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Cooperative Allosteric Ligand Binding in Calmodulin

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Cooperative Allosteric Ligand Binding in Calmodulin COOPERATIVE ALLOSTERIC LIGAND BINDING IN CALMODULIN A dissertation submitted to Kent State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy by Prithviraj Nandigrami December, 2017 c Copyright All rights reserved Except for previously published materials Dissertation written by Prithviraj Nandigrami B.Sc., University of Calcutta, 2006 M.Sc., Indian Institute of Technology Bombay, 2008 M.S., Brandeis University, 2010 Ph.D., Kent State University, 2017 Approved by , Chair, Doctoral Dissertation Committee Dr. John J. Portman , Members, Doctoral Dissertation Committee Dr. Hamza Balci , Dr. Bj¨ornL¨ussem , Dr. Robin Selinger , Dr. Qi-Huo Wei Accepted by , Chair, Department of Physics Dr. James T. Gleeson , Dean, College of Arts and Sciences Dr. James L. Blank Table of Contents List of Figures . vi List of Tables . xviii Acknowledgments . xix 1 Introduction .................................. 1 1.1 Overview ................................. 1 1.2 Protein Structure ........................... 2 1.3 Energy Landscape Theory ...................... 4 1.4 Mechanism of Allostery in Proteins ................ 9 1.5 Calmodulin ............................... 12 1.6 Organization of Dissertation ..................... 15 2 Models of cooperative ligand binding . 18 2.1 Introduction ............................... 18 2.2 The Hill Equation ........................... 19 2.3 Cooperativity for binding two ligands . 21 2.4 Other models of cooperativity ................... 25 2.5 The MWC Model ........................... 27 2.6 Generalized ensemble view of allostery . 31 2.7 Allostery in calmodulin ........................ 33 iii 3 Coarse-grained models for conformational dynamics in proteins . 35 3.1 Introduction ............................... 35 3.2 Native structure-based models for proteins . 36 3.3 An analytic model for CaM ..................... 39 3.4 Simulation model for CaM ...................... 42 3.4.1 Model for conformational transition . 44 3.4.2 Model for Ca2+-binding .................... 45 4 Comparing allosteric transitions in the domains of calmodulin through coarse-grained simulations ................... 49 Abstract .................................... 49 4.1 Introduction ............................... 50 4.2 Methods ................................. 53 4.3 Conformational Transitions of Isolated Domains . 60 4.4 Transition Kinetics .......................... 67 4.5 Discussion ................................ 68 5 Coarse-grained molecular simulations of allosteric cooperativity . 71 Abstract .................................... 71 5.1 Introduction ............................... 72 5.2 Methods ................................. 74 5.3 Simulations of Binding a Single Ligand . 79 5.4 Simulations of Binding Two Ligands . 84 5.5 Binding Cooperativity ........................ 86 5.6 Molecular Description of Ligand Binding . 90 iv 5.7 Concluding Remarks ......................... 93 6 Thermodynamic and kinetic representations of cooperative allosteric ligand binding in intact calmodulin ................... 95 6.1 Introduction ............................... 95 6.2 Methods ................................. 97 6.3 Binding Thermodynamics . 103 6.4 Binding Kinetics ............................ 110 6.5 Concluding Remarks . 117 7 Outlook and future directions . 119 Appendices . 124 A Supplement for Chapter 4 . 125 A.1 Simulated probability of contact formation . 125 B Supplement for Chapter 5 . 127 B.1 Ligand-mediated contact pair distribution . 127 B.2 One-dimensional simulated free energy . 131 B.3 Exploring ligand contact strength and range . 132 v List of Figures 1.2.1 Different levels of hierarchy in protein structure. (A) A protein's pri- mary structure consists of its amino acid sequence. (B) Secondary structure consists of organization of helices and sheets (only helix is shown in the figure). (C) Tertiary structure is illustrated by one of the four polypeptide chains (subunits) of hemoglobin. Here, N-terminal represents amino terminus and C-terminal represents carboxyl termi- nus of the polypeptide chain. (D) Quaternary structure is shown as the arrangement of multiple polypeptide chains to form the functional hemoglobin molecule. Adapted from Branden and Tooze (1991)[20]. 3 1.3.1 Funnel-shaped protein folding landscape. Folding occurs through the progressive organization of ensembles of structures on a free energy landscape. 6 1.3.2 Schematic representation of protein functional motion at the bottom of the folding funnel. At the bottom of the funnel, protein dynamics is sensitive to modulation by binding of ligand. The resulting energy landscape upon binding of a ligand involves redistribution of popula- tion. Also shown is the stabilization of a low energy conformer upon binding a ligand. 