entirely respectively. Such motions were also observed another filtered data. This raises attenuated in TRPM8 / males but remained unchanged in females. Conse- the possibility that there are multiple modes of conformational change in quently, following-mating activation of dopaminergic neurons in the ventral TRPV1. The movement of ligand-evoked conformational changes was slower tegmental area was impaired in TRPM8 / males. Together, these results sug- than the thermal fluctuations. gest that TRPM8 mediates sex-reward mechanism in males and depletion of its function leads to a delayed sexual satiety phenomenon. 2238-Plat The Conformational Wave in Capsaicin Activation of Transient Receptor Potential Vanilloid 1 Ion Channel Platform: Intrinsically Disordered Proteins (IDP) Fan Yang1, Xian Xiao2, Bo Hyun Lee3, Simon Vu3, Wei Yang1, Vladimir Yarov-Yarovoy3, Jie Zheng3. and Aggregates III 1Department of Biophysics, School of Medicine, Zhejiang University, Hangzhou, China, 2Westlake University, Hangzhou, China, 3Department of 2241-Plat Membrane Biology, School of Medicine, University of California, Davis, Tardigrade Intrinsically Disordered Proteins Protect Enzymes from Davis, CA, USA. Desiccation-Induced Inactivation The capsaicin receptor TRPV1 has been intensively studied by cryo-electron Samantha Piszkiewicz1, Kathryn H. Gunn2, Shannon L. Speer1, microscopy and functional tests. However, though the apo and capsaicin- Owen Warmuth1, Aakash Mehta1, Francis J. Lauzier1, Kenny H. Nguyen1, bound structural models are available, the dynamic process of capsaicin Elizabeth Kuhlman2, Saskia B. Neher2, Gary J. Pielak1,2. 1 activation remains intangible, largely due to the lack of a capsaicin- Dept of Chemistry, The University of North Carolina at Chapel Hill, Chapel 2 induced open structural model and the low occupancy of the transition Hill, NC, USA, Dept of Biochemistry and Biophysics, The University of states. Here we report that reducing temperature toward the freezing point North Carolina at Chapel Hill, Chapel Hill, NC, USA. substantially increased channel closure events even in the presence of Protein-based ‘biologics’ — drugs derived from living organisms — are among saturating capsaicin. We further used a combination of fluorescent unnatural the most effective therapeutic treatments on the market. However, biologics are amino acid (fUAA) incorporation, computational modeling, and rate- unstable, have short half-lives and require low temperature storage, making equilibrium linear free-energy relationships analysis (Phi-analysis) to them prohibitively expensive. Although some biologics can be stabilized by derive the fully open capsaicin-bound state model, and reveal how the formulation with excipients, most still require low temperature storage. In channel transits from the apo to the open state. We observed that capsaicin our search for new, more robust excipients, we turned to the tardigrade, a initiates a conformational wave that propagates through the S4-S5 microscopic animal that synthesizes a unique family of intrinsically disordered linker towards the S6 bundle and finally reaching the selectivity filter. proteins (IDPs) that protect its cellular components during desiccation. Encap- Our study provides a temporal mechanism for capsaicin activation of sulating a globular test protein in a tardigrade IDP hydrogel stabilizes the TRPV1. folded state of that protein. These tardigrade IDPs are more effective than
BPJ 9439_9446 Wednesday, March 6, 2019 453a two FDA approved excipients, trehalose and serum albumin, at protecting ism spectroscopy shows no gain of structure upon formation of this large poly- lactate dehydrogenase activity during desiccation. We even observe protection protein complex, as in the case of ProTa-H1. of lactate dehydrogenase upon heating desiccated enzyme/tardigrade IDP mix- A pattern is emerging that chromatin-remodeling complexes must be kept tures to 95 C. In addition, these IDPs protect the restriction enzyme DpnI from amenable of fine-tuning for optimal regulation. Additionally, both ProTa-H1 desiccation-induced inactivation and lipoprotein lipase against both freezing- and ProTa-NUPR1 undergo liquid-liquid phase transition in the micromolar induced and lyophilization-induced inactivation. Work is underway to quantify concentration range, suggesting that disorder, valency and accessibility of these the protection of antibodies against aggregation. In summary, we have demon- complexes might also be important to grant a further level of regulation via strated the ability of these tardigrade IDPs to stabilize a globular protein and phase transition. protect a diverse range of enzymes against desiccation, freezing, and lyophilization. 2244-Plat A Bacterial Biomolecular Condensate Sequesters a Signaling Pathway that 2242-Plat Drives Spatial Regulation of Gene Expression and Asymmetric Cell Division A Novel Molecular Lego Approach to Measure the Marginal Folding Keren Lasker1, Alex von Diezmann2, W.E. Moerner2, Lucy Shapiro1. Cooperativity of Intrinsically Disordered Proteins 1Developmental Biology, Stanford University, Stanford, CA, USA, Suhani Nagpal1, Thinh Luong2, Mourad Sadqi1, Victor Mun˜oz1. 2Chemistry, Stanford University, Stanford, CA, USA. 1Bioengineering, University of California, Merced, Merced, CA, USA, Selective recruitment and concentration of signaling proteins within membrane- 2Chemistry and Chemical Biology, University of California, Merced, less compartments is a ubiquitous mechanism for subcellular organization. We Merced, CA, USA. combined single-molecule tracking and super-resolution microscopy, light- Intrinsically disordered proteins (IDPs) do not fold into a native three- induced spatial mutations, reaction-diffusion modeling, and spatially-resolved dimensional structure, but exist as dynamic conformational ensembles. Their transcriptional profiling to study signal exchange in and out of a <200 nm cyto- flexibility enables IDPs to exhibit sophisticated binding modes, such as binding plasmic microdomain at the cell poles of the asymmetrically dividing bacterium to multiple partners, induced-fit binding, and allosterism. Accordingly, IDPs Caulobacter crescentus. We show that the signaling proteins in a core develop- play key regulatory roles in fundamental biological processes such as transcrip- mental pathway that culminates in the activation of the cell fate transcription fac- tion, cell cycle control and signaling. The key to their functioning is their ability tor CtrA are transiently confined to the polar microdomain. Restricted rates of to fold onto specific structures when bound to their ligand partner(s). The mech- entry into and escape from the microdomain enhance phospho-signaling, leading anistic characterization of IDPs poses a challenge to current methods which to a steep submicron gradient of activated CtrA. Thus, nanoscale protein assem- rely on folding to well-defined structures. blies can modulate signal propagation with fine spatial resolution, and in Caulo- We have devised a novel approach to measure the inherent folding cooperativ- bacter, this modulation serves to prime asymmetric genome readout. ity of an IDP that we term Molecular LEGO. Inspired by the LEGO game in The polar microdomain is defined by self-assembly of the disordered protein which building blocks associate to one another by complementary indentations, PopZ that directly binds signaling proteins in vivo. We show that cytosolic pro- our approach consists on analyzing the conformational properties of fragments teins lacking a microdomain binding partner cannot penetrate the microdomain of an IDP containing each secondary structure element as well as all possible despite being similar in size and charge to proteins that do enter. These combinations of them. Direct comparison of conformational preferences of iso- microdomain-excluded-proteins can be recruited to the microdomain through lated elements, their combinations, and the entire protein, we infer the degree of light-induced dimerization between the excluded protein and PopZ, suggesting energetic coupling between each the various parts. We do this experimentally, that microdomain binding controls entry. We further show that light-induced analyzing conformational properties by circular dichroism and using tempera- recruitment of signaling proteins to the poles of the cell at the wrong time alters ture and TFE as thermodynamic parameters to enhance information, and also the fate of the two daughter cells, leading to loss of asymmetry. Modifying either computationally, by simulating all possible fragments using atomistic Molecu- the charge or composition of the disordered region modulates selectivity proper- lar Dynamics simulations in explicit solvent. Using NCBD as paradigmatic ties, leading to severe phenotypes that are in some cases lethal. Finally, the selec- example of IDP, we observe, although NCBD exhibits an apparently featureless tive properties of PopZ are independent of geometry: In cells with a deformed thermal unfolding profile, the various elements interact with one another, spherical shape, PopZ becomes a droplet that maintains its selectivity properties. biasing the conformational space of the protein in significant ways to populate the native-like partially folded conformations that enable its complex binding 2245-Plat behavior. This approach can generally be applied to any partially disordered Sequence Determinants of Protein Phase Separation of the Intrinsically IDPs thus providing an essential new tool for their analysis. Disordered RGG Domain from LAF-1 Benjamin S. Schuster1, Gregory Dignon2, Craig Jahnke1, 2243-Plat Matthew C. Good1, Daniel A. Hammer1, Jeetain Mittal2. Highly Disordered 10:1 Complex of Two Anti-Apoptotic, Chromatin-Re- 1University of Pennsylvania, Philadelphia, PA, USA, 2Lehigh University, modelling Intrinsically Disordered Proteins Bethlehem, PA, USA. Alessandro Borgia1, Madeleine B. Borgia1, Alain Scaiola2, Robert Best3, Phase separation of intrinsically disordered proteins (IDPs) underlies the for- Benjamin Schuler1. mation of membraneless organelles with prominent biological roles, such as 1Department of Biochemistry, University of Zurich, Zurich, Switzerland, P granules in C. elegans embryos. Determining the sequence-level features 2Department of Biology, Institute of Molecular Biology and Biophysics, responsible for IDP phase separation is important for understanding native bio- ETH, Zurich, Switzerland, 3NIDDK, Laboratory of Chemical Physics, NIH, logical function, protein evolution, and approaches to manipulate phase Bethesda, MD, USA. behavior. However, sequence determinants of IDP condensation are poorly un- The prominence of intrinsically disordered proteins (IDPs) in regulatory roles is derstood because the field lacks a predictive framework for guiding experi- in no small part due to their flexibility, which allows them to interact with many ments. We investigated sequence determinants governing phase separation of partners and be accessible to modifying enzymes. The repertoire of IDPs inter- the RGG domain from the P granule protein LAF-1, using theory and experi- actions has recently been expanded by our discovery and characterization of the ments. We uncovered three features of the LAF-1 RGG domain that mediate first case of an IDP-IDP high-affinity interaction between the linker histone its phase behavior. First, based on a predictive coarse-grained (CG) model of H1.0 and prothymosin-a (ProTa). Mapping these interactions is an inherently IDPs, we identified an unappreciated region of the RGG domain that is also difficult task due to their transient nature, but thanks to single-molecule FRET highly conserved between species. Experiments revealed that deletion of this (smFRET), NMR and molecular simulations, we demonstrated that disorder of region indeed significantly disrupts phase separation, much more than would the interacting proteins is fully maintained in the complex, where the constitu- have been expected from control deletions of other regions. Second, based ent proteins dynamically interconvert within a broad conformational ensemble on CG molecular dynamics simulations, we predicted amino acid replacements with no preferential binding sites. that may dramatically affect phase behavior. We experimentally determined We have now investigated the interaction between the highly acidic ProTa and that mutating Tyr to Phe, or Arg to Lys, dramatically perturbs RGG phase sep- its basic partner NUPR1: both IDPs are involved in cancer progression, aration; these trends agree with recent findings on other proteins, including apoptosis, and, like H1.0, in chromatin remodeling. Multiple intra- and inter- FUS. Finally, we assessed the effect of sequence charge decoration on phase molecular distances measured with smFRET show a progressive addition of behavior, harnessing a new finding relating single-molecule properties and NUPR1 molecules on a single ProTa chain, which is consistent with results phase separation. Experimentally, we found that sequences with large charge from dual-focus FCS showing a continuous increase in hydrodynamic radius segregation have dramatically enhanced propensity to phase separate compared upon titration of ProTa with NUPR1. Using FRET we were able to determine to wild-type RGG, as expected from simulation and theory. Together, these a 10:1 stoichiometric ratio in saturating conditions, which is highly unusual for studies identify key determinants of RGG phase separation, including proteins of roughly similar sizes. However, strikingly, far-UV circular dichro- conserved residues and charge patterning. These results elucidate physical
BPJ 9439_9446 454a Wednesday, March 6, 2019 principles of IDP condensation and its biological implications, while advancing is completely resistant to liquid-liquid phase separation (LLPS) of the eye lens a computational/experimental hybrid framework for studying phase separation. proteins, also known as cold cataract. Despite this observation, a sub-fraction of the D. mawsoni eye lens proteins was found to undergo LLPS above the body tem- 2246-Plat perature of the toothfish. Six g-crystallins were identified from this fraction and The Ins and Outs of Phase Separation in Nucleolar Biology characterized for their LLPS behavior and general features of stability. No obvious 1 1 1 1 Richard Kriwacki , Diana Mitrea , Mylene Ferrolino , Eric Gibbs , relationship could be discerned between the critical temperatures at which these g- 1 1 2 Aaron H. Phillips , Michele Tolbert , Christopher B. Stanley , crystallins undergo LLPS and the various measures of stability. Additionally, Amanda Nourse1, Paulo L. Onuchic3, Priya R. Banerjee3, Ashok A. Deniz3. 1 2 mutagenesis of predicted surface exposed residues was performed to study their Dept Struct Biol, St Jude Childrens Res Hosp, Memphis, TN, USA, Biology role in the LLPS behavior. Mutagenesis revealed that replacing exposed arginine and Biomedical Sciences Group, Oak Ridge National Laboratory, Oak Ridge, 3 for lysine results in a decrease of the phase transition temperature, while replacing TN, USA, Department of Integrative Structural and Computational Biology, lysine with arginine had the opposite effect, but similar magnitude. These finding The Scripps Research Institute, La Jolla, CA, USA. highlights the importance of arginine in increasing attractive protein-protein inter- The nucleolus is a multi-layered, liquid-like membraneless organelle that medi- actions even when the site specific charge is largely unchanged. Furthermore, we ates the multi-step process of ribosome biogenesis and also coordinates signaling have shown that the components of the buffer solution can further modulate the responses to various cellular stresses. Initiation of ribosomal RNA (rRNA) tran- phase transition temperature of these crystallins. scription during interphase of the cell division cycle triggers phase separation with the nucleolar protein, Fibrillarin, forming the Dense Fibrillar Component region of the nucleolus wherein rRNA is sequentially modified and spliced. Platform: General Protein-Lipid Interactions Mature rRNAs flux outwards from the DFC into the surrounding Granular Component (GC) region wherein they phase separate with another nucleolar pro- 2249-Plat tein, Nucleophosmin (NPM1). Utilizing an intrinsically disordered region (IDR) Understanding the Organization and Dynamics of KRAS4b on a Complex displaying alternating acidic and basic tracts, NPM1 independently phase sepa- 8-Lipid Reconstituted Model Membrane using Microscopy and Spectros- rates with ribosomal proteins (rProteins), sequestering them within the GC and copy Methods enabling their assembly with rRNA to form nascent ribosomal subunits. We seek Rebika Shrestha, Thomas Turbyville. to understand how the molecular interactions that independently drive phase sep- RAS Initiative, Frederick National Laboratory for Cancer Research, aration of NPM1 with rRNA and rProteins also enable the molecular handoffs Frederick, MD, USA. that underlie ribosomal subunit assembly. Our studies employ structural and bio- The enrichment of negatively charged phospholipids, particular phosphatidylser- physical methods that probe molecular interactions at the atomic length scale as ine (PS) and phosphoinositides, in the inner leaflet of plasma membrane (PM) is well as single-molecule, scattering and imaging methods that reveal the struc- one of the underlying principle behind recruitment of numerous intracellualr pro- tural and material properties of liquid-phase condensates on the micron length teins to PM, thus regulating key cellular events. One such protein of interest is scale. Only by bridging these disparate length scales can we understand how KRAS4b, a member of Ras superfamily of small GTPases, which is an oncopro- phase separation contributes to the essential process of ribosome biogenesis. tein implicated in 95% of pancreatic cancer, 45% of colorectal cancer, 35% of We will present our latest findings on the liquid phase organization of the nucle- lung cancer and high extent in other forms of cancer. When Ras is active and olus and how this mediates vectorial ribosomal subunit assembly. bound to the membrane, it binds to its downstream effectors and triggers multiple signal cascading pathways that are essential for cell growth, proliferation and sur- 2247-Plat vival. KRAS4b localizes to PM predominantly by virtue of (i) the electrostatic Sequence Determination of Liquid-Liquid Phase-Separated Assemblies of interaction between sixpositively charged lysine residues located in the hypervar- Engineered Disordered Proteins in Living Cells iable region (C-terminal) of KRAS4b and negatively charged phospholipids in Ming-Tzo (Steven) Wei, Clifford P. Brangwynne. PM and (ii) the hydrophobicity of the farnesyl group, post-translational modifica- Chemical and Biological Engineering, Princeton University, Princeton, NJ, tion in C185 amino acid of KRAS4b, which anchors the protein into the disor- USA. dered lipid domains. In this study, we aim to explore the molecular mechanism There is currently a growing interest in understanding biopolymer phase transi- of KRAS4b interaction with the membrane using recombinant protein on an arti- tions, particularly those involving intrinsically disordered proteins/regions ficial supported lipid bilayer. The biomimetic surface is composed of complex (IDPs/IDRs)and RNA.Ithas been determinedthatintracellularliquid-liquidphase 8-lipids composition that closely resemble the lipid composition of the inner separations underlie the assembly of many non-membranous organelles, including leaflet of biological PM. We use a combination of fluorescence-based microscopy RNA/protein assemblies such as P granules, nucleoli, transcription factors, and and single molecule spectroscopy techniques as well as high resolution scanning stress granules. However, little is known about the physics of these organelles, probe microscopy to study the lateral organization and dynamics of KRAS4b as including their internal molecular organization and feedback between their molec- well as the lipid bilayer. Using a very complex reconstituted model membrane to ular and mesoscale properties. Progress on these questions has been hampered by study KRAS4b, our research provides mechanistic insight into RAS biology and the lack of detailed phase diagrams, which could elucidate how molecular interac- possibly a nobel platform for targeting RAS. tions give rise to emergent droplet properties, particularly condensed-protein con- centrations and their physical characteristics. To answer these questions, we 2250-Plat utilized a novel technique, ultrafast-scanning fluorescence correlation spectros- Structural and Mechanistic Bases of Drp1-Cardiolipin Interactions in copy, to measure the inter-molecular interaction strengths and full binodal of a Mitochondrial Fission 1 1 2 1 disordered protein that induces in-vivo phase transitions. These measurements re- Bin Lu , Mukesh Mahajan , Abhishek Mandal , Nikhil Bharambe , 1 2 1 1 vealed that phase-separated protein droplets have unusually low densities with Rihua Wang , Patrick van der Wel , Matthias Buck , Xin Qi , 1 large void volumes. The data demonstrate how sequence-encoded conformational Rajesh Ramachandran . 