Molecular Dynamics Simulations Of Biological Macromolecules

National Heart, Lung, And Blood Institute

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Abstract

pH dependence of a Na channel Sodium channels play an important role in electrical signaling in cells; as such they are the targets of many drugs, as well as naturally occurring toxins from plants and animals. Inhibition and/or improper functioning of sodium channels due to mutation can lead to disease. In bacterial voltage gated sodium channels, the passage of sodium ions through the pore is controlled by a selectivity filter (SF) comprised of four glutamate residues. Previously, for the bacterial channel NavMs, we have shown that the pKa values of the four SF glutamate residues depend on the number of ions bound in the channel, and that the fully deprotonated, singly protonated and doubly protonated states of the SF are all possible at physiological pH. We have also shown that the conductance of the channel decreases with each proton bound to the SF. Thus, the conductance of the channel is pH dependent, and decreases with lowering of pH, in agreement with experiments on similar channels. We now demonstrated that the pKa values of the four SF glutamates are higher in solution of K+ ions than in solution of Na+ ions. Since pKa values are close to the physiological pH, this leads to a predominant population of the fully deprotonated state (high-conductance state) of glutamates in Na+ solution, while protonated states (low-conductance states) are more populated in K+ solution. Thus, we proposed that a significant component of selectivity is achieved through ion-triggered shifts in the protonation state, which favors more conductive states for Na+ ions and less conductive states for K+ ions. This mechanism also suggests a strong pH dependence of selectivity, which has been experimentally observed in structurally similar NaChBac channels. Electrophysiology of homomeric pentameric: nAChR 7: Alpha7 nicotinic acetylcholine receptor (7 nAChR) plays a vital role in cholinergic nerve system in the brain; Alzheimers disease and schizophrenia are associated with malfunction of this channel. Recently, the structure and gating mechanism of the receptor were reported, allowing the channel to be studied using computational models. We develop all-atom MD simulations of the activated state of the channel to understand the ion transfer mechanism and relate statistical models to results. In addition, we study variations in channel conformation and its conductance dependence on electric charges (protonated and deprotonated states) of key residues, changes in channel radius, and position of side chains interacting with ions passing through the channel. The study further extends to investigate the difference in rate and movement of different cations, the hydration number, electrostatic potentials, and charge densities of these ions. During this research, the long-believed selectivity filter switching between cations to anion (GLU-237 residue ring) was also studied by protonating one residue at a time to understand the transition. Finally, the effect of lateral fenestration of ions are negated in the extra cellular domain of this channel which plays a vital role in ion conductance in glycan receptors of similar structural conformation. This study will help understand the receptor better in activated state with high conductance, which otherwise is difficult to understand experimentally to this level of detail. Charged lipids in plasma membrane and their behavior in electric fields: Electric fields can cause cells to move in a process called electrotaxis, which is essential in wound healing and other biological processes such as growth, development and regeneration. However, the mechanism of electrotaxis is not understood. In processes such as chemotaxis (cells moving when detecting certain chemicals), signaling proteins in membranes communicate the presence of particular chemicals present on the outside of the cell. However, no single signaling protein is found for electrotaxis, and thus we seek to explore the hypothesis that cellular membranes are the ones to detect and signal the interior of the cell in the presence of electric fields. To investigate this phenomenon, we develop computer models of plasma membranes with gangliosides glycolipids with long electrically charged headgroups acting as antennas which stick vertically outwards, and hence are plausible candidates to transmit electric signals through the membrane. We also study the behavior of highly charged lipids PI(3,4,5)P3, in different external electric fields. These plasma membrane models are simulated in different lateral and transverse electric fields to investigate the behavior of these charged lipids by studying local clustering, rate of diffusion and electric drift of lipids in plane of the leaflet of the membranes submerged in different electric fields conditions. Calculating binding energy of arginine and phosphate Noncovalent binding interaction between arginine and phosphate, such as the binding of SH2 domain proteins with phosphorylated peptides, is essential for signal recognition in many biological processes. In this regard, we estimate the free energy of several conformations of arginine and phosphate. We utilize two force fields, CHARM36m and CUFIX*, for a simulation, and Free Energy Perturbation and Umbrella Sampling methods to compute free energies. Armed with these methodologies, we run a molecular dynamics (MD) simulation for 1 millisecond and find that arginine and phosphate have multiple binding conformations. We cluster the binding conformations into five groups and compute binding energies for those five representative snapshots. Our goal is to match a computed free energy value derived from the simulation with the outcome of isothermal calorimetry experiments by adjusting the parameters. QM/MM Simulations of target-strand DNA cleavage in CRISPR-Cas9 HNH domain CRISPR-Cas9 technology is currently being tested for the treatment of blood disorders, genetic blindness, and cancers. To assist future development of gene editing applications, there is a need to gain further insights into the molecular mechanisms of DNA binding to Cas9 and subsequent cleavage. We performed QM/MM free energy simulations of the target-strand-DNA cleavage catalyzed by the wildtype S. pyogenes Cas9 (SpyCas9) and a K866A mutant. We found the free energy barrier for the K866A mutant to be 5 kcal/mol higher with respect to wild type SpyCas9, suggesting K866 plays a critical role during the catalytic step. Our free energy results agree well with experimentally measured kinetic rates, and it is also consistent with our energy decomposition analysis of the free energy profile indicating that the K866 residue lowers the free energy barrier. Based on the analysis, we also hypothesize that a nearby hydrogen bond network could be engineered such that H840 is anchored towards the active site, thereby increasing the DNA cleavage kinetics. Our classical MD simulations of K855 variants suggested K855D/K855E improves the stability of the HNH domain. These variants were experimentally tested by our collaborators (Rajan Lab, University of Oklahoma), who found improved sensitivity to DNA mismatches, albeit with much slower observed rates. Work is currently being performed on developing a -machine-learning-potential to accelerate QM/MM simulations for the study of these and new SpyCas9 variants. Modelling of Catalytic Active State of FnCas12a Cas12a can also be programmed to target the human genome with guide-RNA (gRNA) but differ from other Cas-enzymes in its low molecular size, capabilities for DNA multiplex targeting, and mechanism of DNA cleavage. QM/MM simulations are also underway to investigate the DNA cleavage mechanism.