8 vi 1.4.1 Schematic representations of macroscopic and microscopic allosteric binding. (A) In the macroscopic point of view, binding of a ligand sta- bilizes the ligand-bound ensemble of conformations. (B) Microscopic picture is more complex with rates determined by the relative stabi- lization of conformations in the unbound and bound ensembles. 11 1.5.1 Structure of calmodulin in different forms. (A) Ca2+-free structure of calmodulin (PDB id: 1CFD), and (B) Ca2+-bound structure of calmod- ulin (PDB id: 1CLL). Upon binding 4 Ca2+-ions to its binding loops, calmodulin undergoes a large structural change that exposes its hy- drophobic surface, and thereby enabling calmodulin to bind to target proteins. (C) NMR structure of calmodulin bound to smooth muscle myosin light chain kinase (PDB id: 2KOF). The calcium ions are shown as silver circles. This visualization was made using Visual Molecular Dynamics (VMD) software[85]. 14 2.2.1 Schematic representation of Hill equation given by Eq. 2.1 for different values of the Hill coefficient, nH. The sharpness of the curve increases for higher values of nH. The x-axis represents ligand concentration, [X], and y-axis shows fractional occupancy, Y . 20 vii 2.3.1 Thermodynamic cycle showing microscopic schematic representation for binding two ligands of heterogeneous strength. Starting from unli- gated state ligand binding stabilizes the state with both sites occupied by ligand. The two routes via which the binding process proceeds consists of partially bound states where a single site is occupied by a ligand, while the other site is empty. The final state in the cycle is the state with both binding sites occupied by ligand. 22 2.3.2 Dependence of bound probability on the parameter c for a protein with two binding sites of homogeneous strength. The x-axis represents lig- and concentration and y-axis represents probability of states with both sites occupied by ligand (left), and probability of states where only one site is occupied by ligand (right). Non-cooperative binding corresponds to c = 1. The binding curve becomes increasingly cooperative and the peak of population of singly ligated states decreases for higher values of c. .................................... 23 2.5.1 Schematic representation of shift in population in MWC model. In the unbound ensemble of conformations the ligand-free closed state has higher stability in the free energy landscape and it exists in dynamic equilibrium with the open state. Upon ligand binding, the ligand- bound open state is stabilized. The x-axis represents reaction coordi- nate that is used to define the closed and open states of the protein, and the y-axis represents free energy (in arbitrary units). Also shown are representative protein structures in the unbound and ligand-bound ensemble of conformations. 28 viii 2.5.2 States and corresponding statistical weights for a simplified description of MWC model of allostery for a protein with two ligand binding sites. In the unligated ensemble of conformations, the relative stability of the open and closed states is set by the parameter . The singly-ligated ensemble of conformations consists of a ligand bound to either the closed state or the open state. The fully-loaded ensemble comprises of both ligand bound to either the closed state or the open state of the protein. 30 2.6.1 Schematic of ensemble description of models of allosteric cooperativity for a protein with two subunits. (A) The MWC model of allostery, (B) KNF model, (C) A more generalized ensemble allosteric model that ac- commodates all possible microstates of a protein with two binding sites. Green shaded regions correspond to subunit interaction energy. The two different shapes correspond to the closed and open conformations of the protein. Colored shapes correspond to ligand unbound and un- colored shapes correspond to ligand bound conformations. Blue shaded regions show the ensemble of states for each framework. Adapted from the framework developed by Hilser and co-workers[81]. 32 3.3.1 Distribution of strain energy of residues in CaM domain. (a) and (b) Change in strain energy for individual residues along the apo ! holo structural change of nCaM and cCaM. (c) and (d) Residue strain energy distributions at an intermediate state for nCaM and cCaM, respectively. Adapted from the work by Tripathi and Portman[198]. 43 ix 4.2.1 Aligned structures Ca2+-free (closed/apo) and Ca2+-bound (open/holo) native conformations for (a) N-terminal domain and (b) C-terminal do- main of Calmodulin. The closed state (pdb: 1cfd [103] ) is shown in blue, and the open state (pdb: 1cll[34]) is shown in green. The closed (apo) and open (holo) conformations of (a) nCaM (residue index 4{75) consist of helices A, B and C, D with binding loops I and II respectively.
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