Molecular Dynamics Simulations Of Biological Macromolecules

National Heart, Lung, And Blood Institute

Investigators

Linked publications, trials & patents

Abstract

Several projects have been pursued in the reporting period: Self-Guided Molecular Simulation to Enhance Concerted Motion Self-guided (SG) molecular simulation methods, namely self-guided molecular dynamics (SGMD) and self-guided Langevin dynamics (SGLD), enhance conformational search by promoting low-frequency motion. A simple local time averaging scheme is used to extract low-frequency properties with little overhead in computing costs. For molecular processes to form ordered structures like ligand binding and protein folding, it is believed that concerted motions play crucial roles. To enhance the concerted motion in molecular systems, we propose a spatial averaging scheme to extract the concerted motion of a local region. Applying guiding forces based on spatial averaging, self-guided molecular simulations can enhance concerted motion and reach ordered structures more efficiently. Through simulations of amyloid fibril peptides, we demonstrated that the spatial averaging in self-guided Langevin dynamics results in accelerated β-sheet formation. Identify the conformational states of glycine receptor alpha3 through umbrella sampling Ligand-gated ion channels allows cells to respond rapidly to changes in their external environment. The structure change from one state to other states is the key to understand how ion channels function. In many cases due to the limitation in experiment, only some state structures, a closed, open state, or desensitized state, are available. A reliable method is needed to derive the structures of the missing state structures. Even in cases structures of all states are available, it is desired to understand how conformation changes during state transition. This work presents a method that utilizes the umbrella sampling to drive conformation changes from one state to the other state. The trans-membrane domain of glycine receptor is embedded in POPC lipids. A reaction coordinate describing relative orientation of lining substructures of ion channels is proposed and the free energy profile along the reaction coordinates is produced. For glycine receptor alpha-3 pentamer, we find that there are two free energy wells separated by a barrier in the free energy profile. The desensitized state corresponding to one well at a larger reaction coordinate and the closed state corresponding to the other well at a smaller reaction coordinate. The open state locates at the barrier region and the high free energy of the open state make it unstable, which is a main reason that the open state is difficult to be captured in experiment. This free energy profile also explains the observation that the open state structure quickly decays to the other states in some computational studies. Examining the conformations of these state shows that glycine binding produces an expansion movement at the ECD and TMD interface, which opens the ion gate at the middle of the ion channel. Continued opening will result in the structure decaying into the free energy well of the desensitized state. At the desensitized state, the proline residues at bottom of the ion channel close to lock up the channel. The result agrees with the close, open, and desensitized state structures of the glycine receptor alpha1. The free energy profile along the reaction coordinate provides a thermodynamic understanding of the ion channel functional states. Structure analysis and simulation study of microtubule dynamics Microtubules are a structurally and functionally important components of the eukaryotic cytoskeleton that play a crucial role in cell division and intracellular trafficking. Understanding of the mechanism of microtubule dynamics is crucial to control cell proliferation. Disrupting microtubule dynamics is a major strategy in cancer therapy. Through structure analysis of tubulin conformations in microtubules and in unassembled forms, I discover a rotation of the tubulin intermediate domain that switches tubulins from a closed state to an open state. This rotation is responsible for the conformational change of tubulins during microtubule growth. Based on the observation that a GDP shift coincident with the rotation; I propose a hypothesis that the GTP hydrolysis produces a GDP stroke that causes the rotation. Through self-guided Langevin dynamics simulations of tubulin monomers and heterodimers, with and without the GDP stroke, this work proves that the GDP stroke does cause the rotation. At the closed state, tubulins polymerize into a curved protofilament. In the open state tubulins can dock into the open pockets to form a straight protofilament. Lateral interactions between straight protofilaments stabilize microtubules. Based on these results, this work proposes a hydrolysis driven mechanism that can well describe microtubule dynamics. This new mechanism will facilitate new strategies in regulating microtubule dynamics and in development of cancel therapy. Cyclic peptide modeling and Molecular Dynamics simulations Understanding conformational changes in cyclic peptides is crucial for designing libraries with desired permeability and functional properties. In this project, we optimized the Oscillating Drude polarizable force field to model 9-mer cyclic peptides that were experimentally evaluated using in vitro and MDCK assays. These cyclic peptides have different sequences, consisting of both standard and non-standard amino acids , as well as varying stereochemistry. To parameterize the force field, we targeted 2D potential energy surfaces (PES) calculated in MP2/6-311G(d,p) model chemistry by scanning phi and psi dihedral angles in 15° increments. Bayesian optimization with Boltzmann weighting at 600 K was applied using varying energy cutoff, and manual fitting was performed when necessary. To improve agreement with QM target data, CMAP were created for non-standard alanine and non-standard glycine residues. Five highly permeable and five poorly permeable peptides were