Investigation of Protein/Ligand Interactions Relating Structural Dynamics to Function: Combined Computational and Experimental Approaches DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Ryan Elliott Pavlovicz Graduate Program in Biophysics The Ohio State University 2014 Dissertation Committee: Chenglong Li, Advisor Charles E. Bell Michael E. Paulaitis Copyright by Ryan Elliott Pavlovicz 2014 Abstract The use of computers in chemistry has matured significantly since the introduction of the modern personal computer, leading to the development of many tools that may be used to describe phenomena that are difficult or otherwise impossible to observe experimentally. Computational chemistry is becoming an integral component of many research programs, often resulting in the formation of hypotheses that may be tested experimentally. This document details the application of computational tools to study ligand/receptor interactions in two systems: the nicotinic acetylcholine receptor (nAChR) and the retinoic acid receptor (RAR). The nAChR study details how a combination of homology modeling, molecular dynamics (MD), blind docking, and free energy analysis may be used to determine the binding site of a ligand with few clues from experiment to guide the search. Specifically, the binding site for a class of negative allosteric nAChR modulators was successfully identified. The computationally predicted binding site was verified by functional assays performed on receptors that were mutated at the suspected binding site. Additionally, a comparison of structural data from homologous proteins and MD simulations of the receptor in complex with an allosteric modulator lead to a proposed mode of allosteric antagonism that involves inhibition of C loop closure, thereby preventing channel opening. ii In the RAR study, both computation and experiment were applied to characterize the activity of two β-apocarotenoids that have been previously described as antagonists of all-trans retinoic acid (ATRA), the endogenous RAR agonist. The activity of RAR ligands is related to how they influence the interaction between the receptor and coactivator proteins that lead to gene transcription. The results of isothermal titration calorimetry (ITC) experiments indicate that the β- apocarotenoids induce an interaction between the receptor and coactivator that is intermediate in strength between the unliganded and ATRA-bound receptor, indicating that these compounds would be most accurately characterized as partial agonists instead of antagonists. One of the partial agonists, β-apo-13- carotenone, exhibits an unexpectedly high affinity for RAR given its chemical differences from known high-affinity binders. Modeling this compound in the RAR binding site lead to the hypothesis that a covalent interaction may be occurring between the carotenone and a conserved cysteine residue in the binding pocket. While not conclusive, NMR and mass spectrometry experiments suggest that this interaction is indeed occurring. Computational free energy analysis was also performed between the ligand- bound receptors and the coactivator. Using the molecular mechanics Poisson- Boltzmann surface area (MM-PBSA) method applied to microsecond MD simulations, very strong correlation was achieved between the computational binding energies and the experimental ITC data, providing support that the compounds were correctly modeled in the RAR binding pocket. Converged iii binding energy averages that lead to the strong correlation with experiment were contingent upon simulation lengths of ~1 μs, and inclusion of both the calculated PBSA free energy of solvation and entropic components of binding were found to strengthen the correlation. Finally, Chapter 5 includes a study on the parameterization of a new atom type for use in the pair-wise additive AMBER force field. The sulfonium atom type is not included in the current set of parameters since it is relatively uncommon in biology. However, S-adenosylmethionine (SAM), the most common methyl donor in biology, is a notable exception. The development of sulfonium parameters required for the MD simulation of SAM is discussed in detail. iv Dedication This document is dedicated to my family. v Vita 1999 ....................................................... North Royalton High School 2004 ....................................................... B.S. Electrical and Computer Engineering, Ohio State University 2006-2014 .............................................. Graduate Research Assistant, Department of Pharmacy, Ohio State University 2008-2010 ............................................. American Foundation for Pharmaceutical Education Pre-Doctoral Fellowship 2010 ...................................................... NSF East Asian and Pacific Summer Institutes for U.S. Graduate Students Research Fellowship (Institute of Biophysics, Beijing, China) 2010 ...................................................... American Chemical Society Division of Medicinal Chemistry Pre-Doctoral Fellowship 2011 ...................................................... Outstanding Student Achievement Award presented by the Ohio State University Biophysics Graduate Committee 2012 ...................................................... Presidential Fellowship (Ohio State University Graduate School) vi Publications 1.) Frey EN, Pavlovicz RE, Wegman CJ, Li C, Askwith CC. “Conformational changes in the lower palm domain of ASIC1a contribute to desensitization and RFamide modulation”, PLOS ONE, 8(8): e71733, 2013. 2.) Yi B, Long S, González-Cestari TF, Henderson BJ, Pavlovicz RE, Werbovetz K, Li C, McKay DB. “Discovery of benzamide analogs as negative allosteric modulators of human neuronal nicotinic receptors: pharmacophore modeling, rational design, and structure-activity relationship studies”, Bioorganic & Medicinal Chemistry, 21(15):473-43, 2013. 3.) Liu MJ, Bao S, Gálvez-Peralta M, Pyle CJ, Rudawsky AC, Pavlovicz RE, Killilea DW, Li C, Nebert DW, Wewers MD, Knoell DL. “ZIP8 regulates host defense through zinc-mediated inhibition of NF-κB.” Cell Reports, 3(2):386-400, 2013. 4.) Still P, Yi B, González-Cestari TF, Pan L, Pavlovicz RE, Chai H-B, Ninh T, Li C, Soejarto DD, McKay DB, Kinghor AD. “Alkaloids from Microcos paniuclata with cytotoxic and nicotinic receptor antagonistic activities”, Journal of Natural Products, 76(2):243-439, 2013. 5.) Koval OM, Snyder JS, Wolf RM, Pavlovicz RE, Cardona N, Glynn P, Leymaster ND, Dun W, Wright PJ, Qian L, Mitchell CC, Boyden PA, Binkley PF, Li C, Anderson ME, Mohler PJ, Hund TJ. “CaMKII-based regulation of voltage-gated Na+ channel in cardiac disease”, Circulation, 126(17):2084-94, 2012. 6.) Henderson BJ, González-Cestari TF, Yi B, Pavlovicz RE, Boyd RT, Li C, Bergmeier SC, McKay DB. “Defining the putative inhibitory site for a selective allosteric modulator of human α4β2 neuronal nicotinic receptors”, ACS Chemical Neuroscience, 3(9):682-92, 2012. 7.) Henderson BJ, Carper DJ, González-Cestari TF, Yi B, Mahasenan KV, Pavlovicz RE, Dalefield ML, Coleman RS, Li C, McKay DB. “Structure-activity relationship studies of sulfonylpiperazine analogues as novel negative allosteric modulators of human neuronal nicotinic receptors”, Journal of Medicinal Chemistry, 54(24):8681-92, 2011. 8.) Mahasenan KV, Pavlovicz RE, Henderson BJ, González-Cestari TF, Yi B, McKay DB, Li C. “Discovery of novel α4β2 neuronal nicotinic receptor modulators through structure-based virtual screening”, ACS Medicinal Chemistry Letters, 2(11):855-860, 2011. 9.) Pavlovicz RE, Henderson BJ, Bonnell AB, Boyd RT, McKay DB, Li C. “Identification of a novel negative allosteric site on human α4β2 and α3β4 neuronal nicotinic acetylcholine receptors”, PLOS ONE, 6(9): e24949, 2011. 10.) West MB, Wickham S, Quinalty LM, Pavlovicz RE, Li C, Hanigan M. “Autocatalytic cleavage of human gamma-glutamyl transpeptidase is highly dependent on N-glycosylation at asparagine 95”, Journal of Biological Chemistry, 286(33):28876-88, 2011. 11.) Henderson BJ, Pavlovicz RE, Allen JD, González-Cestari TF, Orac CM, Bonnell AB, Zhu MX, Boyd RT, Li C, Bergmeier SC, McKay DB. “Negative allosteric modulators that target human α4β2 neuronal nicotinic receptors”, Journal of Pharmacology and Experimental Therapeutics, 334(3):761-74, 2010. vii 12.) Doddapaneni K, Mahler B, Pavlovicz RE, Haushalter A, Yuan C, Wu Z. “Solution structure of RCL, a novel 2’-deoxyribonucleoside 5’-monophosphate N-glycosidase”, Journal of Molecular Biology, 394(3), 423-434, 2009. 13.) Tiwari R, Mahasenan K, Pavlovicz RE, Li C, Tjarks W. “Carborane clusters in computational drug design: a comparative docking evaluation using AutoDock, FlexX, Glide, and Surflex”, Journal of Chemical Informatics, 49(6), 1581-1589, 2009. 14.) Liu Z, Liu S, Xie Z, Pavlovicz RE, Wu J, Chen P, Aimiuwu J, Pang J, Bhasin D, Neviani P, Fuchs JR, Plass C, Li PK, Li C, Huang THM, Wu LC, Rush L, Wang H, Perrotti D, Marcucci G, Chan KK. “Modulation of DNA methylation by a sesquiterpene lactone pathenolide”, Journal of Pharmacology and Experimental Therapeutics, 329(2), 505-514, 2009. 15.) González-Cestari TF, Henderson BJ, Pavlovicz RE, McKay SB, El-Hajj RA, Pulipaka AB, Orac CM, Reed DD, Boyd RT, Zhu MX, Li C, Bergmeier SC, McKay DB. “Effect of novel negative allosteric modulators of neuronal nicotinic receptors on cells expressing native and recombinant nicotinic receptors: implications for drug discovery”, Journal of Pharmacology and Experimental Therapeutics,