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Modeling Structures and Transport Phenomena of Transmembrane Channels and Transporters

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Modeling Structures and Transport Phenomena of Transmembrane Channels and Transporters Modeling Structures and Transport Phenomena of Transmembrane Channels and Transporters Thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Computational Natural Sciences (Biophysics) by Siladitya Padhi 201166647 [email protected] Center for Computational Natural Sciences and Bioinformatics International Institute of Information Technology, Hyderabad (Deemed to be University) Hyderabad 500032, India October 2016 © Siladitya Padhi 2016 All rights reserved ii iii iv v vi Acknowledgments I must start by thanking my supervisor, Dr. U. Deva Priyakumar, for giving me an opportunity to work with him, and for making all this possible. Having had a similar approach towards work, I was pretty comfortable working with him. He has always been active and cooperative, and I always had the freedom to walk into his office any time and start discussing business. He has given me a lot of opportunity, the most noteworthy one being my visit to Germany for a 10-day workshop at Ruprecht-Karls-Universität Heidelberg, followed by a two-month stay at Westfälische Wilhelms-Universität Münster. I also had a lot to learn working as a teaching assistant with him. There has been a lot to gain from faculty members at the Center for Computational Natural Sciences and Bioinformatics (CCNSB) at IIIT. Dr. Prabhakar Bhimalapuram has constantly given suggestions, right from my initial days, when he explained the basics of enhanced sampling methods to me, to my pre-PhD defense, during which he gave valuable and critical inputs. I have benefited immensely from the well-structured Statistical Mechanics course offered by Dr. Marimuthu Krishnan. The course started with a thorough brush-up of basic stat mech and ended with students writing codes – from scratch – for performing molecular dynamics and Monte Carlo simulations. Working as a teaching assistant with Prof. Harjinder Singh was a very nice experience. I am grateful to Prof. Singh also because he introduced us to our experimental collaborators at ICGEB Delhi as well as RCB Delhi. Working with Dr. Shahid Jameel (formerly at ICGEB Delhi and now CEO, Wellcome Trust/DBT India Alliance) taught me to look at things from an experimentalist’s perspective, and to describe things in a way that would interest an experimentalist. The work being done with Dr. Avinash Bajaj (RCB Delhi) for the last few months was also a nice learning experience. I must seriously thank Dr. Mark Waller, my boss at Universität Münster. His hospitality was admirable, and his patience with me was commendable! I really appreciate the method development work he is doing. vii I am grateful to the Department of Biotechnology, Government of India, and the Department of Atomic Energy, Government of India, for funding our work. I am also grateful to Universität Heidelberg (Prof. Peter Comba, in particular) for funding my visit to Germany. I must now thank the scientific/non-scientific staff and my colleagues at CCNSB, IIIT. Dr. Semparithi Aravindan is awesome: he has a solution for just about anything to do with the clusters. Umesh has been the unsung hero of CCNSB. Tanashree, Sandhya, Siddharth, Indrajit, Navneet, and Chinmayee have been wonderful friends, Aditya has been a funny entertainer, and Suresh and Ramakrishna played a very important role by assisting me with the scripts in my initial days. Friends from other research groups at CCNSB without whom the list would be incomplete: Antarip, Bipin(ji), Broto, Kartheek, Mohan, and the others. Finally, I owe all my gratitude to my mother and my father, who always gave me the freedom to do what I want. The constant support of my parents and my brother (and his family) has been instrumental in making possible whatever I have done. It is their love, patience, and strength that have been my pillars of support. viii Abstract Membrane proteins account for more than half of present day drug targets, but an understanding of structure-function relationships in these proteins continues to be a challenge, owing to the difficulties associated with experimental investigations of these proteins. The work in this thesis makes use of all-atom molecular dynamics simulations to address important problems pertaining to membrane proteins. A major problem in drug design targeting membrane proteins is limited availability of experimental structures. Toward this end, an approach has been proposed here for modeling the structures of -helical membrane proteins. While the approach is able to provide structural models for a number of channel proteins, possible improvements, that would make the approach more suitable for other membrane proteins, are proposed. The other