1Dept Phys/Biophys, Case Western Reserve Univ, Cleveland, OH, USA, fluctuations of IDRs give rise to low overlap volume fractions for driving phase 2 separations. Furthermore, using the inter-molecular interactions of native non- Department of Structural Biology, University of Pittsburgh, Pittsburgh, PA, USA. membranous organelles, we develop an optogenetic platform for light activation Mitochondria are dynamic organelles that undergo regulated cycles of fission and of IDR-mediated phase transitions in living cells. This powerful technique permits fusion. Emergent data indicate that a direct interaction of the cytosolic, mechano- detailed studies of the mechanisms by which IDR sequences determine intracel- chemical GTPase, dynamin-related protein 1 (Drp1) with mitochondrial outer lular phase separation. Such experiments can elucidate not only physiological membrane-localized cardiolipin (CL) catalyzes mitochondrial fission. However, phase transitions but also their links to pathological aggregates. neither the identity of the amino acid residues nor the mechanism by which Drp1 accomplishes specific CL recognition and subsequent membrane remodel- 2248-Plat ing is known. Here using a comprehensive toolkit of structural, biochemical, bio- Uncovering the Role of Surface Residues and Buffer Composition in physical, and cell biological approaches, we reveal the structure, identity, and Liquid-Liquid Phase Separation of Eye Lens Crystallins from an Antarctic mechanism by which the intrinsically disordered Drp1 variable domain (VD) spe- Toothfish cifically binds and remodels membrane CL. We demonstrate that lipid-packing Jan C. Bierma1, Kyle Roskamp1, Aaron Ledray1, Andor J. Kiss2, defects in CL-containing membranes enable specific Drp1-CL recognition inde- C.-H. Christina Cheng3, Rachel W. Martin1. pendent of CL acyl chain composition or shape. We further show that the Drp1 1UC Irvine, Irvine, CA, USA, 2Miami University, Oxford, OH, USA, VD intercalates into the hydrocarbon core of the membrane to specifically restrict 3University of Illinios at Urbana-Champaign, Urbana, IL, USA. CL motion and induce transient CL non-bilayer topological transitions that facil- The Antarctic toothfish, Dissostichus mawsoni, lives in the Southern Ocean itate membrane constriction. We reveal a disorder-to-order structural transition around Antarctica, requiring adaptations to deal with the challenges of living in of the Drp1 VD upon CL binding, and identify critical VD residues, the substitu- a subfreezing environment. One such adaptation is that the D. mawsoni eye lens tion of which selectively reduces Drp1-CL binding affinity. Remarkably, the
BPJ 9439_9446 Wednesday, March 6, 2019 455a expression of mutant VD in Drp1-null cells induces the formation of hitherto un- 2253-Plat seen, ring-like ‘donut’ mitochondria subsequent to mitochondrial fission. Our data Molecular Simulations Reveal the Dynamics of the Band 3 Anion Trans- reveal that a rapid degradation of pro-fission CL to fusogenic phosphatidic acid porter in a Model Native Red Blood Cell Membrane (PA) at the poles of newly divided mitochondria in mutant VD-expressing cells Dario De Vecchis1,2, Reinhart A. Reithmeier3, Antreas Kalli1,2. enable mitochondrial back-fusion and donut formation. These studies firmly 1School of Medicine, Univ Leeds, Leeds, United Kingdom, 2Astbury Centre establish an indispensable role for Drp1 VD-CL interactions in regulating mito- for Structural Molecular Biology, Leeds, United Kingdom, 3Dept chondrial dynamics and morphology both pre- and post-fission. Biochemistry, Univ Toronto, Toronto, ON, Canada. Band 3, the red blood cell anion exchanger (AE1/SLC4A1), is responsible for the 2251-Plat rapid transport of bicarbonate and chloride across the red blood cell plasma mem- Multiple Stochastic Pathways in Forced Peptide-Lipid Membrane brane, a process necessary for efficient respiration. Human Band 3 is comprised of Detachment a cytosolic domain and a membrane domain that contains 14 transmembrane heli- Milica Utjesanovic1, Tina R. Matin2, Krishna P. Sigdel3, Gavin M. King1, ces. Although structural data are available for both isolated domains, the structure Ioan Kosztin1. of the complete Band 3 remains elusive. This is a limiting factor in the study of 1Dept Phys/Astron, Univ Missouri Columbia, Columbia, MO, USA, 2Dept Band 3 anion transport and of its interactions with cytosolic proteins. By inte- Anesthesiology, Weill Cornell Medicine, New York, NY, USA, 3Dept Phys/ grating molecular modelling and molecular dynamics simulations at the coarse- Astron, California State Polytechnic University, Pomona, CA, USA. grained and all-atom resolutions, we have constructed a model of Band 3 that con- We have used high resolution AFM based dynamic force spectroscopy to inves- sists of both the transmembrane and the cytosolic domains. This model enabled us tigate peptide-lipid membrane interactions by measuring the detachment (last- to identify the orientation of the cytosolic domain relative to the transmembrane rupture) force distribution, P(F), and the corresponding force dependent rupture domain and the role of the linker regions that connect these two domains in their rate, k(F), for two different peptides and lipid bilayers. The measured quantities, interactions. Our model was validated using functional data and molecular simu- which differed considerably for different peptides, lipid-membranes, AFM tips lations. Simulations were performed in complex bilayers that resemble the native (prepared under identical conditions), and retraction speeds of the AFM canti- red cell plasma membrane, containing a full complement of phospholipids, sphin- lever, could not be described in terms of the standard theory, according to which gomyelin and cholesterol. Our results provide novel molecular insights into the in- detachment occurs along a single pathway, corresponding to a diffusive escape teractions of the lipid environment with Band 3. Specific lipids, e.g. cholesterol, process across a free energy barrier. In particular, the prominent retraction speed have been found to localize in the Band 3 dimer interface possibly stabilizing dependence of k(F) was a clear indication that peptide-lipid membrane dissocia- the dimer. Specific lipid head groups were also found to interact with the cytosolic tion occurs stochastically along several detachment pathways. Thereby, we have domain regulating its orientation relative to the transmembrane domain. Moreover, formulated a general theoretical approach for describing P(F) and k(F), by our modeling approach identified key residues involved in anion binding. Under- assuming that peptide detachment from lipid membranes occurs, with certain standing the dynamics and interactions of Band 3 in a model native red cell mem- probability, along a few dominant diffusive pathways. This new method was vali- brane provides new insights into the function of this important human transporter. dated through a consistent interpretation of the experimental data. Furthermore, we have found that for moderate retraction speeds at intermediate force values, 2254-Plat k(F) exhibits catch-bond behavior (i.e. decreasing detachment rate with Huntingtin Aggregation Is Modified in the Presence of a Variety of Lipid increasing force). According to the proposed model this behavior is due to the sto- Membranes chastic mixing of individual detachment pathways which do not convert or cross Maryssa A. Beasley1, Sharon E. Groover1, Justin A. Legleiter1,2. during rupture. To our knowledge, such catch-bond mechanism has not been pro- 1C. Eugene Bennett Department of Chemistry, West Virginia University, posed and demonstrated before for a peptide-lipid interaction. Morgantown, WV, USA, 2Blanchette Rockefeller Neurosciences Institute, Work supported by the Burroughs Wellcome Fund (Career Award at the Scien- Robert C. Byrd Health Sciences Center, West Virginia University, tific Interface), the NSF (CAREER Award #: 1054832), and the MU Research Morgantown, WV, USA. Board. The computation for this work was performed on the HPC infrastructure Huntington’s Disease is a fatal neurodegenerative disorder characterized by an provided by RCSS at the University of Missouri, Columbia MO. The HPC expanded polyglutamine (polyQ) region of the huntingtin protein (htt) that leads equipment for the computational work is supported by NSF CNS-1429294. to the formation of toxic aggregates. The first 17 amino-terminal residues of htt (Nt17) have been shown to promote this oligomer formation, and also comprise 2252-Plat a lipid binding domain. The subcellular localization and interaction of htt contain- The Small Heat Shock Proteins, HSPB1 and HSPB6, Have the Ability to ing expanded polyQ domains with membranous surfaces has been well docu- Get Inserted into Lipid Membranes mented and suggests that these interactions play a role in HD pathogenesis. Antonio De Maio1,2, David M. Cauvi1, Ricardo F. Capone1, Nelson Arispe3, While protein/lipid membrane interactions play an important role in a number Wilbert Boelens4. of amyloid-related diseases, the protein represents only one of the participants. 1Dept. of Surgery, University of California San Diego, La Jolla, CA, USA, For example, the size and charge of the lipid headgroups likely influence the inter- 2Dept. of Neurosciences, University of California San Diego, La Jolla, CA, action between Nt17 and lipid membranes. Any effect on lipid binding would also USA, 3Dept Anat/Phy, Uniform Serv Univ Hlth Sci, Bethesda, MD, USA, affect the aggregation and trafficking of htt. Determining factors that regulate the 4Dept. of Biomolecular Chemistry, Institute for Molecules and Materials, affinity of htt for membranes could not only help understand the normal functions Radboud University Nijmegen, Nijmegen, Netherlands. of htt, but could lead to a better understanding of how to modify protein-lipid Increasing evidence has indicated that heat shock proteins (HSP) escape their cyto- interaction for therapeutic purposes. As a result, we undertook a series of exper- solic environment acting as signaling agents in the extracellular fluid. Since the iments to determine the role of specific lipids in modulating the htt/lipid interac- majority of these proteins lack the information necessary for their export via the tion and resulting aggregation. Htt aggregation in the presence of POPC, DOPC, classical secretory pathways, special attention has been directed at alternative DMPC, POPG, POPE, POPS, and total brain lipid extract were investigated via mechanisms for their release. In this regard, a major obstacle for their secretion thioflavin T aggregation assays and other biochemical assays. In addition, the is the crossing of the plasma membrane. A mechanism that appears equally suit- modulating effect of cholesterol was also explored. able for their release is via exosomes. Indeed, several hsp have been detected within exosomes isolated from various sources. Herewith we report that a member 2255-Plat of the small hsp family, HSPB1 (Hsp27), was found to be associated with exo- Characterization of Phosphatidylinositol Phosphate Binding in Lipid Bila- somes and apparently inserted in the vesicle membrane. In order to investigate yers by Solid-State NMR Spectroscopy the potential interaction of HSPB1 and lipid membranes, pure protein was incu- Jacqueline R. Perodeau, Ashley D. Bernstein, Stefany M. Lazieh, bated with liposomes made of various lipid compositions and insertion was ac- Robert D. Palmere, Andrew J. Nieuwkoop. cessed by high-speed centrifugation. We found that HSPB1 interacts with Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ, USA. liposomes made of palmitoyl oleoyl phosphatidylserine (POPS), phosphatidylcho- Phosphatidylinositol phosphates (PIPs) are a seven membered class of lipid that line (POPC) and phosphatidylglycerol (POPG), however, with different character- regulate diverse cell processes in eukaryotic organisms differentiated by the level istics. Another member of the small HSP family, alphaB-crystallin (HSPB5), also of phosphorylation at three sites on the lipid head group. The presence of one or interacts with liposomes, but differently than HSPB1. To address the regions inter- more of these lipids in a membrane serves as a biochemical signal for the local- acting with the membrane, intact proteoliposomes were digested with proteinase K ization and activation of hundreds of intrinsic and peripheral membrane proteins. and the protected domains within the liposomes were detected by mass spectros- Magic-angle-spinning (MAS) solid-state NMR (ssNMR) can provide atomic res- copy. We observed that large parts of HSPB1 and HSPB6 were embedded within olution information about interactions in lipid environments. Direct observation the liposomes. These observations suggest that the interaction with lipid mem- of the lipids and membrane proteins are possible, as well as the interactions be- branes may be part of the mechanisms of export of these proteins. tween them. While ssNMR is capable of investigating all types of lipids, the
BPJ 9439_9446 456a Wednesday, March 6, 2019 existence and importance of up to three additional phosphate groups in PIPs pro- thermodynamic cost imposed by isomerization, and the difference between ATP- vide a distinct advantage for their investigation. We demonstrate sample prepa- and ADP-actin that we calculate exactly matches the values found in experiments. ration and spectroscopic techniques to maximize the sensitivity of 31P spectra of PIP containing liposomes. The location of the PIP binding site on a protein 2258-Plat is important to understand if we seek to detect or modify binding. NMR is Investigations into the Structure and Intermolecular Interface of Human uniquely suited for mapping the binding sites of PIPs to atomic resolution. Mod- Cofilin-2 Assembled on Actin Filaments by Magic Angle Spinning NMR ern ssNMR utilizes very-fast MAS rates (in excess of 100 kHz) and proton detec- Jodi Kraus1, Jenna Yehl1, Elena Kudryashova2, Emil Reisler3, tion to streamline the process of assigning the chemical shifts in moderately sized Dmitri Kudryashov2, Tatyana Polenova1. proteins. PH domains are conserved PIP binding domains found in many different 1Chemisty and Biochemistry, University of Delaware, Newark, DE, USA, proteins. We present progress towards the solid-state assignments of PH domains, 2Chemistry and Biochemistry, The Ohio State University, Columbus, OH, a well characterized class of PIP binding domain. These chemical shifts will be USA, 3Chemisty and Biochemistry, University of California, Los Angeles, used to track the effects of PIP binding on the domain, confirming the binding Los Angeles, CA, USA. site as observed by other techniques. Actin comprises one major component of the eukaryotic cytoskeleton and is one of the most ubiquitous and conserved proteins. The cofilin/ADF family of pro- 2256-Plat teins play an integral role in the regulation of actin polymerization dynamics. Spatial Organization of the Blood Stage Parasitophorous Vacuole of the Specifically, by stimulating severing and depolymerization of actin filaments Malaria Parasite Plasmodium falciparum they potentiate actin recycling in a nucleotide-dependent manner. While actin 1 2 3 4 Matthias Garten , Josh R. Beck , Robyn Roth , Christopher K.E. Bleck , treadmilling is crucial for cell function and is responsible for up to 50% of 1 1 1 John E. Heuser , Tatyana Tenkova-Heuser , Svetlana Glushakova , cellular ATP consumption, there is a lack of atomic-level information regarding Joshua Zimmerberg1, Daniel E. Goldberg3. 1 the structure and dynamics of cofilin/ADF proteins bound to actin filaments. We NICHD/Section on Integrative Biophys, NIH, Bethesda, MD, USA, have previously reported structural characterization of cofilin/actin assemblies 2Department of Biomedical Sciences, Iowa State University, Ames, IA, USA, 3 4 using magic angle spinning (MAS) NMR spectroscopy. Our MAS NMR results Washington University, St. Louis, MO, USA, NIH/NHLBI, Bethesda, MD, for the F-actin binding site agree with the 3.7 A˚ cryo-EM structure of cofilin/ USA. actin complex, with three patches identified on cofilin for F-actin binding. Other The interface of the malaria parasite with its host red blood cell is the parasi- structural changes of cofilin that occur from binding to F-actin will be clarified tophorous vacuole membrane (PVM). The parasite organizes transport of pro- upon the determination of an atomic-resolution structure. Herein, we report in- teins, lipids and small molecules across the PVM. It is unknown how the vestigations into the structure and intermolecular interface of cofilin bound to F- parasite transports lipids across the PVM. Vesicular transport was not reported actin in the ADP and ADP-BeFx nucleotide states. We report 13C and 15N reso- in the PV lumen (the space between the PVM and the parasite plasma mem- nance assignments for cofilin-2 using homo- and heteronuclear MAS NMR brane (PPM)). Curiously, the freely accessible PV lumen is not homogenously experiments acquired at 19.9 T. We also present 13C-13C correlation spectra us- distributed around the parasite indicating regional close apposition of the PVM ing 2-13C and 1,6-13C-labeled cofilin for determination of distance restraints. and PPM. We hence hypothesized that the parasites may establish membrane Paramagnetic relaxation enhancements (PREs) were also used for long-range contact sites for the transport of lipids. distances and the cofilin-actin intermolecular interface. We observe several To explore the protein distribution on the PVM in correlation to the freely peaks disappear and decrease in intensity. Peak intensities are correlated to accessible PV luminal space, we used a parasite line expressing endogenously proximity to unpaired spins, and are used to derive atomic distances. These labeled, PVM resident protein, EXP2-mNeonGreen, and a PV lumen targeted are used in conjunction with our resonance assignments and distance restraints mRuby3. We found that both labels occupy the same PV region, defining from sparse-labeled cofilin to derive the MAS NMR structure of cofilin/F-actin EXP2-rich regions with accessible PV luminal space. We thus hypothesized assemblies, which is currently underway. that EXP2-free regions could be membrane contact sites to facilitate lipid ex- change. Tagging the PPM resident lipid transporter PfNCR1 with GFP, we 2259-Plat found that EXP2-rich and PfNCR1-rich regions anti-correlate to complement Mechanisms for Dendritic Actin Network Formation, Distributed Turn- each other to outline the parasite. over, and Structural Remodeling The data suggests the organization of the PVM and PV lumen in 2 distinct do- Danielle Holz, Aaron Hall, Dimitrios Vavylonis. mains with functions that can be suggested from their resident proteins: EXP2 Physics, Lehigh University, Bethlehem, PA, USA. has been associated with protein export and implicated in nutrient import. It The dendritic network of actin filaments provides the force for lamellipodial pro- may thus define a domain for the exchange of soluble and solubilized constit- trusions, driven by actin filament polymerization and branch generation by the uents and metabolites. PfNCR1 on the other hand may define a domain desig- Arp2/3 complex. Electron microscopy experiments of lamellipodia revealed nated for the exchange of hydrophobic substances. that the network structure of filaments varies with distance to the leading edge. Near the leading edge there is a dense brushwork composed of short filaments. Platform: Actin Structure, Dynamics & Filaments are longer and appear more linear near the center and rear of keratocyte Associated Proteins lamellipodia. Prior modeling of FRAP and single molecule imaging experiments suggested the existence of a diffuse actin oligomer cytoplasmic pool and distrib- 2257-Plat uted turnover of F-actin through the lamellipodium. The precise mechanisms Molecular Dynamics Simulations of G- and F-Actin Explain Aspects of behind network remodeling and the role of the oligomer pool have yet to be deter- Actin Polymerization mined. To answer this question, we created a three-dimensional stochastic model Lauren Jepsen, David Sept. at the filament level that includes rate constants for known mechanisms of poly- Univ Michigan, Ann Arbor, MI, USA. merization, depolymerization, branching, capping, uncapping, severing and de- The proper regulation of actin filament (F-actin) dynamics is key for proper branching. The model reproduces the þ/ 35o orientation pattern when cellular and physiological function. Filament dynamics are regulated through a branching occurs within 10o of the lamellipodial plane as well as the density wide array of actin binding proteins, but the nucleotide state of actin itself is of branches, ends and length distribution near the leading edge for both fibroblast also an important factor. Actin filaments polymerize in a head-to-tail fashion, and keratocyte lamellipodia (that differ in the magnitude of retrograde flow and meaning that the filament is polar and the two filament ends are intrinsically polymerization rates). We show that the simplest implementations of severing, different. The barbed end of the filament polymerizes more quickly, and the debranching, uncapping, and depolymerization from either pointed or barbed pointed end dissociates faster. As of yet, neither a F-actin crystal structure nor ends does not provide mechanisms for network structural remodeling at the level high resolution cryo-EM structures of either filament end are available, limiting observed in electron micrographs. We examine and test several mechanisms that our knowledge of important changes responsible for end-dependent dynamics. include severing and oligomer annealing that may provide an explanation. To explore filament dynamics, we performed extended molecular dynamics sim- ulations of F-actin. We find that the filament’s pointed end is significantly flatter 2260-Plat than the remainder of the filament and is in a conformation not sampled by our G- Different Faces (Phases) of Actin Depolymerizing Factors from Entamoeba actin simulations - this means that incoming monomers would need to undergo Histolytica. significant conformational changes (isomerization) that would both weaken the Pragyan Parimita Rath, Nitesh Kumar, Samudrala Gourinath. binding affinity and slow the rate of polymerization. Conversely, we find that School of Life Sciences, Jawaharlal Nehru University, New Delhi, India. Entamoebahistolytica the barbed end of the filament takes on a conformation nearly identical to that Actin cytoskeleton, the key structural protein present in of the ATP monomer, enhancing ATP G-actin’s ability to polymerize as compared drives the process of phagocytosis, which is an essential requisite for the sur- to ADP G-actin. Applying inverse Boltzmann weighting, we can account for the vival of amoeba. Owing to the importance of actin tread-milling, nature has