then selected for simulated annealing molecular dynamics (MD) simulations in both gas and aqueous phases. The simulations were performed by colling the systems from 900 K to 300 K with 150 K increments. The equilibrated structures obtained at 300 K were analyzed by solvent accessible surface area (SASA) calculations to characterize the physical features governing cyclic peptide permeability. In-silico design of small molecular glues We established a computational protocol for the rational design of molecular glues, exploiting our efficient estimate of cooperativity in ternary complex formation. Our computational pipeline relies on screening large chemical libraries, and employs molecular docking, molecular dynamics, QM/MMGBSA, and free energy perturbation simulations. We established our protocol on the E3 ubiquitin ligase component, Cereblon, targeting members of the Ikaros family of zinc-finger proteins, and are currently working on adapting the pipeline to target oncogenic small GTPases like mutant Ras. We are also incorporating machine learning strategies to allow more efficient and wider screening. Causal inference from biomolecular dynamics We are testing existing and developing novel statistical analysis tools to infer causal relationships between conformational events in molecular dynamics. We generated synthetic data with known underlying causal structure as well as performed simulations on a simple membrane bilayer test system to generate abundant data for benchmarking. We are applying the best-performing tools on protein dynamics to identify the causal structure upon ligand unbinding from the human muscarinic receptor 3. Induced switching mechanism in small GTPases We found that interactions between Ran’s C-terminal tail and a hydrophobic triad at the edge of switch I can distort the triad and drive Ran·GTP into an inactive state. Notably, we were able to reproduce this effect even in the absence of the C-terminal tail by directly perturbing the triad, leading to the same inactivation. We are now testing whether this induced switching mechanism works in other active small GTPases, as well as in oncogenic mutants that remain active even when GDP-bound. Ion Conduction and pH Dependence of Nicotinic Acetylcholine Receptor (nAChR) The α7-nicotinic acetylcholine receptor (α7-nAChR) is a cation-selective member of the superfamily of Cys-loop receptors. Ubiquitously expressed throughout the body of vertebrate animals, this pentameric ligand-gated ion channel participates in a wide range of physiological phenomena — as diverse as synaptic transmission and the control of excessive inflammation — and is an attractive therapeutic target for novel ligands. Although notable efforts have been made to understand this receptor-channel in terms of function and structure, many questions remain unanswered despite the molecular simplicity of its homomeric assembly. Recent cryo-EM studies have provided atomic models of this channel in different conformations, thus enabling the application of atomistic molecular dynamics (MD) simulations to the study of cation conduction. We perform both single-channel patch-clamp recordings and MD simulations on the α7-nAChR. MD simulations of an α7-nAChR model (PDB ID 7KOX) reproduced the measured single-channel conductance and revealed Poissonian ion permeation, which we further modelled as a double-Poisson process incorporating inter-event delay times. We found that cations can enter the channel through lateral fenestrations in the extracellular domain although the probability of ions following this lateral pathway — rather than the axial one — is much lower than observed in simulations of other Cys-loop receptors. We also examined other atomic models (PDB ID 7EKT and 8V80) of the α7-nAChR proposed to represent partially open states of the channel and found them to be non-conductive. This study provides insight into how ions permeate through the pore of the α7-nAChR and offers a detailed analysis of an ion-conductive conformation likely to represent the physiological open state of this receptor-channel. Moving forward, we are investigating the pH dependence and the effect of ionizable residues in the channel's transmembrane domain using Hamiltonian Replica Exchange to calculate free energy profiles. The aim is to reproduce the experimental observations and gain a deeper understanding of the receptor's mechanics. Electrostatic Effects on Charged Lipid Membranes Gangliosides (GMs) are glycosphingolipids that are the only charged lipids found on the outer leaflet of lipid membranes. We are using atomistic MD simulations to investigate the behaviour of these charged lipid membranes in asymmetric membrane bilayers. This study addresses the critical question of how external electric fields and varying ionic conditions influence the organization and local environment of these charged lipids. The primary objective is to understand the mechanisms governing the clustering of GMs. We are systematically exploring how the application of an electric field—a common variable in biological systems, such as during nerve impulses—modifies the electrostatic interactions that drive this clustering. A key part of the investigation is to characterize the specific interactions between the charged GM headgroups (NANA) and different cations, including monovalent (Na+ and K+) and the divalent (Ca2+) ions. By analysing the dynamic behaviour and binding of these cations, we aim to uncover how they screen the negative charges of the GMs and influence their local environment. This analysis aims to provide insights into how ion-specific interactions contribute to the overall membrane organization and fluidity. The findings from this research are expected to provide fundamental insights into the role of electrostatic forces in shaping membrane organization and function. Understanding the clustering of charged lipids and their interactions with a diverse range of ions under an electric field has broad implications for cell signalling, protein-lipid interactions, and the mechanics of membrane microdomains like lipid rafts.