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CONSERVED SOLVENT NETWORKS in GPCR ACTIVATION by ELISE

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CONSERVED SOLVENT NETWORKS in GPCR ACTIVATION by ELISE CONSERVED SOLVENT NETWORKS IN GPCR ACTIVATION by ELISE BLANKENSHIP Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Systems Biology and Bioinformatics Program CASE WESTERN RESERVE UNIVERSITY May, 2016 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of ELISE BLANKENSHIP Candidate for the degree of Doctor of Philosophy* Committee Chair Dr. Marvin Nieman Committee Member Dr. Mark Chance Committee Member Dr. David T. Lodowski Committee Member Dr. Masaru Miyagi Date of Defense March 3rd, 2016 *We also certify that written approval has been obtained for any proprietary material contained therein Table of Contents List of Tables…………………………………………………………………………...iii List of Figures………………………………………………………………………….iv Acknowledgements…………………………………………………………………..vi List of Abbreviations…………………………………………………………………vii Abstract…………………………………………………………………………………ix 1. Introduction…………………………………………………………………………..1 1.1 Rhodopsin in the Retina…………………………………………………….2 1.2 GPCR Structure……………………………………………………………16 1.3 Computational Analysis of Solvent in Protein Structure………………..33 2. Methods……………………………………………………………………………..37 2.1 Rhodopsin Purification from Bovine Retinal Tissue…………………….37 2.2 Crystallization and Structure Determination of Rhodopsin…………….47 2.3 Analysis of Rhodopsin Crystal Structures……………………………….50 2.4 Analysis of Crystal Structures of Class A GPCRs………………………54 3. The Crystallographic Structure of Rhodopsin………………………………..60 3.1 The High-Resolution Structure of Activated Opsin……………………..60 3.2 Analysis of Previously Determined Rhodopsin Crystal Structures……72 i 4. Water Networks in the Rhodopsin Activation Cycle…………………………77 4.1 Constructing the Rhodopsin Catalytic Cycle…………………………….77 4.2 Water Networks in Ground State and Active State Opsin………………84 4.3 Solvent Subtypes in the Rhodopsin Transmembrane Region…………92 5. Allosteric Networks in GPCR Signaling………………………………………..99 5.1 Conserved Solvent in GPCR Structures…………………………………99 5.2 Whole Network Comparisons of GPCR Solvent Networks…………...106 6. Conclusions and Future Directions…………………………………………...114 6.1 Implications of Solvent Networks for GPCR Signaling………………..114 6.2 Computational Modelling of GPCR-Solvent Interactions……………..115 6.3 Modern Approaches to Drug Discovery………………………………..116 Bibliography…………………………………………………………………………118 ii List of Tables Table 1. Examples of Monogenic Retinal Diseases Linked to Protein Malfunction……………………………………………………………………….9 Table 2. Preparation of Sucrose Solutions for ROS Isolation………………………41 Table 3. Preparation of Kühn’s Buffer………………………………………………..42 Table 4. Data Collection & Refinement Statistics for Activated Opsin…………….50 Table 5. Changes in Water Number in Rhodopsin after Refitting Protocol………..54 Table 6. Class A GPCRs Used for Analysis………………………………………….58 Table 7. Structural Superposition of Selected Bovine Rhodopsin Structures…….67 iii List of Figures Figure 1. Retinal structure and function………………………………………………..4 Figure 2. Class A GPCRs, the rhodopsin family…………………………………….10 Figure 3. Conserved motifs in mammalian rhodopsin essential for activation……18 Figure 4. The phototransduction cascade in mammalian rod cells……………….22 Figure 5. Sucrose gradient construction and purification of crude ROS…………40 Figure 6. The ligand binding site of published rhodopsin structures show evidence for occupancy by detergent……………………………………………………56 Figure 7. Detergent stabilizes an activated state like structure of opsin bound to a high-affinity peptide mimetic of the Gtα-CT…………………………………..64 Figure 8. A networks of waters mediates contacts with Gα-CT in the structure of activated opsin………………………………………………………………….66 Figure 9. Docking of full-length Gt onto activated opsin…………………………….68 Figure 10. Occupancy of the density within the chromophore site is better satisfied by a C9G detergent molecule than all-trans-retinal………………………….71 Figure 11. Entry and exit tunnels in activated opsin…………………………………75 Figure 12. Rhodopsin activation cycle constructed for structures available on the PDB……………………………………………………………………………..80 Figure 13. Ordered solvent employs polar contacts with conserved residues in the ground state and activated state of the receptor……………………………81 Figure 14. Waters found in rhodopsin structures form conserved clusters throughout the activation cycle………………………………………………. 