BPJ 9439_9446 Wednesday, March 6, 2019 457a recruited many Actin Binding Proteins (ABPs) for its regulation. More than 200 faster translational and rotational dynamics that the water experiences around types of ABPs have been reported, each with a different binding and regulatory hydrophobic residues offsets the structural contribution to the entropy, and mechanism. We have channelled our focus on Actin Depolymerizing Factors overall a negligible change in water entropy is observed in the water layers (ADF) that makes a family of proteins, responsible for actin filament severing immediately surrounding the protein. and this property makes them extremely important during phagocytic cup for- mation. From our in silico analysis of the E. histolytica proteome, we found 10 2263-Plat proteins harbouring the ADF-homology domain. We have deciphered the high Acetylation of Actin K328 Contributes to a Loss in Tropomyosin-Mediated resolution crystal structure of a couple of them and the others are underway. Inhibition of Myosin Binding One of the proteins, called Ehcoactosin, interestingly was shown to tread on William M. Schmidt, D. Brian Foster, Anthony Cammarato. an off beaten track and does not depolymerize or sever the actin filaments. Dept Medicine, Johns Hopkins Univ Sch Med, Baltimore, MD, USA. Although belonging to the ADF family it rather stabilized the filamentous actin Lysine deacetylases (KDAC’s) catalyze the removal of acetyl groups from lysine (F-actin). EhTwinfilin contains two ADF-H domains and we were successful in side chains, while lysine acetylases (KAT’s) perform the reverse reaction. KDAC inhibition was shown to increase lysine acetylation of muscle proteins, and was determining the crystal structure of the N-terminal ADF domain. The unique- 2þ ness of this protein lies in the differential protein activity shown by the two do- associated with enhanced Ca -sensitivity in skinned fibers and increased relax- mains; N-terminal domain stabilized the F-actin whereas the C-terminal ation rate in isolated myofibrils. Interestingly, the KAT enzyme, PCAF, directly in vitro, domain depolymerized it. We are reporting these for the first time. Here we pre- acetylates myosin and actin has been implicated as a potential target of in vitro, sent a comparative study to understand the structural and functional differences PCAF. Therefore, we attempted to acetylate actin with PCAF, to deter- present in the many forms of the same ADF-homology domain proteins and mine its potential effect(s) on muscle function. PCAF underwent autoacetylation their role in phagocytosis. In this study we are reporting the key structural and bound to actin, yet failed to acetylate it. Chemical treatment of actin with ace- and functional differences observed in the mechanistic details of the same tic anhydride, however, increased lysine acetylation roughly 200-fold. Surpris- in vitro m ADF-H domain when present in different proteins. ingly, motility (IVM) of (100 g/ml myosin concentration) and tropomyosin affinity for acetylated F-actin were indistinguishable from unacety- 2261-Plat lated control. However, IVM of acetylated F-actin, performed at low myosin con- m Reconstitution of Dynamic Actin Cables with Tunable Lengths centration (12.5 g/ml) and in the presence of tropomyosin, revealed a significant < Luther W. Pollard, Salvatore L. Alioto, Mikeal V. Garabedian, 63% increase in motile filaments relative to unacetylated control (p 0.0001), Bruce L. Goode. suggesting that actin acetylation reduces tropomyosin-mediated inhibition of Biology, Brandeis University, Waltham, MA, USA. myosin binding. Since K326 and K328 have been reported as the most reactive in vivo The sizes and functions of many actin-based cellular structures, such as micro- lysines on actin, are acetylated , and pseudo-acetylation of both modulates muscle contraction, we assessed the effect of K326Q and K328Q pseudo- villi, stereocilia, filopodia and polarized actin cables, depend critically on the 2þ precise control of actin bundle length and turnover dynamics. Yeast cells offer acetylated actin on Ca -mediated thin filament activation via regulated IVM. 5 an ideal system in which to study principles of cytoskeletal length control, Although reconstituted thin filaments containing K326Q (pCa50 = 6.36 2þ 5 because they contain discrete linear actin cables that extend to well-defined 0.03) did not alter Ca -sensitivity relative to WT (pCa50 = 6.37 0.03), 2þ D lengths (3-7 mm), matching the cell compartment size, despite undergoing high- K328Q induced a significant leftward shift in Ca -sensitivity ( pCa50 = 0.19; < ly rapid turnover. Yeast is also simpler compositionally and more genetically p 0.0001), consistent with a loss in tropomyosin-mediated inhibition. The amenable compared to most animal cells. Over the past 30 years, the factors data indicate that actin K328 acetylation modulates contractile function, which in vivo involved in yeast actin cable formation have been identified, and many of these makes it a potential mechanism for regulating muscle performance . individual parts have been studied for their biochemical activities. Yet it has 2264-Plat remained unclear how this complex group of proteins works in concert to build Leiomodin and Tropomyosin, Binding at the Pointed End of the Thin Fil- a single actin-based structure of a specified length and architecture. Here, we aments used purified components to reconstitute the formation of yeast actin cables Dmitri Tolkatchev1, Garry E. Smith1, John R. Cort2, Gregory L. Helms3, that dynamically turnover and have steady-state lengths similar to those Alla S. Kostyukova1. in vivo observed . Cables are rapidly polymerized at one end by formins immo- 1Voiland School of Chemical Engineering & Bioengineering, Washington bilized on beads, in a profilin-dependent manner, and disassembled at the other State Univ, Pullman, WA, USA, 2Fundamental & Computational Sciences end by the combined actions of Cofilin, Coronin, and AIP1. Cable length is Directorate, Pacific Northwest National Laboratory, Richland, WA, USA, tuned by Tropomyosin, which decorates the sides of actin cables and antago- 3The Center for NMR Spectroscopy, Washington State Univ, Pullman, WA, USA. nizes the disassembly-promoting factors. Capping protein restricts actin poly- Leiomodin is an important actin-binding protein responsible for the thin filament merization to the formin-coated beads. Bundling proteins with different formation. Mutations in leiomodins were demonstrated to cause myopathies. The properties alter the geometry and dynamics of the cable networks. Together, spectrum of leiomodin functions remains a subject of an intensive debate. One of our results offer new mechanistic insights into the sculpting of actin cables the models proposes that leiomodin and its homolog tropomodulin modulate the through orchestrated assembly, turnover, and crosslinking. length of the thin filaments in muscle sarcomeres. The modulation is thought to function through competition between leiomodin and tropomodulin for the 2262-Plat pointed end of the thin filaments in a tropomyosin-dependent manner. We studied Structural, Kinetic, and Thermodynamic Response of Water to Mechani- complexes formed by peptides representing the tropomyosin-binding site of leio- cal Unfolding of Spectrin Repeats modin 2 (Lmod2s1) and the N-terminal coiled coil fragment of tropomyosin. By Sarah J. Moe, Torvin Rajala, Alessandro Cembran. using a combination of NMR and MDS methods, we determined the mode of Dept Chem/Biochem, Univ Minnesota Duluth, Duluth, MN, USA. Lmod2s1 binding to the N-terminus of tropomyosin. The structure places leiomo- Many common biophysical processes involve mechanical forces acting on and din at the pointed end of thin filaments. This finding provides compelling evi- between biomolecules. Among these processes, the mechanical unfolding of dence in favor of the competition hypothesis and has important implications proteins has been extensively studied with both experimental and theoretical for the role of leiomodin in forming thin filaments in muscle sarcomeres. approaches because it allows gaining insight in the mechanism of function of this class of proteins. Among the proteins that have been subject to mechanical Platform: Protein Assemblies/Enzyme Function, unfolding studies, spectrin repeats occupy a prominent place, as spectrins are the building block of many structural proteins that are subject to deformation. Cofactors & Post-translational Modifications II Although the response of spectrin repeats to external forces has been thor- oughly studied, little is currently known about the role that the solvent has in 2265-Plat the unfolding mechanism. In this work, we have investigated with steered mo- Hydroxylation of Type I Collagen: Effects on Fibrillar Structure and lecular dynamics simulations the mechanical unfolding of spectrin repeat 19 of Mechanics human dystrophin. We measured several observables to characterize spatially Alekhya A. Kandoor1, Michele Kirchner2, Vered Wineman-Fisher1, and temporally the response of water to protein unfolding in terms of its struc- Yujia Xu2, Sameer Varma1,3. tural, kinetic, and thermodynamic properties. Our results show that the 1Department of CMMB, University of South Florida, Tampa, FL, USA, response of water to protein unfolding is dictated by many factors, among 2Dept Chemistry, CUNY Hunter Col, New York, NY, USA, 3Department of which the nature of the exposed residues, the curvature of the protein surface, Physics, University of South Florida, Tampa, FL, USA. and the size of the protein chain play a major role. We also have determined Proline hydroxylation contributes to type I collagen assembly by increasing the that the exposure of hydrophobic residues results in a structural ordering of wa- thermal stabilities of constituent triple helices. Results from recent studies on ter around the protein, which should result in lower water entropy. Yet, the rat type I fibrils suggest another potential role of proline hydroxylation – since
BPJ 9439_9446 458a Wednesday, March 6, 2019 proline hydroxyls make up 21% of the hydrogen bonds between triple helices, phase separation of P-body component DCP1a, which is a part of the eukaryotic proline hydroxyls can also be expected to contribute to the structural integrity mRNA decapping complex. This dynamic phase separation occurs within sec- and the mechanical properties of fibrils. Here we investigate this hypothesis by onds, in contrast to stress granules that form over minutes, and is rapidly revers- carrying out comparative molecular dynamics studies of fibrils in the presence ible over the course of minutes. We show that the extent of DCP1a condensation and absence of hydroxylation. We investigate this in both rat and human type I is independent of the osmolyte used, is correlated with the degree of cellular vol- fibrils. The structure of the human type I fibril structure is constructed using ho- ume decrease, and is promoted by the trimerization domain of DCP1a. Other mology modeling, and its hydroxylation sites are determined using MS/MS multimeric proteins with valency of at least three, but not less, exhibit compara- sequencing. Expectedly, we find that in both human and rat fibrils, the presence ble condensation responses to osmotic compression. Together, these observa- of hydroxylation does alter the contact interface between triple-helices. Specif- tions are consistent with predictions from phase-separation theory and suggest ically, hydroxylation affects hydrogen bonding patterns, but in different ways that multimerization domains are programmed to mediate protein condensation in the two collagens, despite the two collagens sharing 94% sequence identity. in response to sudden cellular dehydration. We propose that self-interacting pro- However, in both rat and human fibrils, the altered local interactions have little teins spontaneously assemble to counteract the entropic challenge of cellular effect on macroscopic structural properties, including D-band lengths and gap- compression. These observations reveal an under-appreciated physiological overlap ratios. Additionally, hydroxylation only marginally increases Young’s context in which proteins undergo phase separation and unveil a hidden role moduli, although the effects of hydroxylation are more pronounced under large of conserved multimerization domains in post-transcriptional gene regulation (> 5%) strains. Overall, this study suggests that although local interactions be- and metabolic pathways that may predate modern cell biology. tween triple helices in fibrils are sensitive to hydroxylation and that there does exist some degree of plasticity in interfacial contacts between triple helices, the 2269-Plat hallmark macroscopic properties of fibrils are not governed by hydroxylation. Photonic Platform for Detailed Physical Characterization of Liquid Pro- tein Droplets 2266-Plat Gheorghe Cojoc1, Timon Beck1, Saeed Ahmed1, Titus Franzmann2, Superresolution Imaging of Amyloid Structures over Extended Times us- Paul Muller€ 1, Mirjam Schurmann€ 1, Raimund Schlu€bler1, Kyoohyun Kim1, ing Transient Binding of Single Thioflavin T Molecules Elisabeth Fischer-Friedrich1, Simon Alberti2, Jochen Guck1. 1 2 3 1 2 Kevin Spehar , Tianben Ding , Yuanzi Sun , Niraja Kedia , Jin Lu , 1Biotec, Dresden, Germany, 2MPI-CBG, Dresden, Germany. George R. Nahass3, Matthew D. Lew2, Jan Bieschke1,3. 1 Protein bodies are membrane-less organelles which form intracellularly by Biomedical Engineering, Washington University in St Louis, St Louis, MO, means of a liquid-liquid phase separation. Protein droplet formation can be USA, 2Electrical and Systems Engineering, Washington University in St 3 sometimes reversible,but frequently droplets undergo a phase transition. Louis, St Louis, MO, USA, Institute of Prion Diseases, UCL, London, Mounting evidence suggests a direct connection between the phase transition United Kingdom. of protein droplets and diseases. In vitro assays such as droplet fusion assisted Oligomeric amyloid structures are crucial therapeutic targets in Alzheimer’s by optical tweezers,fluorescence recovery after photobleaching or particle and other amyloid diseases. However, these oligomers are too small to be tracking are commonly used to detect and quantify changes in the mechanical resolved by standard light microscopy. We have developed a simple and versa- properties. However, direct measurement of visco-elastic properties and decou- tile tool to image amyloid structures using Thioflavin T without the need for pling of the two is difficult and is based on many assumptions. covalent labeling or immunostaining. Dynamic binding of single dye molecules We present a novel photonic platform for selective droplet delivery into the generates photon bursts that are used for fluorophore localization on a nano- analysis region and their physical characterization. Our method combines a meter scale. Thus, photobleaching cannot degrade image quality, allowing dual-beam laser trap with quantitative phase imaging(QPI). In our in vitro as- for extended observation times. Super-resolution Transient Amyloid Binding says,we used phase separated fused in sarcoma(FUS) protein droplets. The me- (TAB) microscopy promises to directly image native amyloid using standard chanical properties of the FUS droplets can then be inferred from the change of probes and record amyloid dynamics over minutes to days. We imaged amyloid the droplet shape after the optical forces are applied, meanwhile QPI gives us fibrils from multiple polypeptides, oligomeric, and fibrillar structures formed information regarding protein concentration/distribution inside the droplets. b during different stages of amyloid- aggregation, as well as the structural re- Our preliminary results show that the mechanical properties of FUS droplets b modeling of amyloid- fibrils by the compound epi-gallocatechin gallate depend on the salt concentration and pH,but not on the protein concentration (EGCG). in the buffer solution. Higher protein concentration results in more and bigger 2267-Plat droplets,but with similar protein concentration inside. Tuning the power and In Vitro Study of the Effect of Insulin on Amyloid b-Protein Assembly and wavelength of the lasers,we can monitor the dissolving rate and stability of Toxicity the FUS droplet as function of the temperature and for different buffer condi- Kaho Long1, Thomas L. Williams2, Brigita Urbanc1. tions. Moreover, we can track changes, in a decoupled manner, of the viscosity 1Physics, Drexel University, Philadelphia, PA, USA, 2Clarivate Analytics, and elasticity over time. By applying oscillatory mechanical and light mediated Philadelphia, PA, USA. stress on the droplets,we can accelerate the process of phase transition. This in vitro Alzheimer’s disease (AD) is the primary source of dementia among elderly, for photonic platform can be used for detailed screening assays of protein which there is no cure. The leading amyloid hypothesis of AD posits that self- droplet properties and aging. assembly of amyloid b-protein (Ab) into oligomers triggers the AD pathology. Substantial evidence based on longitudinal studies also shows a strong correlation 2270-Plat between AD and type 2 diabetes mellitus, whereby disrupted insulin homeostasis Dissection of Protein Function Within a Bacterial Biomolecular Conden- is associated with an increased risk for AD. Although administration of intranasal sate by In Vitro Reconstitution insulin is explored as a potential therapy against AD, the molecular mechanisms Saumya Saurabh, Lucy Shapiro. of insulin activity in the brain remain unknown. We here investigate the effect of Developmental Biology, Stanford University, Stanford, CA, USA. insulin on Ab42 self-assembly in vitro using Thioflavin T fluorescence spectros- Recent work in micron-sized bacteria has highlighted the presence of membrane- copy and atomic force microscopy. We also examine the effect of insulin on the less microdomains that organize protein signaling complexes. In addition to well ability of Ab42 oligomers to disrupt the integrity of biomimetic lipid vesicles understood ‘‘lock and key’’ protein domains that lie at the core of cell signaling, we using the calcein release assay. Our results demonstrate that insulin attenuates are beginning to understand the role of unstructured protein bimolecular conden- both Ab42 fibril formation and the ability of Ab42 oligomers to disrupt mem- sates in intracellular communication. However, the molecular principles that orga- brane integrity in a concentration dependent manner. Our findings elucidate a nize chemical reactions in these domains are poorly understood. In order to plausible molecular basis for the observed correlations between AD and understand these principles, we have used a combination of in vitro reconstitution type 2 diabetes mellitus and offer a new perspective on therapeutic approaches and single-molecule fluorescence techniques to study one of the two cell pole mi- against AD. crodomains in the Gram negative bacterium, Caulobacter crescentus. Caulobacter divides asymmetrically into a sessile stalked cell and a flagellated swarmer cell. 2268-Plat Much of this asymmetric division is regulated through different signaling path- Multimeric Proteins Reversibly form Condensates Upon Osmotic ways that reside in microdomains at the two cell poles. Specifically, at the stalked Compression pole, two intrinsically disordered proteins, PopZ and SpmX, form a stable bimo- Ameya P. Jalihal. lecular condensate. PopZ recruits the lysozyme homolog SpmX, which further re- Cell and Molecular Biology, University of Michigan, Ann Arbor, MI, USA. cruits the kinase DivJ to establish the stalked cell identity. Reconstitution of this Hyperosmotic stress is known to induce the formation of cytosolic phase- signaling complex on supported lipid bilayers revealed that SpmX and PopZ separated structures called P-bodies in eukaryotes. Using live-cell fluorescence can form phase-separated droplets that sequester DivJ and control its kinase activ- microscopy we observed that hyperosmolarity induces rapid and reversible ity. Further, we have identified molecular determinants of SpmX and PopZ phase
