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Computational Prediction and Experimental Validation of Cytochrome C Oxidase Main-Chain Flexibility and Allosteric Regulation of the K-Pathway

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Computational Prediction and Experimental Validation of Cytochrome C Oxidase Main-Chain Flexibility and Allosteric Regulation of the K-Pathway COMPUTATIONAL PREDICTION AND EXPERIMENTAL VALIDATION OF CYTOCHROME C OXIDASE MAIN-CHAIN FLEXIBILITY AND ALLOSTERIC REGULATION OF THE K-PATHWAY By Leann Marie Buhrow A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Cell and Molecular Biology 2012 ABSTRACT COMPUTATIONAL PREDICTION AND EXPERIMENTAL VALIDATION OF CYTOCHROME C OXIDASE MAIN-CHAIN FLEXIBILITY AND ALLOSTERIC REGULATION OF THE K-PATHWAY By Leann Marie Buhrow Comparison of crystallographic structures and deuterium accessibility of different redox states of cytochrome c oxidase (CcO) have suggested conformational changes of mechanistic significance. To predict the intrinsic flexibility and low energy motions in CcO, this work has analyzed available high-resolution crystallographic structures with ProFlex and elNémo computational methods. CcO is predicted to undergo rotational motions on the interior and exterior of the membrane, driven by transmembrane helical tilting and bending, coupled with rocking of the β-sheet domain. Consequently, the proton K-pathway becomes sufficiently flexible for internal water molecules to alternately occupy upper and lower parts of the pathway. At the entrance of the K-pathway, a conserved crystallographically-defined steroid binding site had been previously identified. Binding of diverse amphipathic molecules including detergents, fatty acids, steroids, and porphyrins affect the activity of the Rhodobacter sphaeroides CcO variant E101A, as well as the wild type and bovine enzymes. Detergent inhibition is observed for the E101A variant but may be overcome in the presence of micromolar concentrations of steroids and porphyrin analogs. Computational modeling of lauryl maltoside, bilirubin, and protoporphyrin IX into the conserved membrane site shows energetically favorable binding modes for these ligands and suggests that a groove at the interface of subunits I and II, including the entrance to the K-pathway, mediates competitive ligand interactions involving two overlapping sites. The high affinity and specificity of a number of compounds for this region, and its conservation and impact on CcO activity, support its physiological significance. Physiological ligands, specific for the steroid binding site, were identified by combining three computational approaches: ROCS comparison of ligand shape and electrostatics, SimSite3D analysis of similarity to ligand binding sites in the Protein Data Bank, and SLIDE screening of small molecules by docking. Together, the results suggest several steroids, adenine and guanine nucleotides, NAD+, FAD, and phosphorylated isoprenes as top candidates for interacting at this site, along with bile acids and porphyrins. In vitro oxygen consumption assays support some of these predicted interactions. In the wild type R. sphaeroides CcO, ATP and GDP are mildly inhibitory while the steroidal deoxycholate and fusidic acid ligands are highly inhibitory. Cytochrome c titration assays indicate nucleotides inhibit CcO activity in low cytochrome c conditions, similar to the observed ATP inhibition of mammalian CcO. These finding suggest that nucleotides regulate CcO on the conserved subunit I-III core, potentially at the steroid binding site. Overall this work predicts CcO conformational changes required for catalysis, including the conformational change of the K-pathway, and describes the first report of allosteric regulation of bacterial CcO by nucleotides. These results have been used to understand allosteric regulation by restricting conformational changes, generate a two-site model for lipid and ligand- specific regulation, and propose CcO regulation by arresting the enzyme in a state which cannot produce oxygen radical byproducts. Copyright by LEANN MARIE BUHROW 2012 ACKNOWLEDGEMENTS I would like to thank: • My co-advisors, Shelagh Ferguson-Miller and Leslie Kuhn, for their guidance, support, and direction in both research and life. • Past and present members of the Ferguson-Miller and Kuhn labs including Drs. Carrie Hiser, Jian Liu, Denise Mills, Ling Qin, Jon Hosler, Shujuan Xu, Namjoon Kim, Xi Zhang, and Jeff Van Voost and Fei Li, Matt Tonero, and Nan Liu. I would especially like to thank Carrie Hiser for her technical support, brainstorming sessions, and being a wonderful collaborator on our lipids project. I would also like to thank Ling Qin and Jeff Van Voost for their inspirational drive. Without their crystal structures and SimSite3D methods, this work would not be possible. • My committee members: Drs. Benning, Hausinger, Hegg, and Yan for their suggestions, critiques, and diverse research