TEMPORAL AND STERIC ANALYSIS OF IONIC PERMEATION AND BINDING IN NA+,K+-ATPASE VIA MOLECULAR DYNAMIC SIMULATIONS A dissertation presented to the faculty of the Russ College of Engineering and Technology of Ohio University In partial fulfillment of the requirements for the degree Doctor of Philosophy James E. Fonseca June 2008 2 c 2008 James E. Fonseca All rights reserved 3 This dissertation entitled TEMPORAL AND STERIC ANALYSIS OF IONIC PERMEATION AND BINDING IN NA+,K+-ATPASE VIA MOLECULAR DYNAMIC SIMULATIONS by JAMES E. FONSECA has been approved for the Department of Electrical Engineering and Computer Science and the Russ College of Engineering and Technology of Ohio University by Savas Kaya Associate Professor of Electrical Engineering and Computer Science Dennis Irwin Dean, Russ College of Engineering and Technology 4 Abstract Fonseca, James, Ph.D., June 2008, Electrical Engineering Temporal and Steric Analysis of Ionic Permeation and Binding in Na+,K+-ATPase via Molecular Dynamic Simulations (206 pp.) Director of Dissertation: Savas Kaya Interdisciplinary research has become a mature approach for the development of novel, integrated solutions for many complex problems in basic science and applied technology. The convergence of biology and nanotechnology is particularly promising from an engineering perspective. This dissertation will use computer simulations to investigate the structure-function of the P-type ATPases, a class of vital biological transmembrane proteins. A detailed understanding of protein function at the atomic level and associated time scale is not only important for biomedical research but also vital for the design and development of engineering applications, such as self- assembling molecular devices. This work’s methodology will show that significant insight into the structure-function relationship of ion-motive ATPases can be derived by a combination of simulation tools and analysis techniques including molecular dynamic trajectories, steric pathway analysis, and electrostatic calculations. Approved: Savas Kaya Associate Professor of Electrical Engineering and Computer Science 5 My dissertation is dedicated to my grandfathers, Ernest Hart and James Fonseca, and dissertation mentor, Dr. Bob Rakowski. 6 Acknowledgments I would like to thank my graduate advisor, Dr. Savas Kaya, for his guidance and commitment to my research and education over the past six years. He has nurtured my interest in semiconductor devices, numerical methods, and nanotechnology, cul- minating in my current research. I hope that his energy, attention to detail, and enthusiasm have been instilled in me. Dr. Bob Rakowksi was an established authority on the Na+,K+-ATPase. His interest in exploring new methods of investigation of this protein led to the interdis- ciplinary collaboration from which this work resulted. I am greatly indebted to him for his advice and mentorship during my Ph.D. research. I am grateful to my dissertation committee for their helpful questions and advice as well as their time invested. I would like to thank my parents, my girlfriend, Monica, and my friends for their support in this endeavor. I am very appreciative for the support I have received from the EECS departmen- tal, GSS, and CMSS. I would like thank Dr. Scott Hooper for the use of his parallel computing system. I would also like to thank Tammy Jordan, EECS graduate secre- tary, for her help over the course of my graduate studies. The majority of the computational time was provided by the Ohio Supercomputer Center. Supported by NIH Grant NS-022979 and NSF Grant NSF-0622158. 7 Table of Contents Page Abstract ...................................... 4 Dedication ..................................... 5 Acknowledgments ................................. 6 List of Tables ................................... 14 List of Figures ................................... 15 1 Introduction .................................. 17 1.1 Scope of Work ............................... 17 1.2 Motivation: Biomimetic Devices ..................... 18 1.2.1 Beyond Silicon .......................... 19 1.2.2 Advantages ............................ 21 1.2.3 Challenges ............................. 22 1.2.4 Molecular Devices: ATPase Proteins .............. 23 1.2.5 Structure-Function ........................ 24 1.2.6 Biomedical Relevance ....................... 25 1.2.7 Computational Angle ....................... 26 1.3 The Na+,K+-ATPase ........................... 28 1.3.1 Biophysical Background ..................... 28 1.3.2 Physiology ............................. 30 1.4 Objectives of this Research ........................ 33 8 1.5 Contributions of this Research ...................... 35 1.6 Chapter Summaries ............................ 37 1.6.1 Glossary and Appendicies .................... 