BPJ 9439_9446 Wednesday, March 6, 2019 459a separation that control the material properties and enzyme kinetics in this conden- N-terminal where previous X-ray structures, transmembrane (TM) predicting sate. Our results underscore the relationship between IDR mediated interactions programs, and molecular dynamic (MD) simulations placed it. This is a ma- that govern the physical environment around signaling proteins, and demonstrate jor result that re-frames the nature of the lipid-protein interactions in MscS the applications of disordered scaffolds to modulate biochemical reactions in vivo. and forces us to reexamine years of experimental and computational data and reassess existing mechanistic gating models. In a second major observa- 2271-Plat tion, the structures also reveal that all available MscS crystallographic Effect of Resultant Dipole Moment on Mechanical Stability of Protein- models are in fact, incomplete: Detergent-solubilized MscS is missing an Peptide Complexes anchoring interfacial domain, comprised of the N-terminal end and three 1 2 1 3 Maksim Kouza , Anirban Banerji , Andrzej Kolinski , Irina Buhimschi , additional turns of TM1. In contrast, bilayer-embedded MscS extends >12 Andrzej Kloczkowski2. ˚ 1 2 A towards the outer leaflet of the bilayer when compared to X-ray structures. Faculty of Chemistry, University of Warsaw, Warsaw, Poland, Battelle The N-terminal domain (NTD) forms a novel fold, and we hypothesize, plays Center for Mathematical Medicine, Nationwide Childrens Hospital, 3 a significant role in channel activation. This was further confirmed an alanine Columbus, OH, USA, Center for Perinatal Research, Nationwide Childrens scan of the NTD assessed by cell survival assays together with high-speed Hospital, Columbus, OH, USA. pressure clamp electrophysiology. The present observations reshape our un- Protein-peptide interactions play essential roles in many cellular processes and derstanding of the mechanism of force-from-lipids gating and suggest that their structural characterization is the major focus of current experimental and allosteric interactions between TM segments play a fundamental role in theoretical research. Two decades ago, it was proposed to employ the steered mo- MscS gating. lecular dynamics to assess the strength of protein-peptide interactions1. The idea behind using steered molecular dynamics simulations is that the mechanical sta- 2274-Plat bility can be used as an efficient alternative to computationally highly demanding 2,3 Structures and Simulations of Membrane Adjacent Fragments of estimation of binding affinity and aggregation rate . However, mechanical sta- Protocadherin-15 bility defined as a peak in force-extension profile depends on the choice of the Pedro De-la-Torre1, Yoshie Narui1, Deepanshu Choudhary1, pulling direction. Here we propose an uncommon choice of the pulling direction Raul Araya-Secchi2, Marcos Sotomayor1. along resultant dipole moment vector, which has not been explored in simulations 1Chemistry and Biochemistry, Ohio State University, Columbus, OH, USA, so far. Using explicit solvent all-atom MD simulations, we apply steered molec- 2Structural Biophysics, Section for Neutron and X-ray Science, Niels Bohr ular dynamics technique to probe mechanical resistance of protein-peptide sys- 4 Institute, University of Copenhagen, Copenhagen, Denmark. tem pulled along two different vectors . A novel pulling direction, along the The tip link, a fine filament that connects neighboring stereocilia of inner-ear resultant dipole moment vector, results in stronger forces compared to commonly hair cells, is made of two unique, non-classical cadherins essential for hear- used peptide unbinding along center of masses vector. Our results demonstrate ing. One half of the tip link is protocadherin-15 (PCDH15), which is that resultant dipole moment is one of the factors influencing the mechanical sta- comprised of 11 extracellular cadherin (EC) repeats followed by a mem- bility of protein-peptide complex. brane adjacent domain (MAD12). Here, we report the structures of pig Science 1) H. Grubmuller et al., 271, 997-999 (1996) PCDH15 EC10-12 and mouse PCDH15 EC9-MAD12, which revealed a J Chem Phys 2) M. Kouza et al., 148, 215106 (2018) bent EC9-10 configuration as well as parallel homodimerization mediated Physical 3) M. Kouza, A. Banerji, A. Kolinski, I. Buhimschi, A. Kloczkowski, by both EC11 and a unique MAD12 that adopts a ferredoxin-like fold. Chemistry Chemical Physics , 19, 2990-2999 (2017) Steered molecular dynamics simulations indicate that when force is applied Mole- 4) M. Kouza, A. Banerji, A. Kolinski, I. Buhimschi, A. Kloczkowski, to both ends of the protein, the bent EC9-10 linker fully extends before un- cules 23, 1995 (2018) folding of MAD12 occurs. Simulations also suggest that MAD12 is mechan- 2272-Plat ically weak and may unfold before tip-link rupture. In addition, sequence Coarse-Grained and Atomistic Simulations of the Mature HIV Capsid and analyses and structural modeling predict the existence of similar domains Related Restriction Factors in cadherin-23, protocadherin-24, and the ‘‘giant’’ FAT and CELSR cadher- Alvin Yu1, Barbie K. Ganser-Pornillos2, Owen Pornillos2, Gregory A. Voth1. ins, indicating that some of them may also exhibit MAD-induced parallel 1Chemistry, University of Chicago, Chicago, IL, USA, 2Univ Virginia, dimerization. Together, these results provide key information about the Charlottesville, VA, USA. elastic properties of the tip link and give insight into the architecture of The human immunodeficiency virus (HIV) uses enclosing capsid (CA) proteins the full-length PCDH15 homodimer. to package and release its genetic information into newly infected host cells. CA forms fullerene cones that compartmentalize viral RNA. TRIM5a is an 2275-Plat important antiretroviral restriction factor found in rhesus macaques that blocks Understanding Piezo1’s Relationship with Lipids 1 2 3 4 HIV infection. Recent cryo-electron microscopy analysis demonstrate cyto- Amanda Buyan , Charles D. Cox , Jonathan Barnoud , Boris Martinac , 5 1 plasmic TRIM5a self-assembles into hexagonally patterned nets that encage Siewert-Jan Marrink , Ben Corry . 1Res Sch of Biology, Australian National Univ, Acton, Australia, 2Victor mature capsids, with a symmetry and spacing that matches the underlying 3 CA lattice. In this work, we use atomistic simulations to probe structural and Chang Cardiac Res Inst, Sydney, Australia, University of Groningen, Groningen, Netherlands, 4Dept Molec, Victor Chang Cardiac Res Inst, thermodynamic properties of the HIV capsid, and top-down coarse-grained 5 modeling to understand the mechanisms of how TRIM5a recognizes and as- Darlinghurst, Australia, Dept Biophys Chem/Molecular Dynamics, Univ sembles on CA lattices. Groningen, Groningen, Netherlands. The sensations of touch and hearing, along with many other physiological pro- cesses, require cells to be able to sense and react to mechanical stimuli. One Platform: Mechanosensation way this is done is via membrane embedded mechanically gated channels. These channels can detect forces through deformation of the lipid bilayer, deemed the 2273-Plat ‘‘force-from-lipids’’ principle. Bacterial homologues, such as MscL and MscS, High-Resolution Structures of MscS in a Lipid Bilayer: Reinterpreting exemplify this principle and have been studied for the past couple of decades, and ‘‘Force from Lipids’’ Activation in Mechanosensitive Channels have contributed greatly to our understanding of mechanically gated channels. Bharat Reddy1, Allen Lu1, Navid Bavi1, Allen Hsu2, Mario Borgnia2, However, understanding the underlying molecular force sensing mechanisms, Eduardo Perozo1. and how similar bacterial and eukaryotic mechanosensitive channels are in terms 1Univ Chicago, Chicago, IL, USA, 2GISBL/DIR/NIEHS/NIH, Research of their gating mechanisms, remains an open question. Triangle Park, NC, USA. The recent discovery and structure elucidation of the first eukaryotic mechan- Prokaryotic mechanosensitive (MS) channels are some of the best character- ically gated channels, named the PIEZO family, allows for the mechanisms of ized force-activated ion channels. Given the critical role of lipid-protein in- mechanical gating to be studied in higher organisms. Since their discovery, teractions in defining force through lipid channel gating, this is a question of PIEZO channels have been implicated in many cellular processes, but the paramount importance in the field. We have determined several high- gating mechanism and the role that lipids play in PIEZO’s mechanics remain resolution cryo-EM structures of MscS in nanodisc (ND) environments that elusive. To this end, we are using a combination of electrophysiology and mo- varied the membrane thickness, charge, size, and rigidity. Compared to pre- lecular simulations to understand protein-lipid interactions between Piezo1 and viously solved crystal and low-res EM structures, the key observation from relevant lipids in model mammalian bilayers, and ultimately the role that lipids our MScS ND structures is the unexpected location of the lipid bilayer in have on Piezo1’s activation. We are able to show that piezo has specific inter- relation to MscS TM segments: We find the bulk of the ND-containing lipid actions with a number of membrane components that likely play a role in medi- annulus surrounding the channel to be almost a full bilayer leaflet towards the ating the bilayer-protein interaction.
BPJ 9439_9446 460a Wednesday, March 6, 2019
2276-Plat Piezo1 significantly reduced the nuclear shrinkage under shear stress. Activa- Dietary Fatty Acids Fine-Tune Piezo1 Activity tion of Piezo1 with Yoda1 caused similar nucleus shrinkage without shear Luis O. Romero1, Alejandro Mata-Daboin1, Andrew Massey2, stress. These results demonstrate that Piezo1 channel is accountable for trans- Francisco J. Sierra Valdez3, Chauhan C. Subhash2, Julio F. Cordero-Morales1, mitting the shear force input to nuclei via Ca2þ signaling. To assess the relative Valeria Vasquez1. contributions of Ca2þ and cytoskeleton reorganization on force transduction, 1Physiology, UTHSC, Memphis, TN, USA, 2Pharmaceutical Sciences, we examined F-actin reorganization under shear stress and static conditions, UTHSC, Memphis, TN, USA, 3Science, ITESM, Memphis, TN, USA. and showed that reorganization is not necessary for the nucleus shrinkage. Mechanosensitive ion channels rely on membrane composition to transduce These results emphasize the role of the mechanosensitive channels as primary physical stimuli into electrical signals. The Piezo1 channel is a mechanosen- transducers in force transmission to the nucleus. sitive channel that regulates physiological processes, including vascular architecture and remodeling, cell migration, and erythrocyte volume. Muta- tions in Piezo1 are associated with dehydrated hereditary stomatocytosis, a 2279-Plat hemolytic anemia characterized by increased cation permeability and dehy- Structural Relationship between the Putative Hair Cell Mechanotransduc- drated erythrocytes. While it is widely agreed that Piezo1 is activated by tion Channel TMC1 and TMEM16 Proteins 1 2 membrane tension, the identity of the membrane lipids that modulate Piezo1 Angela Ballesteros Morcillo , Maria Cristina Fenollar-Ferrer , 1 function and the molecular mechanisms by which they do so remain un- Kenton J. Swartz . 1Molecular Physiology and Biophysics Section, NINDS NIH, Bethesda, MD, known. Dietary fatty acids are among the membrane lipid components that 2 regulate ion channel function. The most common fatty acids in membrane USA, NIDCD-NIMH, Bethesda, MD, USA. lipids have 14 to 22 carbon atoms and carry one unsaturation, and as Our senses of touch, hearing, balance, and proprioception depend on mechan- many as six double bonds. These chemical features confer cells the capacity ically gated ion channels, which transduce mechanical energy into electrical to control membrane thickness and fluidity. We use lipidomics and electro- signals that are transmitted to the brain. Previous studies on the mechanisms physiology to demonstrate that Piezo1-mediated mechano-currents are of hearing have elucidated the biophysical properties of the mechanotransduc- modulated by dietary fatty acids in different cell types. We found that tion (MET) channel essential for hearing, yet its molecular identity and struc- margaric acid (MA), a saturated fatty acid, inhibits Piezo1 activation cur- ture remain elusive. The trantasmembrane channel-like 1 protein (TMC1) rents, whereas u-3 and u-6 polyunsaturated fatty acids (PUFAs) modify localizes to the site of the MET channel, interacts with the tip-link responsible its inactivation. Differential scanning calorimetry and atomic force for mechanical gating, and genetic alterations in TMC1 alter MET channel microscopy experiments show that membranes enriched in MA are more properties and cause deafness, supporting the hypothesis that TMC1 forms rigid and display higher bending stiffness than to those containing PUFAs. the MET channel. We generated a model of TMC1 based on X-ray and cryo- Our findings demonstrate that saturated and polyunsaturated fatty acids EM structures of TMEM16 proteins, revealing the presence of a large cavity contained in the plasma membrane modulate the mechanical response of near the protein-lipid interface that also harbors two TMC1 mutations Piezo1. (hTMC1 p.M418K and p.D572N/H) that cause autosomal dominant hearing loss, suggesting that it could function as a permeation pathway. We also find 2277-Plat that hair cells are permeable to 3 kDa dextran labeled with Texas Red, and Enantiomeric Ab Peptides Inhibit the Fluid Shear Stress Response of that dextran permeation requires TMC1/2 proteins and functional MET chan- PIEZO1 nels, supporting the presence of a large permeation pathway and the hypothesis Philip A. Gottlieb1, Mohammed M. Maneshi1, Frederick Sachs1, that TMC1 is a pore forming subunit of the MET channel complex. Susan Z. Hua2. 1Dept Physiol/Biophys, State Univ New York Buffalo, Buffalo, NY, USA, 2Dept Mech & Aerospace Eng, State Univ New York Buffalo, Buffalo, NY, 2280-Plat USA. Cysteine Substitution Reveals the Pore-Forming Region of TMC1 in Hair Using precision fluid shear stress as a stimulus on a cell line overexpressing Cell Sensory Transduction Channels 1 2 1 1 PIEZO1, we show that Ab peptide monomers inhibit PIEZO1 at fM to pM con- Bifeng Pan , Nurunisa Akyuz , Xiao-Ping Liu , Yukako Asai , 1 3 2 1 centrations. The Ab aggregates were much less potent. The effect of Abson Carl Nist-Lund , Kiyoto Kurima , Bruce Derfler , Bence Gyo¨rgy , 2 4 4 PIEZO gating did not involve peptide-protein interactions since the D and L Walrati Limapichat , Sanket Walujkar , Lahiru Wimalasena , b Marcos Sotomayor4, David Corey2, Jeffrey R. Holt1. enantiomers had similar effects. A peptides co-localized with PIEZO1 as 1 b Otolaryngology, Harvard Medical School/Boston Children’s Hospital, seen with a fluorescent derivative of A and a fluorescently tagged PIEZO1. 2 b Boston, MA, USA, Neurobiology, Harvard Medical School, Boston, MA, To better understand the inhibitory effects of A , we examined the effects in 3 4 a wound healing assay. Simply over-expression of PIEZO1 in HEK293 cells USA, Otolaryngology, NIH/NIDCD, Bethesda, MD, USA, The Ohio State increased cell migration velocity 10-fold, and both enantiomeric Ab peptides University, Colubmus, OH, USA. and GsMTx4 inhibited migration. We examined the correlation of PIEZO1 Molecular identification of ion channels that mediate sensory transduction function with the tension in F-actin using a genetically encoded fluorescent has been a major focus in the field of sensory biology for many years, yet, stress probe. Ab peptides increased resting stress in F-actin, correlated with the proteins that form sensory transduction channels required for hearing the Ab block of PIEZO1-mediated Ca2þ influx. Ab inhibition of PIEZO1 in and balance have not been definitively identified. TMC1 and TMC2 have the absence of stereospecific peptide-protein interactions shows that Ab pep- been proposed as possible pore-forming subunits, but the pore region itself tides modulate both cell membrane and cytoskeletal mechanics to control has not been identified. We generated 17 unique cysteine substitutions in PIEZO1-triggered Ca2þ influx. TMC1 and packaged the mutant sequences into AAV2/1 vectors. The vectors were injected into the inner ears of P1 mice that lacked expression of endog- 2278-Plat enous Tmc1 and Tmc2. Injected ears were dissected at P7 and the organ of Piezo1 Mediated Ca2D Signaling Causes Nuclear Shrinkage under Fluid Corti was cultured for an additional 8 to 10 days. We used rapid hair bundle Shear Stress deflections and the whole-cell, tight-seal technique to record sensory trans- Deekshitha Jetta1, Philip A. Gottlieb2, Frederick Sachs2, Susan Z. Hua3. duction currents from 566 inner hair cells. We applied four different cysteine 1Dept of Mech Eng, University at Buffalo, Buffalo, NY, USA, 2Dept Phys modification reagents methanethiosulfonate (MTS) to native mouse sensory and Biophys, University at Buffalo, Buffalo, NY, USA, 3Dept Phys and hair cells. We identified thirteen TMC1 residues that altered the biophysical Biophys/Mech Eng, University at Buffalo, Buffalo, NY, USA. properties of hair cell mechanosensory transduction. Five residues were Nuclei of cells respond to local mechanical cues with changes in their size, rapidly modified by MTS reagents to confer changes in whole-cell current morphology, and motility, to maintain tissue homeostasis and proper functions. amplitudes. Three sites examined in detail, caused changes in single- Previous studies indicated that the external forces are coupled to nuclei through channel current amplitudes. Eleven sites altered calcium selectivity and the surrounding cytoskeleton network linked to the nuclear envelope, but the cysteine substitution at one site blocked current almost entirely. The data molecules that directly interact with outside environments are unclear. Here support a revised TMC1 topology with ten transmembrane domains and iden- we show that the fluid shear stress applied to MDCK cells caused the nucleus tify four TMC1 transmembrane domains that line the permeation pathway of to shrink through a Ca2þ dependent signaling pathway. Inhibiting Ca2þ perme- mechanosensory transduction channels in mammalian auditory and vestibular able mechanosensitive Piezo1 channels, with the specific inhibitor GsMTx4, hair cells. We conclude TMC1 is a major pore-forming subunit of the hair eliminated the nuclear changes. Piezo1 knockdown with miRNA that targets cell sensory transduction channel.