interests. • The Cell and Molecular Biology and Quantitative Biology Graduate Programs at MSU for allowing me to focus on protein structure and function and expand my interests into computational structural biology. I would especially like to thank Dr. Susan Conrad for her support and guidance. I would also like to thank Helen Geiger and Becky Mansel for their friendship and endless technical assistance. • My advisors at the University of Wisconsin-Parkside: Drs. MacWilliams, Lewis, and Wood for their mentoring and encouragement to attend graduate school. • My fellow students at MSU and UW-Parkside for their friendship throughout the years. Without your laughter and shenanigans, my life would be a more serious and boring experience. • My family, especially John, Terri, and Emily Buhrow. I love you! v TABLE OF CONTENTS LIST OF TABLES vii LIST OF FIGURES viii CHAPTER 1: Rhodobacter sphaeroides cytochrome c oxidase as a model system for understanding energy coupling and allosteric regulation in membrane proteins 1 References 27 CHAPTER 2: From static structure to living protein: computational analysis of cytochrome c oxidase main-chain flexibility 40 Introduction 41 Materials and Methods 44 Results and Discussion 52 Conclusions 77 References 78 CHAPTER 3: Structural predictions and functional consequences of porphyrin, steroid, and detergent ligands binding to the cytochrome c oxidase steroid binding site 84 Introduction 85 Materials and Methods 93 Results and Discussion 96 Conclusions 109 References 110 CHAPTER 4: Three-pronged computational approach to predict regulatory ligands of cytochrome c oxidase 115 Introduction 116 Materials and Methods 119 Results and Discussion 124 Conclusions 178 References 179 CHAPTER 5: Perspectives on CcO K-pathway conformational change and allosteric regulation 186 References 193 vi LIST OF TABLES TABLE 2.1: CcO structures analyzed by ProFlex and elNémo 46 TABLE 2.2: Comparison of oxidized and reduced two-subunit RsCcO structures 47 TABLE 2.3: Diverse membrane protein folds analyzed by ProFlex and elNémo 60 TABLE 2.4: elNémo percentage of atoms significantly displaced and relative frequencies of normal modes 60 TABLE 2.5: CcO helical conformational changes in elNémo low-energy modes 64 TABLE 3.1: RsCcO and BtCcO steroid binding site characterization 98 TABLE 3.2: Effects of bile acids on E101A mutant and WT RsCcO 101 TABLE 4.1: ROCS predicted ligands 125 TABLE 4.2: SimSite3D predicted binding sites 134 TABLE 4.3: Percentage of protein binding site matches between the CcO steroid binding site and diverse Binding MOAD proteins 143 TABLE 4.4: SLIDE docked ligands and their protein interactions 145 vii LIST OF FIGURES FIGURE 1.1: The electron transport chain 8 FIGURE 1.2: Structure of bacterial and mammalian cytochrome c oxidases 10 FIGURE 1.3: Cytochrome c oxidase oxygen reduction mechanism 15 FIGURE 2.1: Architecture of the two-subunit structure essential for RsCcO activity 42 FIGURE 2.2: Dependence of CcO flexibility on thermal energy increase, analyzed by ProFlex hydrogen bond dilution profiles 45 FIGURE 2.3: Mean squared displacement values within RsCcO trans- membrane helices 49 FIGURE 2.4: ProFlex flexibility comparison the of two and four subunit crystal structures of RsCcO 52 FIGURE 2.5: ProFlex prediction of main-chain flexibility and stability in RsCcO 54 FIGURE 2.6: Thermal denaturation of the RsCcO structure by ProFlex 55 FIGURE 2.7: elNémo simulation of low frequency motions in RsCcO 58 FIGURE 2.8: Relative flexibility of membrane proteins assessed using ProFlex 61 FIGURE 2.9: RsCcO, β2 adrenergic receptor, KcsA potassium channel, and VDAC residue displacement 61 FIGURE 2.10: Planes of relatively stationary residues in the CcO trans- membrane helices 67 FIGURE 2.11: Comparison of RsCcO normal modes and crystallographic temperature factors 69 FIGURE 2.12: Water molecule movement in the K-pathway 71 FIGURE 2.13: Conformational gating in the RsCcO oxygen channels 74 viii FIGURE 3.1: Bile acid structures 86 FIGURE 3.2: Deoxycholate resolved near the K-pathway entrance of RsCcO 89 FIGURE 3.3: RsCcO and BtCcO steroid binding site with conserved residues and bound water molecules 98 FIGURE 3.4: The steady-state activities of the detergent-solubilized RsCcO E101A 99 FIGURE 3.5: Potential binding orientations of known lipidic ligands in the RsCcO steroid binding site 103 FIGURE 3.6: RsCcO K-pathway rigidification upon ligand binding 107 FIGURE 4.1: Structure of bacterial and mammalian cytochrome c oxidases 117 FIGURE 4.2: ROCS predicted 2D ligand structures 130 FIGURE 4.3: ROCS aligned crystallographic deoxycholate and predicted ligands 132 FIGURE 4.4: SimSite3D predicted analogous chemical points between the RsCcO steroid binding site and sites found in Binding MOAD 142 FIGURE 4.5: Similar binding modes were predicted by SimSite3D aligned binding site and SLIDE docked ligands 147 FIGURE 4.6: Ethanol-soluble
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