39 2 Molecular Dynamics .............................. 40 2.1 Introduction ................................ 40 2.2 Molecular Dynamics Algorithm ..................... 41 2.3 Topology .................................. 42 2.4 Force Field ................................ 43 2.4.1 Parameterization ......................... 46 2.5 Periodic Boundary Conditions ...................... 46 2.6 Thermodynamic Ensemble ........................ 48 2.7 Energy Minimization ........................... 49 2.8 Water Model ............................... 50 2.9 Lipid Model ................................ 50 3 Modeling the Na+,K+-ATPaseinaLipidBilayerSystem ......... 52 3.1 Overview .................................. 52 3.2 Na+,K+-ATPase Structure ........................ 53 3.3 Homology Modeling ............................ 54 3.4 Choice of Template: SERCA ....................... 54 3.5 Sequence Alignment ........................... 55 3.5.1 Sequence Offset .......................... 60 9 3.5.2 Na+,K+-ATPase N-Domain ................... 62 3.5.3 Model Creation .......................... 63 3.5.4 P-loop Optimization ....................... 64 3.6 Lipid Bilayer Creation .......................... 66 3.6.1 Hole Creation ........................... 67 3.7 Solvation and System Equilibration ................... 68 3.8 Na+,K+-ATPase X-Ray Crystal Structure ............... 72 3.9 Summary ................................. 73 4 MD Simulation and Analysis of the Ca2+ ATPase (SERCA) ........ 74 4.1 Introduction ................................ 74 4.2 Simulations Performed .......................... 75 4.3 Methods and Analysis .......................... 76 4.4 Results ................................... 79 4.4.1 Cytoplasmic Pathway as Determined by CAVER ....... 79 4.4.2 Pathways to the Lumenal Space Predicted by CAVER .... 81 4.4.3 Occlusion Site Connection as Determined by CAVER ..... 85 4.4.4 Summary of Cytoplasmic and Lumenal Pathways as Deter- mined by CAVER ......................... 85 4.4.5 Ca2+ Ion Movement from Site II ................. 86 4.4.6 Ca2+ Ion Occlusion at Site I ................... 89 10 4.4.7 MD Simulations of Ca2+ Ions Placed Along the Putative Lu- menal Pathway .......................... 91 4.4.8 Valence Analysis of Ca2+ Occlusion Sites ............ 93 4.4.9 Electrostatic Calculations of the Transmembrane Environment 94 4.5 Summary ................................. 95 5 MD Simulation and Analysis of the Na+,K+-ATPase ............ 97 5.1 Introduction ................................ 97 5.2 Na+ Binding Sites as Determined by E1 Simulation .......... 98 5.2.1 Na+ Ion Binding without Water ................. 101 5.2.2 Na+ Site I Binding Site ...................... 101 5.2.3 Na+ Site II Binding Site ..................... 105 5.2.4 Na+ Site III Binding Site ..................... 108 5.3 Na+ Ion Binding with Water in E1 Conformation ........... 110 5.3.1 Na+ SitesIandIIwithWater .................. 110 5.3.2 Na+ Site III Binding with Water ................ 112 5.3.3 Water Involvement in Na+ Binding Sites ............ 112 5.4 K+ Binding Sites Determined by Simulation of E2 Conformation ... 114 5.4.1 K+ Site II Binding ........................ 118 5.4.2 K+ Site I Binding ......................... 119 5.5 Steric Investigation of Ion Permeation Pathways ............ 121 5.5.1 E1 Conformation ......................... 122 11 5.5.2 E2P Conformation ........................ 123 5.6 Electrostatic Analysis of E1 and E2P Models .............. 125 5.6.1 Electrostatic Pathway Analysis ................. 126 5.6.2 Electrostatic Binding Site Analysis ............... 129 5.7 Summary ................................. 130 6 Conclusions and Future Work ........................ 133 6.1 Summary and Conclusions ........................ 133 6.2 Future Work ................................ 138 6.2.1 Na+,K+-ATPase X-ray Structure ................ 139 6.2.2 SERCA X-ray Structure with Open Lumenal Pathway .... 140 6.2.3 Computational Outlook ..................... 140 Bibliography .................................... 142 A Publications .................................. 162 A.1 Journal Articles .............................. 162 A.2 Conference Presentations ......................... 163 B Glossary .................................... 164 C Methodology .................................. 170 C.1 Overview .................................. 170 C.2 Homology Modeling ............................ 171 C.2.1 Automated Sequence Alignment ................. 172 C.2.2 Manual Alignment ........................ 173 12 C.2.3 N-Domain Superposition .................... 173 C.2.4 Homology Model Building .................... 174 C.2.5 Homology Model Optimization ................. 175 C.3 System Preparation ............................ 177 C.3.1 Membrane