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Symposium: Membrane Organization and and experiments. Molecular dynamics (MD) simulations of an isolated subunit of the pore domain of the Kv1.2 and KcsA channels in a lipid bilayer were Sculpting by Proteins carried out, followed by a Markov State Model (MSM) analysis. The results show that the monomer of these channels can adopt a very wide range of con- 2281-Symp formations while the two transmembrane (TM) helices and the pore helix Reverse Topology Membrane Scission by the ESCRTs retain their secondary structure. This view is supported by NMR spectroscopy James H. Hurley. showing that the TM helices of single monomers of the KcsA channel Molecular and Cell Biology, University of California, Berkeley, Berkeley, embedded in nanodisc or bicelle are partially disordered. A KcsA mutant CA, USA. construct with a disulphide bridge engineered between the two TM helices The narrow membrane necks formed during viral, exosomal and intra- (A29C-A109C) shows an HSQC spectrum with well-dispersed peaks and endosomal budding from membranes, as well as during cytokinesis and limited heterogeneity on NMR time-scale, indicating that the monomer can related processes, have interiors that are contiguous with the cytosol. be locked into a ‘‘native-like’’ conformation by reducing the disorder of the Severing these necks involves action from the opposite face of the membrane 2 TMs helices. The refolding and assembly of the tetrameric channel in lipo- as occurs during the well-characterized formation of coated vesicles. This somes is examined using a gel-based refolding assay and fluorescent energy ‘‘reverse-topology’’ membrane scission is carried out by the endosomal sort- transfer (FRET) experiments. The results suggest that the wild-type monomers ing complex required for transport (ESCRT) proteins, which form filaments, are partially disordered in the bilayer, with a high proclivity to misfold due to flat spirals, tubes and conical funnels that are thought to direct membrane re- stable non-native helical tetramerization, and that re-folding is limited by a modelling and scission. Their assembly, and their disassembly by the slow concentration-independent unimolecular event. We speculate that the ATPase vacuolar protein sorting-associated 4 (VPS4) have been intensively slow step could correspond to the re-orientation of the monomers and the studied, but the mechanism of scission has been elusive. To probe the bio- insertion of the pore helix into a disordered tetrameric assembly of 8 TM physical mechanism of membrane scission, we encapsulated a minimal helices. ESCRT module consisting of ESCRT-III subunits Snf7, Vps24, and Vps2, þ and the AAA ATPase Vps4 such that membrane nanotubes reflecting the 2284-Symp correct topology of scission could be pulled from giant vesicles. Upon Capturing Sequential Steps of Dynamin-Mediated Fission by Cryo-EM ATP release by photo-uncaging, this system was capable of generating forces Jenny E. Hinshaw1, Leopold Kong1, Kem A. Sochacki2, Huaibin Wang1, within the nanotubes in a manner dependent upon Vps4 catalytic activity and Bertram J. Canagarajah1, Andrew D. Kehr1, William J. Rice3, Vps4 coupling to the ESCRT-III proteins. Imaging of scission revealed Snf7 Marie-Paule Strub2, Justin W. Taraska2. and Vps4 puncta within nanotubes whose presence followed ATP release, 1LCMB, NIH, Bethesda, MD, USA, 2NHLBI NIH, Bethesda, MD, USA, correlated with force generation and nanotube constriction, and preceded 3New York Structural Biology Center, Simons Electron Microscopy Center, scission. Implications for the scission mechanism and remaining open ques- New York, NY, USA. tions will be discussed. Dynamin, a large GTPase, mediates membrane fission by assembling around and cleaving the necks of budding vesicles. The molecular details of 2282-Symp dynamin-driven membrane scission, however, remain elusive. Here, we report Membrane Curvature and the ABC Transporter BmrA: A Yin & Yang a 3.75 A˚ resolution cryo-EM structure of the constricted, membrane- Story associated helical polymer of human dynamin-1 in the GMPPCP bound state. Patricia M. Bassereau1, Ajay K. Mahalka1, Su-Jin Paik1, Giovanni Manzi1, Compared to the soluble tetramer, membrane-associated dynamin self- Andrew Callan-Jones2, Daniel Levy1. assembles across the helical rungs via the GTPase domain, and interacts 1PhysicoChimie Curie Lab., Institut Curie, Paris, France, 2Laboratoire with the lipid membrane with its Pleckstrin homology domain. These differ- Matie`re et Syste`mes Complexes, University Paris Diderot, Paris, France. ences translate to a severely bent bundle signaling element (BSE), which con- Matching protein shape to membrane curvature provides a possible targeting nects the GTPase domain with the rest of the protein. The BSE conformation mechanism for membrane proteins. However, how much membrane curvature is asymmetric across the inter-rung GTPase interaction, and is unique can affect membrane protein function and reciprocally, how protein conforma- compared to all known nucleotide bound states of dynamin. Mutations that tional change modulates its localization has not been investigated. We have disrupt the BSE kink impaired endocytosis but not localization of the protein. studied BmrA, a bacterial homologue of the human ABC (ATP Binding Furthermore, the cryo-EM structure refines the membrane-bound orientation Cassette) transporter P-glycoprotein, to understand the feedback between pro- of the dynamin stalk, which mediates most of the oligomeric interactions tein function, conformation and the physicochemical properties of the sur- that were validated by cell-based assays, and rationalizes previously published rounding membrane including lipid composition and membrane curvature. biochemistry. In addition, the new model also fits well into the 10 A˚ angstrom Upon ATP binding and hydrolysis, BmrA switches conformation between an resolution cryo-EM map of a super-constricted GTP-bound dynamin polymer, apo- and a post-hydrolytic state. The protein was reconstituted in small and gi- confirming that the new interfaces are sufficient to bring the lipid bilayer close ant liposomes (GUVs). We could first demonstrate with small liposomes that to the hemi-fission state. These results reveal molecular snapshots of the bio- different negatively charged and PE lipids stimulate the protein ATPase activ- logically active form of dynamin during the fundamental process of mem- ity. Furthermore, we showed that the activity is reduced by at least two-fold brane constriction. when decreasing the diameter of the liposomes from 150 nm down to 30 nm. Next, from the analysis of fluorescent proteins’ enrichment in nanotubes pulled Symposium: Molecular and Transcriptional from GUVs containing BmrA at surface fraction z 1%, we could demonstrate that the trans-membrane domain of BmrA exhibit 2 contrasted spontaneous cur- Regulation of Cardiac E-C Coupling 1 vature Cp: a high positive Cp in the apo-state, and a negative Cp ( 1/7.5 nm ) in the post-hydrolytic state. In the apo-state, BmrA sorting kinetics is very slow 2285-Symp but leads to a gradual constriction of the tube at constant membrane tension, Acute Loss of cMyBP-C Induces Auto-Oscillatory Contractions in Per- down to tube radius z 30 nm and an enrichment factor of the order of 30. Even- meabilized Cardiomyocytes: Implications for Reverse E-C Coupling? tually, analysis of protein sorting in the presence of ATP suggests that BmrA Samantha P. Harris. remains a large fraction of its cycle in the post-hydrolytic state. Globally, Cellular and Molecular Medicine, University of Arizona, Tucson, AZ, USA. this shows that localization of this type of proteins in cell membranes should Mutations in cardiac myosin binding protein-C (cMyBP-C) are a frequent cause strongly depend on their activity, which itself is modulated by membrane of hypertrophic cardiomyopathy (HCM), affecting 1 in 500 and the most com- shape. mon cause of sudden cardiac death in young people. Unlike mutations in myosin, the vast majority of mutations in cMyBP-C are truncation mutations 2283-Symp that lead to reduced cMyBP-C expression (haploinsufficiency). However, the Structural Dynamics of Potassium Channel Monomer in a Membrane mechanism(s) by which loss of cMyBP-C affects contraction and how muta- Environment and Tetrameric Assembly tions in sarcomeric proteins ultimately lead to arrhythmias are not well under- Benoit Roux, Kevin Song, Young Hoon Koh, Eduardo Perozo, stood. Here we investigated effects of cMyBP-C loss using a novel ‘‘cut and Tobin R. Sosnick. paste’’ approach in detergent permeabilized myocytes from gene-edited Department of Biochemistry and Molecular Biology, University of Chicago, ‘‘Spy-C’’ mice developed exclusively in our lab. Spy-C mice express Chicago, IL, USA. cMyBP-C with a TEV protease site and a ‘‘SpyTag’’ sequence embedded be- The folding of a potassium channel monomer and its assembly into a tetramer tween domains C7 and C8. Permeabilized Spy-C myocytes were first treated was investigated using a multi-disciplinary approach combining computations with TEV protease to ‘‘cut’’ cMyBP-C and then recombinant C0-C7 domains
BPJ 9447_9453 462a Wednesday, March 6, 2019 were ‘‘pasted’’ back via ‘‘SpyCatcher’’ which covalently binds SpyTag. Re- compromised the gain of E-C coupling. In contrast, we found that the sults showed that similar to genetic knockout of cMyBP-C, acute loss of LCC-RyR signaling efficiency and the E-C coupling gain were increased cMyBP-C after TEV treatment caused an apparent increase in cross-bridge significantly during hibernation in ground squirrels. The structural consoli- 2þ cycling rates (ktr) and reduced Ca sensitivity of tension. Strikingly, there dation of LCC-RyR signaling apparatus was in parallel with increased was also a dramatic increase in auto-oscillatory contractions propagated across expression of JPH2 and caveolin-3 (CAV3), the interaction between which multiple sarcomeres. Oscillations were especially evident at sub-maximal anchors the SR to TTs. Interestingly, the promoters of both JPH2 and CAV3 2þ [Ca ] near the pCa50 of force generation and were sustained for seconds to mi- exhibited binding sites for the serum response factor (SRF), which initiated nutes despite conditions of constant Ca2þ. Oscillations abruptly ceased upon JPH2 and CAV3 transcription by binding the transcription co-factor myocar- ligation of new recombinant cMyBP-C N’-terminal domains via SpyCatcher. din. During hibernation, the expression of myocardin was increased while Results suggest a previously unrecognized role of cMyBP-C to dampen me- that of microRNA-24, which mediates post-transcriptional suppression of chanical activity originating from contracting sarcomeres and raise the JPH2 expression, was decreased. Adenovirus-directed overexpression of intriguing possibility that loss of cMyBP-C through haploinsufficiency could myocardin, but not that of SRF, increased JPH2 and CAV3 expression, elicit mechanical feedback that influences electrical activity through reverse improved LCC-RyR signaling efficiency and enhanced E-C coupling gain E-C coupling in intact cardiomyocytes. This work supported by NIH in ground squirrel cardiomyocytes. The myocardin-mediated conjunct regu- R01HL080367 and AHA IRG 17IRG33411051. lation of JPH2/CAV3 transcription explained the cardiac specificity of JPH2 expression, and revealed a stoichiometry-optimized mechanism for E-C 2286-Symp coupling regulation. This finding not only elucidated the mechanism for cBIN1: From T-Tubule Folds to Dyad Organization, to Microparticles and maintained cardiac contractility in hibernation, but also provides new ideas Clinical Use for studying the ‘‘loss-of-function’’ remodeling in heart diseases, such as Robin Shaw. heart failure. Smidt Heart Institute, Cedars-Sinai Medical Center and UCLA, Los Angeles, CA, USA. Platform: Protein Dynamics and Allostery II BIN1 is a membrane curvature protein which is richly spliced. We have identified a cardiac muscle splice variant (BIN1þ13þ17) now named 2289-Plat cBIN1. In the past decade, it has been found that cBIN1 is responsible for pH-Driven Conformational Reorganization of Proteins: NMR Spectros- calcium channel trafficking to T-tubules as well as organizing microfolds copy Study with Buried Lys Residues of cardiac T-tubule membrane. The folds affect the local flow of ions and Christos M. Kougentakis1, Ananya Majumdar2, Jamie L. Schlessman3, help arrange cardiac dyads between L-type calcium channels and ryanodine Bertrand Garcia-Moreno1. receptors. In heart failure, cBIN1 is transcriptionally decreased, affecting 1Department of Biophysics, Johns Hopkins University, Baltimore, MD, USA, T-tubule architecture, and negative impacting calcium transients as well 2Biomolecular NMR Center, Johns Hopkins University, Baltimore, MD, as cardiac function. We recently explored the possibility that the cBIN1- USA, 3Chemistry Deptartment, US Naval Academy, Annapolis, MD, USA. microfolds are externally released from cardiomyocytes. Using electron The pH sensitivity of proteins is essential for biological energy transduction microscopy imaging with immunogold labeling, we found that cBIN1 exists and for cellular and physiological homeostasis. The thermodynamic basis of in membrane vesicles about 200 nm in size, which is consistent with the pH effects in proteins is well understood but detailed mechanistic under- size of microparticles (MPs). In mice with cardiac-specific heterozygous standing of how changes in pH drive conformational reorganization in pro- Bin1 deletion, cBIN1-MPs are appropriately reduced in plasma. In human teins is not. This was addressed systematically using NMR spectroscopy to plasma, cBIN1 detection by an enzyme-linked immunosorbent assay characterize pH-dependent conformational changes coupled to the ioniza- (ELISA), is decreased in humans with heart failure, a condition with tion of residues buried in the hydrophobic core of a protein. The study reduced cardiac muscle cBIN1. The clinical utility is significant. A cBIN1 used 25 variants of staphylococcal nuclease with buried Lys residues. The Score (CS) based on plasma cBIN1 levels was studied in patients with heart pKa values of 19 of 25 of these internal Lys residues are depressed relative failure. We find that CS outperforms current biomarkers for the diagnosis of to the normal pKa of Lys in water, some by as much as 5 pH units. This shift heart failure and also can accurately predict future hospitalization in heart in pKa ensures that the ionizable moiety stays neutral at pH values below failure patients. The data indicate that cBIN1 is released from T-tubule 10 when it is buried, consistent with what is expected from the unfavorable membrane in MPs, and that based CS as a blood test can serve as a clinical transfer of a charge from water to a hydrophobic environment. In most biomarker of cardiomyocyte remodeling, aiding in the management of heart cases, ionization of the buried Lys shifts the conformation of the protein failure patients. into a state where the charged Lys can contact water. This study contributes a detailed description of the location, amplitude and time scale of 2287-Symp reorganization coupled to the ionization of buried Lys residues. The struc- Regulation of the RyR2 Calcium Release Channel by SPEG tural details of the conformational response were characterized with back- Xander H.T. Wehrens. bone chemical shift perturbation analysis, titrations of the buried Nz Cardiovascular Research Institute, Baylor College of Medicine, Houston, atom, and in some cases even X-ray crystallography. At least in this protein, TX, USA. the anomalous pKa values of buried groups are governed by the propensity It is well established that altered sarcoplasmic reticulum (SR) Ca handling of the protein to reorganize. These results demonstrate that computational plays a key role in the pathogenesis of heart failure and cardiac arrhythmias. modeling of pH dependent processes in proteins will require prediction Altered post-translational regulation of the ryanodine receptor (RyR2) is of alternative conformational states and accurate calculation of free en- thought to promote aberrant SR Ca release; however, it remains controversial ergies; this remains a significant challenge even under the most favorable which kinases and phosphatases underlie disease-associated changes. Recent circumstances. studies from our lab have demonstrated that ‘striated muscle preferentially ex- pressed gene’ (SPEG) - a serine/threonine protein kinase - binds to RyR2 and 2290-Plat regulates its activity. In this presentation, the effects of SPEG on RyR2 and SR Rhodopsin Hydration Dynamics Studied by Solid-State Deuterium NMR Ca handling will be discussed. Spectroscopy Nipuna Weerasinghe1, Suchitranga M.D.C. Perera1, Trivikram R. Molugu1, 2288-Symp Andres M. Salinas1, Michael F. Brown1,2. Conjunct Upregulation of Junctophilin-2 and Caveolin-3 Transcription 1Chemistry and Biochemistry, Univ Arizona, Tucson, AZ, USA, Enhanced Excitation-Contraction Coupling Efficiency in Hibernating 2Department of Physics, University of Arizona, Tucson, AZ, USA. Ground Squirrels The physiological properties of proteins strongly depend on the dynamics of Rong-Chang Li, Lei Yang, Yi-Chen Li, Bin Xiang, Li-Peng Wang, water in the hydration shell. Knowledge of hydration water dynamics is vital Xiao-Ting Wang, Jing-Hui Liang, Shi-Qiang Wang. for comprehending protein function [1]. Developing methods for the study Peking University, Beijing, China. of hydration dynamics of membrane proteins such as GPCRs is crucial for Cardiac excitation-contraction (E-C) coupling is controlled by the signaling pharmaceutical applications. We choose rhodopsin as a prototype for our in- between L-type Ca2þ channels (LCCs) in the cell membrane/T-tubules vestigations of GPCRs, where the role of water in its activation mechanism (TTs) and ryanodine receptor (RyR) Ca2þ release channels in the sarco- has been explored using quasi-elastic neutron scattering (QENS) methods plasmic reticulum (SR). In heart failure, decreased expression of [2]. First, we developed methods for generating powdered samples, notable junctophilin-2 (JPH2) decreased the efficiency of LCC-RyR signaling and for exceptionally high protein content while retaining photochemical
BPJ 9447_9453 Wednesday, March 6, 2019 463a functionality. The powdered GPCR samples are suitable for biophysical ex- experimental free energies affirm an accurate and robust approach. Thus, periments to studying the dynamic process of protein activation [3], where alchemical free energy calculations provide a powerful tool for the analysis the protein is hydrated using vapor phase absorption in a controlled manner. of correlated amino acid mutations and their interaction over large distances. We hypothesized that the internal protein dynamics (b-fluctuations) are In fact, the access to accurate mutation free energies and nonadditivities coupled to the solvent shell while large-scale protein domain motion (a-fluc- potentially enables monitoring of the involved interaction pathway throughout tuations) are coupled to the bulk solvent. To gain a comprehensive hold on the entire protein. functional water interactions, we studied rhodopsin hydration shell dy- namics using solid-state 2H-NMR spectroscopy at various hydration levels 2293-Plat and temperatures [4]. From the spectral features at 200 K, we established Towards Comprehensive Control and Design of Targeted Signalling in two dynamically distinct hydration regions. The broad quadrupolar splitting Allosteric Regulation of Protein Activity about 180 kHz is due to bulk water; however, the anisotropic narrow compo- Enrico Guarnera1, Wei-Ven Tee1,2, Zhen Wah Tan1, Igor N. Berezovsky1,2. nent is a result of water molecules dynamically perturbed by rhodopsin. 1Bioinformatics Institute, A*STAR, Singapore, Singapore, 2Department of Additional solid-state 2H-NMR relaxation investigations of molecular dy- Biological Sciences, National University of Singapore, Singapore, Singapore. namics of the rhodopsin-bound water shell are in progress. Our future focus We developed a computational approach combining protein harmonic model is on a comparative study of water dynamics in the light activated state and with statistical mechanical formalism in a perturbation-based approach, which the dark state. [1] M.T. Rao et al. (2009) J.Phys.Chem.B 113, 6994. [2] allows one to evaluate per-residue allosteric free energy as a result of ligand Shresta et al. (2016) J.Phys.Chem.Lett. 7, 4130. [3] S.M.D.C. Perera et al. binding and/or mutations. The model was implemented in the AlloSigMA (2016) J.Phys.Chem.Lett. 7, 4239. [4] T.R. Molugu et al. (2017) Chem.Rev. web-server that provides an interactive framework for estimating allosteric 117, 12087. free energy, can help to find potential latent regulatory exosites and candidates for allosteric mutations. Because of the critical role of global protein dy- 2291-Plat namics in allosteric signaling, we hypothesized reversibility of allosteric Using Histone H1 Derived Peptides to Investigate Binding Affinity and communication, according to which allosteric sites can be detected via the Inter-Domain Dynamics in Human Pin1 perturbation of the functional sites. Validating the ‘‘reversibility hypothesis’’, Dinusha Jinasena1, Jerrano Bowleg1, Robert Simmons1, Yue Zhang2, we also show that, in addition to known allosteric sites, perturbation of func- Steven R. Gwaltney1, Nicholas C. Fitzkee1. tional sites unravels rather extended protein regions, which can host latent 1Department of Chemistry, Mississippi State University, Mississippi State, regulatory exosites. These protein parts that are dynamically coupled with MS, USA, 2National Cancer Institute, National Institutes of Health, functional sites can also be used for inducing and tuning allosteric communi- Frederick, MD, USA. cation. Defining generic characteristic of the allosteric effect of mutation, the Pin1 is an essential Peptidyl-prolyl isomerase (PPIase) that catalyzes cis- modulation range, we build Allosteric Signaling Maps (ASMs) of proteins on trans prolyl isomerization in proteins containing pSer/pThr-Pro motifs. It the basis of residue-by-residue scanning of mutations. ASMs allow one: (i) to has an N-terminal WW domain that targets pSer/Thr-Pro motifs and a C-ter- establish a relationship between mutations allosterically modulating protein minal PPIase domain that catalyzes isomerization. In this study, we report a activity and those that mainly change the protein stability; (ii) to observe a novel series of chimeric peptides that has been designed to optimize binding role of domain/subunit in protein allosteric communication via the distance- across the inter-domain interface of Pin1. The peptide series was designed dependent mode switching in allosteric signaling; (iii) to complement and based on the binding site of the Pin1 and optimal peptide sequences analyzed tune already existing allosteric signaling and to design new elements of in earlier studies. Specifically, we started from a sequence derived from the regulation. human Histone H1.4 sequence (KATGAApTPKKSAKW). Then, we de- signed a series of chimeric peptides based on this H1.4 sequence. NMR ti- 2294-Plat trations were performed for each peptide using both full-length Pin1 as Integration of an Electrostatic Network and Disorder-to-Order Transi- well as the WW domain alone. An analysis of KD values reveals complexity tions in Protein Allostery in the energetics of interaction between the peptide substrate, the PPIase Riya Samanta, Jingheng Wang, Dorothy Beckett, Silvina Matysiak. domain, and the WW domain. 