83 iv Figure 15. Waters in rhodopsin structures are found near residues modified in radiolytic footprinting experiments……………………………………………84 Figure 16. Molecular switch regions in activated opsin are bound to ordered water molecules that connect the ligand binding site to G protein binding site…..87 Figure 17. Remodeling of the ionic lock forms a nucleus for organization of water molecules providing a binding site for activation and Gt binding…………..89 Figure 18. Reorganization of the TM solvent network upon attainment of the activated state………………………………………………………………….92 Figure 19. Changes in the network of ordered water molecules stabilize structural changes in the activated state………………………………………………...94 Figure 20. Conserved waters in GPCR structures…………………………………104 Figure 21. Conservation of hydrogen bonding networks connecting conserved motifs important to GPCR activation………………………………………..110 Figure 22. Water mediated hydrogen bonding networks in Class A GPCRs……111 v Acknowledgements My advisor, Dr. David Lodowski, for his continual support and mentorship over the course of my graduate career. My undergraduate advisor, Dr. Nancy Lill, without whom I would not have begun a career in research. My thesis committee and collaborators, Dr. Marvin Nieman, Dr. Masaru Miyagi, Dr. Mark Chance, Dr. Ardeschir Vahedi-Faridi and Dr. Krishna Vukoti, for guidance, feedback, and reassurance. Faculty, staff, classmates and friends at The Ohio State University and Case Western Reserve University, who helped me to learn and grow. My family and Thomas, for unconditional love and support. vi List of Abbreviations Cyclic guanosine monophosphate (cGMP) Correlation coefficient (CC) Double electron-electron resonance spectroscopy (DEER) Dithiothreitrol (DTT) Endoplasmic reticulum (ER) Extracellular loop region (EC) Electron paramagnetic resonance spectroscopy (EPR) Ethylene diamine tetra acetic acid (EDTA) Feature Enhanced Maps (FEMs) Ganglion cell layer (GCL) G protein coupled receptors (GPCRs) Guanosine 5’triphosphate (GTP) Gα transducin C-terminal mimetic peptide (Gα-CT) Lecithin retinol acyltransferase (LRAT) Lipidic cubic phase (LCP) Intracellular loop region (IC) Inner nuclear layer (INL) Inner plexiform layer (IPL) Metarhodpsin II state (MII) Molecular weight cut off (MWCO) Nuclear magnetic resonance spectroscopy (NMR) Outer nuclear layer (ONL) vii Outer plexiform layer (OPL) Protein Data Bank (PDB) Retinal dehydrogenase enzymes (RDHs) Retinal pigment epithelium (RPE) Root mean square deviation (RMSD) Rod outer segment (ROS) Real space R-value z-score (RSRZ) Transmembrane (TM) Time-resolved wide angle x-ray scattering (TR-WAXS) X-ray free-electron laser (XFEL) n-β-D-nonyl-glucoside detergent (C9G) n-β-D-octyl-glucoside detergent (C8G) n-β-D-dodecyl-maltoside (DDM)/(C12M) 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) 2-(N-morpholino)ethanesulfonic acid (MES) viii Conserved Solvent Networks in GPCR Activation Abstract by ELISE BLANKENSHIP Rhodopsin, the light-activated G protein coupled receptor (GPCR), has served as a model receptor for GPCRs and their activation, and previous analyses have suggested productive roles for conserved solvent in the transmembrane region. A new structure of photoactivated opsin, stabilized by a peptide corresponding to the Gα C-terminus was determined as a basis for this study. In this structure, an extensive water-mediated hydrogen bond network linking the chromophore binding site to the site of G protein binding is observed, providing connections to conserved motifs essential for GPCR activation. Comparison of this extensive solvent-mediated hydrogen-bonding network with the positions of ordered solvent in earlier crystallographic structures of rhodopsin reveals both static (structural) and dynamic (functional) water-protein interactions present during the activation process. Comparison to structures of other Class A GPCRs reveals waters that are present in conserved locations regardless of activation states, while whole network analysis reveals waters common only to GPCRs in a particular activation state. These analyses strongly support an integral role for the dynamic ordered water network in class A GPCR activation. ix Chapter 1: Introduction To regulate the numerous systems that comprise the body, signals must be sent and received between cells. Integral in this process are cellular receptors, proteins that receive signals and elicit a response. G protein coupled receptors (GPCRs) are membrane-spanning cellular receptors that transduce signal across the plasma membrane in response to a wide variety of ligands. A number of molecules act upon these receptors to communicate between cell types and across tissues (Hofmann and Palczewski, 2015). This ligand binding to receptor regulates a variety of processes, and is responsible for responses ranging from a change in blood pressure to memory storage to vision information processing. GPCRs represent the largest classes of proteins in the human genome, and as such, compounds targeting these receptors to modulate their activity make up close to 35% of all current non-antibiotic therapeutics (Salon et al., 2011). The GPCR rhodopsin is found in the mammalian
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