aspect of membrane proteins that has been difficult to investigate experimentally is an atomistic level understanding of the conduction and selectivity mechanism in channels and transporters. This thesis provides insights into the mechanism of transport in a number of channels and transporters, including two viral ion channels, a mammalian aquaporin, and a bacterial urea transporter. In the first part of the thesis, it is shown that the protein Vpu from human immunodeficiency virus type 1 (HIV-1) exists in a pentameric state, and a structural model for the oligomeric form of the protein is proposed. Free energy calculations performed on the structural model are able to provide a thermodynamic basis for the weak ion channel activity of the protein. In the next part of the thesis, a structure modeling approach is described that attempts to predict the structures of -helical membrane proteins by minimizing unfavorable contacts. The approach works well for -helical channels, and can be extended to all -helical membrane proteins. This is followed by an investigation of ion conduction and selectivity in the p7 channel from hepatitis C virus (HCV). It is shown that a hydrophobic stretch in the channel preferentially allows the singly charged K+ rather than the doubly charged Ca2+ to pass through, and that interionic repulsion is crucial for ion conduction through the pore. Water transport through the human aquaporin AQP2 is then examined, revealing an almost barrierless permeation profile for water transport. The implications of the restriction of water orientation in the middle region of the pore are discussed. ix Finally, the mechanism of urea transport through the bacterial urea transporter dvUT is studied, and it is shown that permeating urea molecules are able to overcome a hydrophobic barrier arising from the existence of pore-facing phenylalanine residues by forming stacking interactions with these residues. While the work on structure modeling reveals some ―rules‖ that membrane proteins follow at the time of oligomerization, the studies on transport across membrane proteins suggest that transport proteins have their unique mechanisms for determining selectivity, depending on what chemical species they conduct. x Contents SECTION PAGE Acknowledgments vii Abstract ix Contents xi List of figures xvii List of tables xxv 1 Introduction 1 1.1 Membrane proteins could be helices or barrels 1 1.1.1 Helical structures 2 1.1.2 -barrels 2 1.2 Functional classification of membrane proteins 3 1.2.1 Transport proteins 3 1.2.2 Receptors 4 1.2.3 Enzymes 5 1.2.4 Other membrane proteins 5 1.3 The lipid bilayer 6 1.3.1 Bilayer structure and explicit bilayer models 6 1.3.2 Implicit membrane models 7 1.4 Proteins studied in this thesis 8 1.4.1 Viroporins 8 1.4.2 Channels for polar solutes 8 References 9 2 Methods 12 2.1 Force fields 12 2.1.1 Bond stretching 13 xi 2.1.2 Angle bending 13 2.1.3 Dihedrals 13 2.1.4 Electrostatic interactions 14 2.1.5 van der Waals interactions 15 2.1.6 Other terms 15 2.2 Integration algorithms 15 2.3 Periodic boundary conditions 17 2.4 Ensembles 18 2.5 Enhanced sampling methods 19 2.5.1 Replica exchange 19 2.5.2 Umbrella sampling 20 2.6 Setting up membrane protein simulations 21 2.6.1 Membrane setup 21 2.6.2 Equilibration and production 21 References 21 3 Oligomeric State of HIV-1 Vpu 24 3.1 Introduction 24 3.2 Methods 26 3.2.1 Modeling of TMD and replica-exchange molecular dynamics 26 3.2.2 Selection of representative structures and further equilibration 28 3.2.3 Molecular dynamics in a fully hydrated lipid bilayer 28 3.3 Results and Discussion 30 3.3.1 Higher oligomers display reduced tilt 30 3.3.2 Tetramer, pentamer, and hexamer are possible oligomeric states 31 3.3.3 Explicit membrane MD simulations reveal the pentamer to be the most 33 stable oligomeric state 3.3.4 The helices in the pentamer are held together by strong van der Waals 34 interactions 3.3.5 The hydrophilic and basic residues in the TMD interact with lipid 38 headgroups xii 3.3.6 A hydrophobic region occurs around the middle of the channel 38 3.3.7 The structural model 39 3.4 Conclusions 42 References 43 4 Ion Conduction through Vpu 49 4.1 Introduction 49 4.2 Methods 51 4.3 Results and Discussion 53 4.3.1 Atomistic structure of TM domain of Vpu, and features of the lumen of the 53 pore 4.3.2 Free energy profiles indicate weak ion conduction 55 4.3.3 Extent of solvation governs ion transport kinetics 58 4.3.4 Absence of concerted ion transport explains the slow kinetics 60 4.3.5 Perspectives 63 4.4 Conclusions 64 References 65 5 Modeling Structures of Helical Membrane Proteins 71 5.1 Introduction 71 5.2 Methods 73 5.3 Results and Discussion 76 5.3.1 χssembling -helices based on the minimum unfavorable contacts 76 approach 5.3.2 Multiple simulations and REMD 79 5.3.3 Geometry-based selection 81 5.3.4 Clustering structures
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