15N relaxation and residual dipolar couplings University of Maryland College Park, College Park, MD, USA. (RDCs) were used to monitor the degree to which peptide binding induced Although allostery-in which action at one site alters the function at another inter-domain interactions. When combined with molecular simulations, our site -is integral to many biological regulatory processes, the molecular mech- results suggest a structural basis for how substrate binding can alter the anism of this phenomenon still eludes our understanding. Transcription inter-domain dynamics. Finally, we investigated whether our chimeric se- repression by E. coli biotin repressor, BirA, is allosterically modulated by 1 1 quences could alter catalysis (kex)using H- H EXSY NMR experiments. activation of dimerization via binding of the small molecule effector bio- Interestingly, no relationship was found between kex and either peptide affin- tinoyl-5’-AMP. Previous studies have shown that disorder-to-order transi- ity or inter-domain interaction, suggesting a lack of allosteric control for this tions in loops on the distant dimerization and effector binding surfaces series of peptides. Thus, while our results suggest that peptide binding can communicate in BirA allostery. We hypothesize that an electrostatic network alter the interaction between the PPIase and WW domains, altering the is key to this communication. In this work, combined experimental and inter-domain interaction by itself does not appear to modulate catalysis in computational methods have been applied to investigate the role of this the PPIase domain. network in BirA allostery. Energy Network Analysis predicts that the elec- trostatic network residues participate in a larger allosteric network, while 2292-Plat ITC measurements support network formation in solution upon bio-5’- Thermodynamic Coupling - Free Energy Calculations of Correlated AMP binding. Thermodynamic measurements reveal the importance of the Amino Acid Mutations network for both effector binding and BirA allostery. Force Distribution Martin Werner, Bert L. de Groot. Analysis combined with Energy Network Analysis show coupling between Max Planck Institute for Biophysical Chemistry, Go¨ttingen, Germany. the electrostatic network and disorder-to-order transitions in BirA. The Interactions between combined amino acid mutations play a key role in pro- experimental and computational studies support an allosteric mechanism in tein engineering affecting properties such as cooperativity and allostery. The which an electrostatic network enables communication, via a global popula- effect of a double mutation on properties like thermostability or binding af- tion shift, between disorder-to-order transitions at two distant functional finity can be significantly different from the sum of effects of the separate sites. single mutations. Such a situation is revealed by a nonadditivity of the cor- responding free energies and indicates the mutations involved to be thermo- 2295-Plat dynamically coupled. While this type of epistatic interaction is expected for Graph Spectral Properties of the Sidechain Networks of Protein Struc- residues in close spatial contact, additivity of the effects of both single amino tures: Implications to Allostery and Structure Comparison acid mutations is widely observed for distant pairs. Indeed, even mutations Saraswathi Vishveshwara1, Anasuya Dighe2, Vasundhara Gadiyaram1. on opposite sides of a protein can exhibit significant nonadditivity in their 1Molec Biophys Unit, Indian Institute of Science, Bangalore, India, 2IISc free energies indicating a correlation of both mutations persisting over large Math Int, Indian Inst Sci, Bangalore, India. distances. The investigation of network metrics such as communities and paths of Molecular dynamics simulations with alchemical amino acid mutations grant communication are shown in literature to yield valuable information on protein access to the free energies and nonadditivities of double mutants via different structure, function and folding. Recently, our laboratory has adopted graph pathways. The comparison of the resulting thermodynamic couplings to spectral approach to study the sidechain interactions in protein structures in
BPJ 9447_9453 464a Wednesday, March 6, 2019 detail (https://doi.org/10.1093/comnet/cnw016; https://doi.org/10.1002/prot. 2298-Plat 25332) by transforming all-atom coordinates into a weighted normalized Lap- Understanding the Biophysics of Protein-Surface Interactions lacian network, since the spectra of a graph is known to capture network fea- Gabriel Ortega1, Martin Kurnik1, Philippe Dauphin Ducharme1, Hui Li2, tures with minimal loss of information. A weighted network represents Netzahualcoyotl Arroyo-Curras3, Bishal Gautam4, Kevin Plaxco1. accurate data and many of the important phenomena such as allostery involving 1Center for Bioengineering, University of California Santa Barbara, Santa long distance communication take place through the global effect of local Barbara, CA, USA, 2Engineering Research Center of Nano-Geomaterials, changes in edge weights. Further, the spectra of normalized Laplacian allows China University of Geosciences, Wuhan, China, 3Pharmacology and a comparison of multiple networks in a rigorous manner. The networks can Molecular Sciences, Johns Hopkins University School of Medicine, be from different proteins or different states of the same protein. The edge Baltimore, MD, USA, 4Chemistry and Biochemistry, University of California weight differences provide one component of the scoring scheme. More impor- Santa Barbara, Santa Barbara, CA, USA. tantly, the clustering of interacting residues is a property uniquely elucidated Despite the biological and biotechnological importance of protein-surface in- from spectral studies and it provides the information on changes at global level teractions, our understanding of how and why they occur is still limited: due to local changes in interactions. What are the thermodynamic consequences of the interaction of proteins Methodology and two applications of the above formalism will be presented: a) with surfaces? Why do proteins generally remain folded and functional on Investigation of the graph spectra of GPCR structures and b) Validation of pro- biological surfaces and cell membranes, but often unfold, adhere to, and tein structure models through network scoring scheme (NSS). As part of (a) The inactivate on artificial surfaces? To respond to these questions we need to clustering of interacting residues in adrenergic receptors-ligand complexes will understand the biophysical origins of protein-surface interactions, however be discussed. Distinct patterns emerge from the apo, agonist, and antagonist the absence of experimental methods to measure the thermodynamics of bound structures. The spectral results are discussed in terms of their possible such interactions has so far precluded quantitative analysis. In response, function such as allostery. As part of (b) A large scale comparison of protein we have developed an approach to measure the free energy of protein- structure models from earlier CASP experiments and from the ensemble of surface interactions, which we have employed to explore the extent to which structures obtained from molecular dynamics simulation will be presented. attachment to a specific, macroscopic surface alters the thermodynamic sta- bility of protein L. We have achieved so by modifying protein L with the 2296-Plat redox reporter methylene blue and then covalently tethering its N-terminus Allostery is Highly Tunable by Amino Acid Substitutions at Long-Range to a gold electrode passivated with a hydroxyl-terminated alkanethiol mono- Rheostat Positions layer. Denaturant-induced unfolding of the surface-attached protein alters the Liskin Swint-Kruse, Aron W. Fenton. ease with which the methylene blue reporter transfers electrons to the surface Dept Biochem Molec Biol, Univ Kansas Med Ctr, Kansas City, KS, USA. of the electrode, and therefore electrochemical techniques, such as square- One mechanism by which organisms evolve and adapt to new niches involves wave voltammetry, allow monitoring the unfolding to extract protein stabil- amino acid changes that ‘‘fine-tune’’ protein function. For several proteins, ity. Comparing the thus obtained stability of the surface-attached protein to we previously showed that one group of nonconserved positions contribute to that of the same protein in bulk solution we find that surface-attachment sta- functional tuning by behaving as evolutionary ‘‘rheostats’’. That is, when a va- bilizes the protein due to excluded volume effects that restrict the conforma- riety of amino acids were substituted at a given rheostat position, functional out- tional entropy of the unfolded state. We have also explored the role of comes sampled a wide range of values. In these prior studies, the rheostat macromolecular crowding, solvent composition and electrostatics to find outcomes could be considered ‘‘short-range’’. For example, DNA binding affin- that their surface biophysics are markedly different from those in bulk solu- ity was tuned by mutating positions near the DNA binding site of LacI homo- tion. We believe that our studies refine our understanding of the biophysics logs. In a second example, properties of allosteric regulation (apparent underlying protein-surface interactions, which may in turn improve the effector binding ‘‘Kix’’ and coupling ‘‘Q’’) were tuned by mutating positions design of protein-surface pairs for protein-incorporating biotechnologies in and near the two distinct allosteric binding sites on pyruvate kinase (PYK). such as protein-based sensors. Here, we used a quantitative score for tunability to show that the same PYK substitutions also exerted ‘‘long-range’’ rheostat effects: Mutations at rheostat 2299-Plat positions in the alanine allosteric site tuned both K and Q for fructose-1,6- ix Tie Up Cytoskeleton to Inhibit Ovarian Cancer Metastasis bisphosphate activation (‘‘FBP’’); mutations at rheostat positions in the Ye Zhang. FBP allosteric site tuned Q for alanine inhibition, but had only modest effect Bioinspired Soft Matter Unit, Okinawa Institute of Science and Technology, on K . For Q ,Q , and K , mutational outcomes sampled the range ix,ala ala FBP ixFBP Kunigami-gun, Japan. from ‘‘better’’ than wild-type to ‘‘dead.’’ The ranges observed from long- Cells modify the migration mechanism in response to their surroundings, range tuning were quantitatively equivalent to those from short-range tuning. which sets the challenge for cancer therapy targeting metastasis through Several amino acid positions contributed to tuning multiple functional parame- signaling pathways. To cope with the diversity and complexity, we have ters. Strikingly, for all mutated positions in this study, the long-range effects on developed a mechanical approach that captures membrane dynamicity to sup- Q tunability were larger than the long-range effects on K . This supports the ix press cancer cell migration and invasion. We designed and synthesized a hypothesis that the functional roles of binding and allostery can be controlled ruthenium (Ru)-complex-based peptidic molecule that selectively initiates by distinct sets of amino acid positions. self-assembly into extracellular nanofibrils on lipid rafts of ovarian cancer cells by reacting with glycosylphosphatidylinositol-anchored placental alka- Platform: Engineering and Detecting Cellular line phosphatase (GPI-anchored PLAP). The growing nanofibrils glue the lipid rafts and chain them into large clusters, leading to reinforced focal adhe- (dys) Faction sion suppressing cell migration. The molecular self-assembly constantly ex- erts mechanical stimuli to raft-associated protein receptors, provoking 2297-Plat opposing cell migration against focal adhesion with enhanced motility. The Hydrogel Engineering with Widefield Patterned Illumination contradictory motions generate a mechanical force transferred through the Aurelien Pasturel1,2, Pierre-Olivier Strale2, Vincent Studer1. 1 2 actin cytoskeleton. When the force increases, the restricted ovarian cancer CNRS UMR 5297, Bordeaux, France, ALVEOLE, Paris, France. cell is ruptured. Hydrogels are the material of choice to emulate the cellular micro-environment thanks to their high hydric content and tunable visco-elastic properties. In field such as cancer research, toxicology or tissue engineering, enabling technolo- 2300-Plat gies to control the structural and biochemical properties of hydrogels are Improvement of Maturation State of Human Induced Pluripotent Stem much sought after. Indeed, structuring and functionnalizing hydrogels could Cell-Derived 3D Cardiac Microtissues by Defined Chemical Factors unlock the design of more advanced organotypic models but existing solutions Chen Yu Huang1, Rebeca Joca2, Chin Siang Ong2, Ijala Wilson2, usually lack the simplicity and flexibility to adapt to the day to day flow of mod- Roald Teuben2, Gordon F. Tomaselli2, Daniel H. Reich1. 1 ern research. Here by harnessing generic chemistry with digitally processed UV Department of Physics and Astronomy, Johns Hopkins University, 2 widefield illumination we achieve additive or subtractive manufacturing and Baltimore, MD, USA, Division of Cardiology, Johns Hopkins Hospital, decoration for common hydrogels such has PEG, Matrigel, Agar or Poly- Baltimore, MD, USA. acrylamide. We grew neurons and cell lines onto chemically and topographi- Recent advances in the understanding and use of pluripotent stem cells have cally complex PEG gels, inside engineered Matrigel structures or within micro- produced major changes in the approach to the diagnosis and treatment of hu- fluidic chambers demonstrating that simple principles may unlock engineering man disease. An obstacle to the use of human pluripotent stem cell-derived car- for hydrogels that lack dedicated chemistry. diomyocytes (hiPSC-CMs) for regenerative medicine, disease modeling and
BPJ 9447_9453 Wednesday, March 6, 2019 465a drug discovery is their immature state relative to the adult myocardium. In this sulted in a low fluorescence, which increased over 4 times in the unfolded research, 3D cardiac microtissues (CMTs) were generated using hiPSC-CMs to state (Caþ2 free) of the protein, resulting in a large change in the fluorescence recapitulate the structural, functional and metabolic properties of normal and signal. The successful in-vitro detection of Caþ2 ions confirms the eligibility diseased adult ventricular myocardium. CMTs were treated with ontologically of the ‘‘folding coupled to binding’’ strategy as the next generation sensor defined biochemical interventions (thyroid hormone, dexamethasone and designing methodology. insulin-like growth factor, TDI) to promote the maturation of hiPSC-CMs. The effects of TDI treatment on both structural and functional (biomechanical, 2303-Plat þ Ca2 handling and electrophysiology) properties at the tissue level were char- pH Sensitive Peptide Functionalized High Stability Polymeric Nanopar- acterized. Also, the molecular correlates of maturation of the hiPSC-CMs in the ticles for Mitochondria Targeted Cancer Drug Delivery CMTs were studied by gene expression, proteomics studies. Our data demon- Palanikumar Loganathan, Mona Kalmouni, Sumaya Al Hosani, strate that TDI treatment improves both the structure and function of CMTs. Mazin M. Magzoub. Structurally, the hiPSC-CMs show improved alignment and longer sarcomere Biology Program Division of Science, New York University Abu Dhabi, Abu length, as shown by immunofluorescence and confocal microscopy. Function- Dhabi, United Arab Emirates. ally, CMTs’ static and dynamic force both increase following TDI treatment, A major focus of cancer nanomedicine is to achieve a high therapeutic index and cardiac electrophysiology assessed by optical mapping showing that the for cancer therapy. Despite the unprecedented growth in the development of CMTs are electrically coupled. TDI-treated CMTs exhibited both chronotropic polymeric self-assembled nanosystems, only a few of them end up in clinics and inotropic response to isoproterenol treatment. The enhanced functional (e.g. Doxil) as a result of (i) poor stability, (ii) drug leakage and (iii) disso- properties that this maturation approach yields have the potential to yield ciation kinetics due to the strong influence of blood components in vivo.The improved hiPSC derived cardiac model systems that can advance both mecha- most common method for improving nanocarrier stability is to tune the drug nistic studies and the development of new therapies for the treatment of cardiac hydrophobicity and polymer matrix miscibility under complex chemical re- diseases. action conditions, that leads to poor drug cytotoxicity and attenuation of therapeutic efficacy. To address these challenges, we have designed a simple and robust poly (lactide-co-glycolide) (PLGA) and bovine serum albumin 2301-Plat (BSA) hybrid nanoparticles to provide high stability and trigger drug release Protein Detection in Blood with Single-Molecule Imaging inside cancer cells. The hybrid nanoparticles were prepared by covalently Shih-Chin Wang, Chih-Ping Mao, Yu-Pin Su, T.C. Wu, Chien-Fu Hung, wrapping BSA over the surface of drug-loaded PLGA nanoparticles using Jie Xiao. simple carbodiimide-chemistry in mild reaction conditions. To increase the Johns Hopkins Univ, Baltimore, MD, USA. stability, the BSA shells were crosslinked. The BSA shells can be dissociated The ability to accurately identify and characterize proteins in blood samples in the high redox microenvironment of the tumor and within the cancer cells. would facilitate disease diagnosis and monitoring. Current blood tests, Subsequently, the drugs loaded into the PLGA core can be released by accel- including ultra-sensitive assays, depend on ensemble measurements which erated degradation (hydrolysis) due to the low pH conditions in the tumor are hampered by background detection errors. Single-molecule imaging ap- microenvironment and lysosomal compartments. The cancer cell targeting proaches, by contrast, directly inspect individual target proteins, offering ability of the hybrid NPs was achieved by coupling of the amino groups much greater reliability than ensemble methods. However, the detection limit of the BSA shell with a pH-sensitive peptide (acidity-triggered rational of single-molecule imaging has thus far been confined to the picomolar (10 12 membrane peptide, ATRAM). The insertion of the ATRAM-BSA-PLGA M) range, and these approaches have not been previously translatable to clin- nanoparticles into cancer cell membranes occurs at the peritumoral pH ical samples. Here we describe Single-Molecule Augmented Capture (6.5). In vitro studies showed highly-efficient pH-dependent uptake and (SMAC), a single-molecule imaging technique to visualize and quantify indi- remarkable cytotoxicity of doxorubicin triphenylphosphine loaded ATRAM- vidual proteins-of-interest down to the sub-femtomolar (<10 15 M) range BSA-PLGA NPs. Our results demonstrate that the novel pH-sensitive pep- with minimal detection errors, even in complex fluids such as blood. We tide-functionalized high stability BSA-PLGA nanoparticles are a highly demonstrate SMAC in a wide variety of applications with human blood sam- promising nanoplatforms. ples, including the analysis of secreted proteins (prostate-specific antigen and anti-p53 autoantibodies), membrane proteins (programmed death-ligand 1), and rare intracellular proteins (mutant p53). SMAC opens the door to the 2304-Plat use of single-molecule imaging in non-invasive disease profiling. This plat- Protein Phase Transition: From Biology Towards New Protein Materials 1 € 1 1 2 form can be adapted to multiplex or high-throughput formats to characterize Miriam Linsenmeier , Andreas Kuffner , Lenka Faltova , Maria Hondele , Karsten Weis2, Paolo Arosio1. heterogeneous biochemical and structural features of circulating proteins-of- 1 interest. Chemistry and Applied Biosciences, ETH Zurich, Zurich, Switzerland, 2Dept Biology, ETH Zurich, Zurich, Switzerland. Many cellular organelles can form via phase separation of proteins and nu- 2302-Plat cleic acids. Yet, the molecular mechanisms that govern the lifetime, the Recombinant Protein Based CaD2Ion Sensor Designing; an In-Vitro Test of composition and the size of these membrane-less compartments remain Folding Coupled to Binding Hypothesis largely elusive. Here, we apply methods based on polymer physics and micro- Abhigyan Sengupta, Mourad Sadqi, Victor Mun˜oz. fluidic technology to analyze the physical chemical processes underlying the Bioengineering, University of California Merced, Merced, CA, USA. phase separation of biological proteins, and we use these lessons to induce Recombinant protein-based sensor designing is a burgeoning topic of interest controlled self-assembly of soluble proteins in biotechnology. In particular, due to its potential in-vivo applications. Proteins are particularly suitable for we focus on the phase separation of the DEAD-box protein ATPase Dhh1, sensor designing as they have high cell viability, negligible cytotoxicity, inter- which is strongly associated with the formation of processing bodies (P- action with the substrate with a large three-dimensional surface and binding bodies) in yeast. We identify the role of ATP and RNA in triggering the with high affinity. But protein-based sensors are scarcely available as engi- nucleation and growth of the protein-rich droplets, as well as in maintaining neering protein as a functional sensor is very challenging. I will describe the protein dense phase in the liquid state. These results reveal molecular the designing of a recombinant protein-based Caþ2 ion sensor using ‘‘folding mechanisms that cells have plausibly developed to accurately control the coupled to binding’’ strategy. The Caþ2 ion binding domain of a natural pro- reversible assembly and the biophysical properties of P-bodies. Moreover, tein is used as a template and site-selective mutations are utilized to destabi- we demonstrate the possibility to mimic these mechanisms and induce similar lize the protein structure and couple its folding-unfolding kinetics with the behaviours in soluble proteins by conjugating low complexity domains to sol- ligand association equilibrium. In the coupled equilibrium, the apo (Caþ2 uble globular regions (Faltova L., Kuffner€ A. et al, ACS Nano, 2018, https:// free) form of the protein folds only in presence of the target analyte and doi.org/10.1021/acsnano.8b04304). We show that these molecular adhesives the structural changes in the protein is exploited as a transducer for the enable? sensitive, controlled self-assembly of these proteins into supramolec- biosensor. We have utilized both FRET and PET-based techniques to estimate ular? architectures via a? multistep process. This multistep pathway involves the ligand-induced changes in protein structure. To probe less than 2 nm dis- an initial liquid-liquid phase transition, which creates protein-rich droplets tance change between folded and unfolded states of the protein, photo-induced that mature into protein aggregates over time. These protein aggregates electron transfer (PET) is exploited as an alternative to FRET. An organic flu- consist of permeable structures that maintain activity and release active solu- orophore is covalently attached to one site of the protein and at another site, a ble proteins. We further demonstrate that this feature, together with the dy- selected amino acid is mutated to tryptophan. In folded state (Caþ2 bound) the namic state of the initial dense liquid phase, allows one to directly intramolecular electron transfer from the tryptophan to the fluorophore re- assemble different globular domains within the same architecture, thereby
BPJ 9447_9453 466a Wednesday, March 6, 2019 enabling the generation of both static multifunctional biomaterials and dy- 2307-Plat namic microscale bioreactors. Enhanced Crossbridge Binding with 2-Deoxy-ATP Results from Increased Electrostatic Interactions between Myosin and Actin in Cardiac Muscle Chen-Ching Yuan1, Joseph D. Powers1, Kimberly J. McCabe2, Platform: Cardiac Muscle Mechanics, Structure, Jason D. Murray3, Morhan Saffie1, Castillo Romi1, Zuzek Carla1, Weikang Ma4, Andrew D. McCulloch5, Thomas C. Irving6, and Regulation II Michael Regnier1. 1Dept BioEngineering, Univ Washington, Seattle, WA, USA, 2UC San 2305-Plat Diego, San Diego, CA, USA, 3Dept Physiol & Biophys, Univ Washington, Stretch-Induced Activation of the Myosin Motors on the Thick Filament in Seattle, WA, USA, 4BioCAT, Argonne, IL, USA, 5Bioengineering, UC San Rat Cardiac Trabeculae Diego, La Jolla, CA, USA, 6Dept Biology, Illinois Inst Tech, Chicago, IL, So-Jin Park-Holohan, Elisabetta Brunello, Thomas Kampourakis, USA. Martin Rees, Malcolm Irving, Luca Fusi. We have demonstrated that 2-deoxy-ATP (dATP) enhances muscle contraction Randall Centre for Cell and Molecular Biophysics, King’s College London, by increasing cross-bridge binding and cycling rates. Our recent molecular dy- London, United Kingdom. namics study (Nowakowski 2017, Protein Sci 26:749-62) suggested that dATP The contractility of cardiac muscle is regulated by thick-filament based increases the positive charge of the actin binding surface of myosin, thus mechanisms in addition to the classical calcium/thin-filament mediated enhanced binding may result from increased electrostatic interactions with actin mechanisms. Here we studied the role of structural changes of the thick in resting heart muscle. To test this hypothesis we employed multiple biophys- filament in length-dependent activation in cardiac muscle. We used fluores- ical approaches. X-ray diffraction analysis comparing dATP vs. ATP was per- cence polarization from bifunctional sulphorhodamine probes on the N- and formed on rat cardiac muscle in physiological (170mM) and low (100mM) C-lobes of the myosin regulatory light chain (RLC) to monitor changes in ionic strength (m) solutions at two sarcomere lengths (SL). In 170mM m relaxing the orientation of the myosin motors induced by increasing sarcomere length solution (pCa9) with dATP, cardiac muscle had a significantly higher I1,1/I1,0 ra- in relaxed and partially calcium-activated demembranated trabeculae. tio, larger M3 spacing (SM3) and lattice spacing (d1,0) at both SL. These results Under relaxing conditions at near physiological temperature and lattice suggest that, compared with ATP, dATP in resting cardiac muscle causes spacing the myosin motors are roughly parallel to the filament axis, consis- myosin to move towards actin filaments, and the space between adjacent crowns tent with the OFF structure of the thick filament. Cooling of the relaxed is similar to the activated myosin conformation (pCa5.2). These effects were trabecula induced a more perpendicular orientation of the myosin motors reduced or eliminated at m=100mM where surface charge is increased (with indicating disruption of the OFF-state. Application of a staircase stretch ATP). This suggests that increased positive charge on the actin binding surface protocol in the sarcomere length range 2.0-2.3 mm(25C, 3% dextran of myosin with dATP may indeed be responsible for enhanced crossbridge bind- T500) at pCa 9.0 also induced a more perpendicular orientation of the ing. Brownian Dynamics simulations predicted the myosin.dADP.Pi structure myosin motors, but despite an increase in passive tension to 30% of has a significantly higher association rate to actin (than myosin.ADP.Pi) over T the maximum active force ( 0), the orientation change was only 4% of a wide range of reaction distances, and this results primarily via an increase in that associated with full calcium activation. Larger stretch-induced orienta- positive (myosin) and negative (actin) contact pairs. Consistent with this, as tion changes were observed at pCa 7 ( 7%) and at pCa 6.6 ( 20%), the the m of solutions in the in vitro motility assay was increased, more actin fila- T latter accompanied by an increase in active force of 0.05 0. RLC phos- ments remained on cardiac myosin-coated surfaces with dATP compared with phorylation further increased the stretch-induced orientation changes to ATP. Combined these data suggest that dATP enhances electrostatic interac- 14% and 33% respectively, and the active force response at pCa 6.6 tions that facilitate increased weak myosin-actin binding, leading to increased T increased to 0.2 0. These results indicate that increasing sarcomere length crossbridge binding and contraction in cardiac muscle. in the physiological range at diastolic calcium concentration induces activation of the myosin motors and that this effect is enhanced by RLC 2308-Plat phosphorylation, suggesting that changes in thick filament structure may Basic Amino Acids within the C-Terminal 16 Residues of Troponin T mediate length-dependent activation. Supported by Wellcome Trust and Modulate Calcium Sensitivity and the Distribution of Actin States BHF, UK. Dylan Johnson, Li Zhu, Joseph M. Chalovich. Biochemistry and Molecular Biology, Brody School of Medicine at East 2306-Plat Carolina Univ, Greenville, NC, USA. Cardiomyopathy Mutation at End-End Overlap of Alpha - Tropomyosin We previously presented evidence that the highly conserved C-terminal 14 res- Influences Cooperative Activation and Calcium Sensitivity idues of cardiac troponin T has two regulatory functions. 1) Formation of the 1 2 2 SaiLavanyaa Sundar , Michael J. Rynkiewicz , William Lehman , inactive state (B or 10) does not occur when the last 14 residues are removed þ Jeffrey R. Moore3. from troponin T. 2) Those last 14 residues limit Ca2 activation to 33% of 1University of Massachusetts Lowell, Lowell, MA, USA, 2Dept Physiol/ full activation in solution. Deletion of the last 14 residues leads to doubling þ Biophys, Boston Univ, Boston, MA, USA, 3Dept Biological Sciences, Univ of Ca2 activation by. We recently showed that stepwise deletion of the last Massachusetts Lowell, Lowell, MA, USA. 14 residues of Troponin T changes both regulatory functions in a graded Tropomyosin (Tpm) polymerizes head-to-tail along the long-pitch helix of manner. We proposed that ionic interactions between basic residues within F-actin to form a coiled-coil super-helical uninterrupted cable structure the C-terminal of Troponin T and acidic residues elsewhere in the thin filament that wraps around the complete length of actin filaments. Muscle contraction are important for these functions. We tested this hypothesis by substituting Ala is governed by the ability of Tpm to azimuthally shift between three states for each of 5 Lys and 2 Arg residues within the last 16 residues (to substitute an on F-actin (Blocked, Closed and Open) in response to calcium-binding to additional Lys) of human cardiac Troponin T. We also engineered a C-terminal troponin and acto-myosin cross-bridge formation. It is also known that the Cys residue into both the mutant and wild type troponin T for coupling to þ end-to-end bonds formed between the N- and C- terminus of adjacent probes. In the virtual absence of Ca2 the ATPase rate of actin filaments con- Tpm molecules defines Tpm continuity, plays a critical role in the ability taining that mutant troponin T was slightly elevated with respect to wild type. of Tpm to cooperatively bind to actin and for Tpm conformational switching Also, the fluorescence change characteristic of forming the inactive B state was þ to cooperatively propagate along F-actin. We hypothesize that a missense missing with the mutant troponin T. At saturating Ca2 the ATPase rate was mutation, A277V, in this critical overlap region, associated with dilated car- approximately double that of wild type. Thus the mutant Troponin T behaved diomyopathy, will alter tropomyosin binding, thin filament activation by sta- like Troponin T that lacked the C-terminal 14 residues. We named this mutant bilizing the overlap structure. Here, we used co-sedimentation assays and in HAHA Troponin T (High Alanine-High Activity Troponin T). We plan to use þ vitro motility assays to determine how the mutation alters Tpm binding to HAHA troponin T to explore the mechanism of modulation of Ca2 activation. actin, and its ability to regulate acto-myosin interactions. The A277V Tpm exhibited enhanced actin - Tpm binding affinity and cooperativity, 2309-Plat consistent with overlap stabilization. However, regulated thin filament On the Functional Assessment of Hypertrophic Cardiomyopathy-Causing motility measurements exhibited a decrease in both, cooperativity and Mutations in Human b-Cardiac Myosin and the Role of Myosin Binding Ca2þ sensitivity, when compared to wild-type. The observed effects are Protein-C consistent with a mutation-induced disruption of the blocked-closed equilib- Darshan V. Trivedi1,2,*, Saswata S. Sarkar1,2,*, Arjun S. Adhikari1,2,*, rium, an increased Tpm-Tpm end-end bonding and a decrease in overlap Makenna M. Morck1,2, Kristina B. Kooiker1,3, Daniel Bernstein2,3, stiffness. Kathleen M. Ruppel1,3, James A. Spudich1,2.
BPJ 9447_9453 Wednesday, March 6, 2019 467a
1Dept. of Biochemistry, Stanford Univ, Stanford, CA, USA, 2Stanford variants in desmosomal genes, however the molecular mechanisms underlying Cardiovascular Institute, Stanford Univ, Stanford, CA, USA, 3Dept. of AC remain unclear. We identified a novel variant (R451G) in the desmoplakin Pediatrics (Cardiology), Stanford Univ, Stanford, CA, USA. (DSP) gene; heart autopsy revealed pronounced fibrofatty scarring and a sig- *Equal Contribution nificant loss of DSP protein at the intercalated disc (ID). Engineered heart tis- Human b-cardiac myosin and cardiac myosin binding protein-C (MyBP-C) sues (EHTs) from induced-pluripotent stem cells generated from a harbor the majority of hypertrophic cardiomyopathy (HCM)-causing point symptomatic R451G-positive family member showed a significant loss of mutations which lead to a hypercontractile heart with systolic and diastolic DSP protein compared to control EHT. Notably, the R451G-variant sits in a defects. Previous studies looking at the effects of HCM mutations on the pocket containing several clinical DSP variants and we show that endogenous force, velocity and ATPase activity of the catalytic domain of human b-car- DSP is cleaved via a calpain-dependent mechanism. From this, we hypothe- diac myosin have not shown clear trends that establish a causal link to hyper- size that mutant DSP is more susceptible to calpain cleavage, resulting in contractility. However, recently, a regulatory, off-state of myosin with its cellular haploinsufficiency. In previous work, we defined a unifying mecha- heads folded back onto its own coiled-coil tail (probably the interacting heads nism for AC-linked variants by assessing the biomolecular properties of motif or IHM described by others) has been experimentally demonstrated to DSP variants. While these variants did not significantly perturb the structure be one of the primary states that yields the very slow turnover of ATP (called of DSP, there were significant changes to stabilizing intramolecular interac- the super relaxed state or SRX) typically observed in muscle fibers. This reg- tions of select variants, which correlated with augmented calpain degradation ulatory off-state is in equilibrium with the on-state of myosin which is avail- due to increased exposure of an auto-inhibited calpain-cleavage site. We have able to interact with actin and generate force. Our model posits that HCM now shown that by changing the molecular environment using site-directed mutations destabilize this SRX state, thus leading to a premature release of mutagenesis, we can ameliorate the calpain sensitivity of clinical variants. myosin heads from the thick filament backbone causing clinically observed Taken together we have identified a unifying mechanism for select AC- hypercontractility. Using IHM as the model for the folded-back state, we linked variants of DSP grounded in increased susceptibility to calpain- have previously presented binding data demonstrating that HCM mutations cleavage leading to decreased functional DSP. DSP protein expression was at the head-tail interface weaken this interaction and possibly destabilize also decreased in end-stage failing human hearts compared to control donor the IHM state. Here we present functional data showing that six separate hearts. The physiological and biochemical impact of this loss is being further HCM mutations located at the myosin head-tail and head-head interfaces of evaluated to define the role DSP haploinsufficiency plays in the fundamental the IHM lead to a significant increase in the number of heads functionally disease mechanism of AC. accessible for interaction with actin. Interestingly, one of the mutations also ablates the binding of myosin with the N-terminal C0C7 fragment of 2312-Plat MyBP-C hinting at a role of MyBP-C in regulating the availability of myosin Determining the In Vivo Role of Microtubule Detyrosination in Healthy heads for contraction of the cardiac muscle. and Diseased Myocardium Christina Yingxian Chen1, Matthew A. Caporizzo1, Kenneth Bedi2, 2310-Plat Michael P. Morley2, Kenneth B. Margulies2, Benjamin L. Prosser1. Development of an Imaging Pipeline to Model and Predict the Integrated 1Dept Physiology, Univ Pennsylvania, Philadelphia, PA, USA, 2Dept Localization of Organelles in hiPSC-Derived Cardiomyocytes Medicine, Univ Pennsylvania, Philadelphia, PA, USA. Melissa Hendershott, Susanne Rafelski. Detyrosinated microtubules provide mechanical resistance that can impede Allen Institute for Cell Science, Seattle, WA, USA. cardiomyocyte motion. Cardiomyocytes from patients with heart failure The Allen Institute for Cell Science is developing an imaging pipeline to study are characterized by a dense, heavily detyrosinated microtubule network changes in cellular organization and gene expression during differentiation of that is associated with increased myocyte stiffness and impaired contractility. human induced pluripotent stem cells (hiPSCs) into cardiomyocytes. We Pharmacological or genetic suppression of detyrosinated microtubules have used CRISPR/Cas9 to generate endogenous fluorescently tagged hiPSC lowers the viscoelasticity and improves contractile function in failing human lines (www.allencell.org), each expressing a monoallelic EGFP-tagged protein myocytes. These data demonstrate that a modified cytoskeletal network im- that localizes to structures including adhesions, actin and microtubule cytoskel- pedes contractile function in cardiomyocytes from failing human hearts and eton, mitochondria, nuclear envelope, desmosomes, endoplasmic reticulum, that targeting detyrosinated microtubules could represent a new inotropic and the Golgi. In addition, we have developed methods for scarless GFP- strategy for improving cardiac function. However, whether an increase in de- tagging of late expressing cardiomyocyte genes, including ACTN2, ssTNNI1, tyrosinated microtubules is sufficient to induce cardiac dysfunction in vivo is MYL2, MYL7 and TTN, to study the organization and morphogenesis of the unknown. To test this, we generated a cardiac-specific tubulin tyrosine ligase contractile apparatus. Furthermore, we have developed an optimized, scalable, knockout (TTL-cKO) mouse model to study the in vivo function of detyro- and robust protocol for differentiation of hiPSCs into cardiomyocytes, proto- sinated microtubules. In TTL-cKO hearts, detyrosinated tubulin increases cols for freezing/thawing cells, and methods for preparing cells for imaging, 4-fold compared to that of the control mice, mimicking similar levels of in- including dyes that label the cell membrane and nucleus. To quantify the crease seen in failing human hearts. Baseline echocardiography shows a sig- live, high-resolution 3D fluorescence images of hiPSC-derived cardiomyo- nificant increase in LV mass/body weight ratio in 3-4 months old TTL-cKO cytes, we developed image-based assays and segmentation algorithms that mice. Interestingly, TTL-cKO male and female hearts appear to undergo enable single-cell analyses of structure localization in differentiated cardio- different types of remodeling at this age. TTL-cKO females show purely myocytes. We have applied this imaging workflow to hundreds of cells for compensatory hypertrophy with increased wall thickness, mild changes in 20 different intracellular structures. These data were used to develop a ma- chamber diameter and no change in LV ejection fraction (LVEF), while chine learning neural net algorithm that can predict the location of cardiomyo- TTL-cKO males demonstrate a dilated remodeling with decreased septal cyte structures from transmitted light images. We are now extending our thickness, increased chamber diameter and a trend of declining LVEF. imaging workflow to incorporate fluorescence in situ hybridization (FISH); Ongoing echocardiography on older mice and mice with transverse aortic this will allow us to combine image data with gene expression profiles on a constriction (TAC) will inform whether remodeling and cardiac function is single-cell level. This imaging pipeline will be scaled up to generate an image perturbed with age and pressure overload induced stress. This will yield database of high-resolution, high-replicate image data of the fluorescently deeper insight on the in vivo functions of detyrosinated microtubules in tagged structures with a goal of generating an integrated image ‘‘state space’’ health and disease. of intracellular reorganization during cardiomyocyte differentiation. Platform: Protein-Nucleic Acid Interactions/ 2311-Plat Defining a Unifying Mechanism for Select Cardiomyopathy-Linked Vari- Chromatin and the Nucleoid II ants of Desmoplakin Heather R. Manring1, Ronald Ng2, Taylor Albertelli3, Trevor Dew1, 2313-Plat Tyler L. Stevens1, Ahmet Kilic4, Paul M.L. Janssen1, Nathan T. Wright3, Shelterin Components Modulate the Phase-Separation Propensity of Telo- Stuart Campbell5, Maegen A. Ackermann1. meres 1Dept Physiol & Cell Biol, Ohio State Univ, Columbus, OH, USA, 2Yale Andrea Soranno, Jeremias Incicco, Paolo De Bona, Eric Tomko, Eric Galburt, Univ, New Haven, CT, USA, 3Dept Chem/Biochem, James Madison Univ, Roberto Galletto. Harrisonburg, VA, USA, 4Ohio State Univ, Columbus, OH, USA, 5Dept Biochemistry and Molecular Biophysics, Washington University School of Physiol & Cell Biol, Yale Univ, New Haven, CT, USA. Medicine, Saint Louis, MO, USA. Arrhythmogenic cardiomyopathy (AC) affects 1 in 2000 Americans every Telomeres are nucleoprotein complexes that provide a mechanism to shelter the year and segregates with sudden cardiac death (SCD). AC is linked to genetic ends of chromosomes and are essential to maintain chromosome stability in
BPJ 9447_9453 468a Wednesday, March 6, 2019
Eukaryotes. In cells, individual telomeres are bound by the six-protein shelterin facilitating the qualitative and quantify describe the non-covalent interac- complex (TRF2-hRap1-Tin2-TRF1-TPP1-Pot1) and form distinct condensed tions inside membrane channels. globules of finite size. Interestingly, incells that maintain their telomeres via an alternative lengthening mechanism, multiple telomeric DNAs and shelterin 2316-Plat proteins coalesce in membrane-less promyelocytic leukemia (PML) nuclear Disordered RNA Chaperones Enhance Nucleic Acid Folding via Local bodies. The mechanism controlling this dual nature of telomere organization Charge Screening 1 2 3 4 is currently not understood. Here, we propose that shelterin proteins modulate Erik D. Holmstrom , Zhaowei Liu , Daniel Nettels , Robert B. Best , 3 the phase-separation propensity of telomeres, leading to either the condensation Benjamin Schuler . 1University of Kansas, Lawrence, KS, USA, 2University of Basel, Basel, of single DNA chains or the coalescence of multiple ones. In particular, we use 3 4 a combination of optical and fluorescence microscopy and single-molecule ex- Switzerland, University of Zurich, Zurich, Switzerland, NIH, Bethesda, periments to quantify the contribution of shelterin components to phase- MD, USA. separation and condensation of single DNA chains. Nucleic acid chaperones are an emergent class of proteins that facilitate for- Our experiments reveal that TRF2 promotes phase-separation of TRF2-DNA mation of natively folded RNAs and DNAs. These chaperones are involved mixtures. Even though the N-terminal basic domain and the dimerization in many nucleic acid-dependent processes, including critical steps in the life domain of TRF2 are sufficient to promote phase-separation, the C-terminal cycles of many viruses. Interestingly, many of these proteins are intrinsically DNA-binding domain imparts specificity for telomeric DNA repeats. More- disordered, raising the fundamental question of how such a chaperone func- over, addition of shelterin components, such as hRap1, provides a fine-tuning tions without a well-defined three-dimensional structure. One such example of the properties of the resulting dense and de-mixed phase. is the nucleocapsid protein of the hepatitis C virus, which catalyzes genome dimerization. Recently, we have uncovered many of the structural and 2314-Plat dynamical aspects of chaperone activity using a variety of smFRET tech- RNA Binding Mode Regulates PKR Activation niques. This positively charged intrinsically disordered protein functions as Stephen J. Hesler, Bushra Husain, Matthew Angeliadis, James L. Cole. a chaperone by acting as a flexible macromolecular counterion that locally Dept Molec Cell Biol, Univ Connecticut, Storrs, CT, USA. screens repulsive electrostatic interactions within the nucleic acid. The re- The antiviral kinase PKR is activated by RNA. The enzyme contains two sulting compaction biases the unfolded nucleic acid towards compact confor- dsRNA-binding domains, dsRBD1 and dsRBD2, and a kinase domain. Co- mations that are primed for folding, thereby increasing the folding rate localization of the kinase domains upon binding of two or more PKRs to a constant. Finally, molecular simulations that treat the nucleic acid and chap- single dsRNA enhances dimerization and subsequent activation. However, erone as a simple polyelectrolytes reproduce the experimental observations multiple lines of evidence indicate that the mode of RNA binding regulates and support the proposed molecular mechanism of these disordered viral PKR activation beyond simply modulating the overall binding affinity. proteins. dsRBD2 binds RNA weakly and engagement of this domain by an RNA is 2317-Plat correlated with enzymatic activation. In order to define the role of dsRBD2 Eukaryotic Transcription Factors Can Track and Control their Target in PKR activation we have systematically mutated residues in this domain Genes using DNA Antennas that typically mediate dsRBD-RNA interactions and probed the consequences Victor Munoz. for PKR activation and overall binding affinity. Several mutations inhibit PKR Bioengineering, Univ Calif Merced, Merced, CA, USA. activation and reduce RNA binding affinity in parallel. However, mutation of Eukaryotic transcription factors (TF) function by binding to short, 6-10 bp outward-facing residues on helix 1 that typically engages the RNA minor DNA recognition sites located near their target genes, which are scattered groove dramatically reduce PKR activation while only slightly decreasing through vast genomes. Such process surmounts enormous specificity, effi- overall RNA binding affinity. Steady-state fluorescence anisotropy analysis ciency and celerity challenges using a molecular mechanism that remains of PKR constructs containing a single tryptophan in either dsRBD1 or poorly understood. Combining biophysical single-molecule fluorescence ex- dsRBD2 indicate that both domains undergo a significant decrease in mobility periments, theory and bioinformatics analysis, we dissected the interplay be- upon RNA binding. These anisotropy changes are not markedly affected by tween the DNA-binding domain of Engrailed, a Drosophila TF, and the the helix 1 mutations, implying that the interaction of dsRBD2 with RNA per- regulatory regions of its target genes. Remarkably, Engrailed binding affinity sists. We propose that instead of interacting with RNA, helix 1 in dsRBD2 is enormously amplified by the DNA regions flanking the recognition site, engages in an intramolecular interaction that regulates kinase activity based which contain long tracts of degenerate recognition-site repeats. Such on RNA binding mode. DNA organization operates as an antenna that attracts TF molecules in a pro- miscuous exchange between myriads of comparatively weaker binding sites. 2315-Plat The antenna ensures a local TF supply, enables gene tracking and fine con- A Single-Molecule Interaction Spectrum for Non-Covalent Interaction In- trol of basal site occupancy. This mechanism illuminates puzzling gene side Membrane Protein Channel expression data, and suggests novel engineering strategies to control gene 1 1 1 2 1 Meng-Yin Li , Yi-Lun Ying , Wei Tong , Yong-Jing Wan , Yi-Tao Long . expression. 1Key Laboratory for Advanced Materials & School of Chemistry and Molecular Engineering, East China University of Science and Technology, 2318-Plat Shanghai, China, 2School of Information Science and Engineering, East Multiple Interaction Modes of the Nucleosomal Histone H3 N-Terminal China University of Science and Technology, Shanghai, China. Tail Revealed by High Precision Single-Molecule FRET Dynamic non-covalent interactions of biomolecules are ubiquitous in almost Kathrin Lehmann1,2, Suren Felekyan2, Ralf Kuhnemuth€ 2, Mykola Dimura2, all biological process. In particular, it underlies the successive transport of Katalin To´th1, Claus A.M. Seidel2. ions or target carriers by membrane protein, controlling many key cellular 1Biophysics of Macromolecules, German Cancer Research Center, functions. However, few experimental approaches can directly define the Heidelberg, Germany, 2Dept Molec Phys Chem, Heinrich Heine Universit€at multiple non-covalent interactions within the channel. Since all the dynamic Dusseldorf,€ Dusseldorf,€ Germany. non-covalent dynamic interaction inside membrane protein have been all re- Nucleosomes, as the basic unit of chromatin, are the key element of chromatin corded but hide in the traditional single-channel recording of ionic current, compaction and gene accessibility. The nucleosomal disassembly process, its here, we present a rational strategy in the combination of experiments, dynamics and the effects of posttranslational modifications have been widely MD simulations and frequency-energy spectra of ionic flow to quantify studied. However, little is known about the actual conformation and dynamics and quality a role of non-covalent interaction for membrane protein channel of the protruding histone tails. Here, we focus on the N-terminal tail of histone in transporting the carriers. We employed wild-type aerolysin transporting of H3 (H3NtT) to augment our recently published model on nucleosome methylcytosine and cytosine as a model to detailed explore the dynamic disassembly. ionic signatures with the non-stationary and non-linear frequency analysis. In this FRET study salt dependent conformational changes of reconstituted Wi- The results demonstrate that The main frequency peak position in dom 601 mononucleosomes with a DNA length of 170 bp, or 210 bp were frequency-energy spectrum represents the main ion mobility inside mem- measured from the perspective of the labeled histone tails by systematic vari- brane protein while its energy suggests the vibration amplitude of the related ation of the DNA labeling positions. Both, ensemble FRET experiments using ions. The sudden spikes in the frequency-energy considered as the fingerprint a Typhoon multimode scanner, and single-molecule experiments using multi- spectra for potential characterization of non-covalent interaction. The dimensional confocal fluorescence spectroscopy with pulsed interleaved exci- frequency-energy spectrum of ions flowing inside membrane channels tation (MFD-PIE) were performed. constituting a Single-Molecule Interaction Spectrum, which bridges the Our results reveal that the highly dynamic H3NtT is located within 10 nm dis- gap in-between traditional ionic current recording and the MD simulations, tance from the nucleosomal dyad axis and the linker DNA featuring multiple
BPJ 9447_9453 Wednesday, March 6, 2019 469a interaction modes. We resolved three DNA associated and one histone core Studies of multi-domain proteins and multi-protein machines necessitate a associated conformations and found that the H3NtTs are following the DNA deeper understanding of dynamic structure and transient conformations. There- motions during the disassembly process. The two major steps of the proposed fore, conventional structure determination methods need to be integrated into NaCl induced nucleosome disassembly process: 1) unwrapping of the weakly dynamic detection approaches. bound DNA side and 2) weakening of the more stable DNA side coincide We developed a hybrid method that integrates x-ray structure information into with the loss of the respective H3NtT:DNA interactions. Those results comple- self-consistent distance networks based on single-molecule Fo¨rster resonance ment our earlier model on the nucleosome disassembly with hitherto unknown energy transfer (FRET) [1,2]. By analyzing time-correlated distance distribu- intermediate steps obtained from the perspective of the labeled histone tails and tions globally, we can separate real protein dynamics and fluctuations from emphasize the pivotal role of the H3NtT for nucleosome integrity. modelling uncertainties, and ultimately generate time-correlated structural en- sembles. On top of that, we reveal correlated small- and large-scale intra-mo- 2319-Plat lecular fluctuations from time-resolved, color- and polarization-sensitive Yeast Pioneering Transcription Factors Rely on Slowed Dissociation Ki- fluorescence measurements. netics to Efficiently Target Nucleosomal Sites We applied our approach to the heat shock protein Hsp90. The chaperone ac- 1 2 1 2 2 Benjamin T. Donovan , Hengye Chen , Caroline Jipa , Chao Yan , Lu Bai , tivates large sets of signal transduction proteins often assisted by co-chaperones Michael G. Poirier1. 1 2 [3]. These conformation- and nucleotide-dependent processes are not yet Physics, Ohio State Univ, Columbus, OH, USA, Penn State Univ, comprehensively understood. Our approach resembled the x-ray structure of University Park, PA, USA. Hsp90’s closed conformation with an RMSD of 2.8 A˚ . Beyond that, we Nucleosomes are key regulators of transcription factor (TF) occupancy by re- resolved the previously unknown dynamic open structure of this multidomain stricting binding and accelerating dissociation rates, while pioneer transcrip- protein. The large-scale fluctuations on the lower millisecond timescale might tion factors (PF) somehow circumvent nucleosome regulation. Here, we be the basis of a general regulation mechanism. investigate the essential S. cerevisiae TF Reb1, which binds nucleosome Finally, I want to show how FRET can be orthogonally integrated into cross- entry-exit sites with high occupancy. We show that Reb1 binds its site within disciplinary platforms such as a microfluidics [4] and others, enabling the nucleosomes, trapping a partially unwrapped state without histone eviction. simultaneous detection of dynamic structure and low-affinity interactions. Reb1 binds nucleosomes and DNA with the same affinity, while surprisingly [1] Hellenkamp B, Wortmann P, Kandzia F, Zacharias M, Hugel T. Multido- the binding and dissociation kinetics are 50-fold slower at nucleosome sites main structure and correlated dynamics determined by self-consistent FRET relative to DNA. We then found similar results for a separate S. cerevisiae networks. Nature Methods 14(2), 174-180 (2017) TF, Cbf1. From these results, we propose that Reb1 and Cbf1 are pioneer fac- [2] Hellenkamp B, Schmid S et al. Precision and accuracy of single-molecule tors that target sites within nucleosome entry-exit regions by decreasing FRET measurements—a multi-laboratory benchmark study Nature Methods their dissociation rates to compensate for reduced nucleosome binding. This 15, 669-676 (2018) allows these PFs to induce nucleosome unwrapping and reside at nucleosome [3] Taipale, M et al. Quantitative Analysis of Hsp90-Client Interactions Re- entry-exit sites for minutes to facilitate the recruitment of transcription veals Principles of Substrate Recognition. Cell 140(5), 987-1001 (2012) coactivators. [4] Hellenkamp B, Thurn J, Stadlmeier M, Hugel T., submitted 2320-Plat 2322-Plat Role of RNA-Binding Activity of Hu in Chromosomal Organization Probing Structural States in Fast Exchanging Proteins by FRET and 1 2 2 2 Kelsey E. Bettridge , Xiaoli Weng , Subhash Verma , Sankar Adhya , Computational Methods 1 Jie Xiao . Hugo Sanabria. 1Biophysics and Biophysical Chemistry, Johns Hopkins Sch Med, Baltimore, 2 Dept Phys/Astro, Clemson University, Clemson, SC, USA. MD, USA, National Cancer Institute, National Institutes of Health, Fo¨rster Resonance Energy Transfer provides distance information between Bethesda, MD, USA. spectroscopic labels that are chemically coupled to the biomolecule of The E. coli chromosome must condense over a thousand fold to fit into the interest. A series of designed positions could provide sparse distance infor- micron-sized cell. This compaction is spatially organized by several factors, mation to describe the topology of the system. Given the fact that the motion including a group of proteins termed nucleoid-associated proteins (NAPs). of the spectroscopic labels is decoupled from the dynamics of the backbone The most highly conserved NAP in eubacteria is HU, comprised of two sub- of biomolecules, we can identify limiting conformational states even when units that can form homo- or heterodimers, which binds to the bacterial these exchange at rates in the microsecond timescales. Complementary in- DNA with no sequence preference. While not lethal, deletion of both subunits formation is provided by independent molecular dynamic information that of HU results in changes to replication, transcription, and chromosomal archi- fill in the missing degrees of freedom of biomolecular motion. Combining tecture. Puzzlingly, the biochemical properties of HU suggest it’s DNA- molecular dynamic simulations and a set of FRET-derived distances, exper- binding activity is particularly poor, in the millimolar range. Recently, it was iments allows one to create structural models of fast exchanging conforma- discovered that HU can bind to various RNAs inside the cell, in particular small tions. We apply this to resolved previous diverging structural models of the non-coding RNAs from a repeat extragenic palindromic (REP) sequence, re- N-terminal PDZ1-PDZ2 tandem of the Postsynaptic density protein of 95 named nucleoid-associated RNAs (naRNAs). Deletion of this REP element kDa (PSD-95). We identified two conformations: an open-like conformation hinders the ability of HU to bridge chromosomal contacts, and this ability with a small contact interface stabilized by salt bridges, and a closed-like can be restored by expression of just one of the naRNAs. This suggests a mech- conformation with a larger contact interface stabilized by surface-exposed anism by which HU binds naRNAs and bridges interactions in the chromosome hydrophobic residues. Both interfaces were independently probed. The through the naRNAs themselves. Here, we investigate the dynamics of HU us- low-energy barrier between conformations allows sub millisecond dy- ing superresolution single molecule tracking. We find that HU has two diffusive namics, which were time-averaged in previous diverging models. previous states corresponding to DNA-bound and freely diffusing, and that HU rapidly diver. Moreover, the small contact interfaces were likely overridden by lat- switches between the two states, confirming prior biochemical data. Addition- tice contacts as crystal structures were rarely sampled in simulations. This ally, HU dynamics and localization are drastically altered in both general RNA hybrid approach can identify transient inter domain interactions, which depletion, and specific naRNA depletion. Finally, our data suggests HU must be are abundant in multidomain proteins yet often obscured by dynamic able to bind both HU and the DNA in order to execute its function. Together, averaging. our data supports a model in which HU mediates chromosomal contacts through naRNAs. 2323-Plat Integrative Dynamic Structural Biology with Fluorescence Spectroscopy Platform: Member Organized Session: Claus A.M. Seidel1, Mykola Dimura2, Hugo Sanabria3, Katherina Hemmen4, Integrative Structural Modeling Using Thomas-Otavio Peulen5, Dmitro Rodnin1, Holger Gohlke6. 1Chemistry, Heinrich Heine University, Duesseldorf, Germany, 2Heinrich Information from Spectroscopic Lables Heine Univ, Dusseldorf,€ Germany, 3Dept Phys/Astro, Clemson University, Clemson, SC, USA, 4Rudolf-Virchow-Zentrum fur€ Experimentelle 2321-Plat Biomedizin, University Wuerzburg, Wuerzburg, Germany, 5Physical Single Molecule FRET - A Multi-Environment Ruler for Determining Chemistry II, Duesseldorf, Germany, 6Inst Pharm Med Chem, Univ Structure and Dynamics Duesseldorf, Duesseldorf, Germany. Bjorn Hellenkamp. Fo¨rster Resonance Energy Transfer (FRET) studies on the ensemble and single- Columbia University, New York, NY, USA. molecule level probe biomolecular structure and dynamics and identify
BPJ 9447_9453 470a Wednesday, March 6, 2019 coexisting conformational states. Integrative structural biology solves bio- measured distances with a modeling approach best suited for the specific sys- molecular structures by combining data from different sources, which indi- tem under investigation. As such, the integrative experimental/computational vidually would be insufficient. Here, we combine FRET experiments with approach described here represents a hybrid method for determining all-atom computer simulations to overcome their sparsity. I will discuss recent meth- models based on experimentally-derived distance measurements. odological developments in integrative structural modelling by satisfying spatial restraints on networks of FRET pairs (hybrid-FRET). We introduce 2326-Plat new procedures for: (i) an automated FRET experiment design tool deter- Dynamic Enzyme: An NMR Study of USP7 mining most informative FRET pairs for structural modeling using prior Irina Bezsonova, Gabrielle Valles, Dmitry M. Korzhnev. structural knowledge (https://github.com/Fluorescence-Tools), (ii) a protocol Molecular Biology and Biophysics, Univ Connecticut Health, Farmington, for efficient FRET-assisted computational structural modelling at multiple CT, USA. scales (http://nmsim.de), (iii) choice of appropriate models for describing Ubiquitin-specific protease 7 (USP7) is a deubiquitinating enzyme (DUB) that the dye behavior and consequences for designing appropriate labels, (iv) plays a pivotal role in multiple oncogenic pathways and therefore is a desir- use of all eight characteristic fluorescence parameters as a vectorial local able target for new anti-cancer therapies. It is a cysteine protease that belongs and dynamic structure information, and (v) a new quantitative quality esti- to the USP family of DUBs. USP7 is unique among other USPs in that its mate for judging the accuracy of determined structures. I will present appli- active site is catalytically incompetent in the apo-state and can rearrange cations simulations and real experiments of our techniques to proteins such into a productive conformation upon substrate binding. Such substrate- as T4 Lysozyme (T4L). We used 33 variants with single-FRET pairs to induced structural rearrangements likely play a role of a ‘‘safety switch’’ resolve three T4L conformers via their characteristic distance sets. Screening that turns the enzyme ‘‘on’’ only when it is engaged with a specific ubiquiti- the known T4L structures, revealed that T4L in solution mainly adopts the nated substrate. Although USP7 has been crystallized in both apo- and known open and closed states in exchange at 4 ms. A newly found minor ubiquitin-bound forms that highlighted the structural differences between state, undisclosed by at present more than 500 crystal and NMR structures the active and inactive protein conformations, these structures represent static of T4L and sampled at 230 ms, may be actively involved in the product snapshots of the enzyme. Whether USP7 samples these conformations in so- release step in catalysis. Importantly, we released together with the PDB lution and the role USP7 conformational dynamics play in its function remain the initial version of the fluorescence (FLR) dictionary extension (https:// unknown. Using the latest developments in Nuclear Magnetic Resonance github.com/ihmwg/FLR-dictionary) on the Integrative/Hybrid Modeling (NMR) spectroscopy we have observed and characterized the conformational (IHM) working group GitHub site. Now, fluorescence-restrained structural dynamics of USP7 in solution. Our data suggest that apo-USP7 has two models can be deposited at PDB-Dev. distinct sites of conformational exchange. Remarkably, they are located in two key functional sites of the enzyme. 2324-Plat 2327-Plat Proteins’ Dynamics, Hydration and Conformational Changes Studied by Specific 13CH3 Labeling and NMR Reveal the Role of Structural Dy- EPR namics to Enzymatic Function Enrica Bordignon. Mioara Larion1, Alexandar Hansen2, Lei Bruschweiler-Li2, Chemistry and Biochemistry, Ruhr University Bochum, Bochum, Germany. Vitali Tugarinov3, Rafael Bruschweiler€ 2, Brian Miller4. Site-directed spin labeling EPR enables detection of conformational changes 1Neuro-Oncology Branch, NIH/NCI, Bethesda, MD, USA, 2Chemistry and in proteins with almost no restriction in the environmental conditions. Key in- Biochemistry, The Ohio State University, Columbus, OH, USA, 3Laboratory formation for structural analysis is provided by changes in the dynamics of of Chemical Physics, NIH/NIDDK, Bethesda, MD, USA, 4Chemistry and spin-labeled sites and by interspin distances between selected pairs of labels. Biochemistry, Florida State University, Tallahassee, FL, USA. Dynamics are detected by continuous wave EPR, interspin distances by pulse To understand the catalytic function of glucokinase, an enzyme that catalyzes dipolar techniques (DEER or PELDOR being the most common). The long- glucose during glycolysis following a kinetic cooperative mechanism, we em- range EPR distance constraints, combined with existing structural data at ployed 13CH3 labeling of methyl groups and NMR. We showed that the origin atomic level for one state of the protein, enable the creation of coarse- of kinetic cooperativity is rooted in intramolecular protein dynamics using kinetic grained models of complex protein rearrangements in their physiological CPMG-NMR data of 17 isoleucine sidechains distributed over all parts of GCK. milieu. Each protein conformational transition due to oligomerization, ligand Residues of glucose-free GCK located in the small domain display a distinct ex- binding, transfer from a water to a membrane environment, etc. is also tightly change behavior involving multiple conformers that are substantially populated coupled to rearrangements in the hydration water surrounding the different (p > 17%) with a kex = 509 5 51 s–1, whereas in the glucose-bound form these protein interfaces. Changes in local hydration dynamics and water accessi- exchange processes are quenched. This exchange process directly competes with bility can be monitored directly and with high precision at physiological tem- the enzymatic turnover rate at physiological glucose concentrations, thereby perature using Overhauser dynamic nuclear polarization (ODNP), which has generating the sigmoidal rate dependence that defines kinetic cooperativity. several advantages with respect to other EPR techniques which provide water accessibility data. We will show examples of structural studies in which the 2328-Plat changes in the protein and its surrounding water environment are observed. Decomposing NMR Ensemble with the Assistance of Single Molecule Examples will be given on ABC transporters, Bcl-2 proteins, and light FRET receptors. Chun Tang. Wuhan Inst Phys/Math, Chinese Academy of Sciences, Wuhan, China. 2325-Plat Proteins are inherently dynamic, and the dynamics allow the protein to fulfill An Integrated Spin-Labeling/Computational-Modeling Approach for specific functions. NMR is exquisitely sensitive to protein dynamics. In partic- Mapping Global Structures of Nucleic Acids ular, paramagnetic relaxation enhancement (PRE) has an