Van A. Ngo

Mapping Ryanodine Binding Sites in the Pore Cavity of Ryanodine Receptors

Ryanodine (Ryd) irreversibly targets ryanodine receptors (RyRs), a family of intracellular calcium release channels essential for many cellular processes ranging from muscle contraction to learning and memory. Little is known of the atomistic details about how Ryd binds to RyRs. In this study, we used all-atom molecular dynamics simulations with both enhanced and bidirectional sampling to gain direct insights into how Ryd interacts with major residues in RyRs that were experimentally determined to be critical for its binding. We found that the pyrrolic ring of Ryd displays preference for the R4892AGGG-F4921 residues in the cavity of RyR1, which explain the effects of the corresponding mutations in RyR2 in experiments. Particularly, the mutant Q4933A (or Q4863A in RyR2) critical for both the gating and Ryd binding not only has significantly less interaction with Ryd than the wild-type, but also yields more space for Ryd and water molecules in the cavity. These results describe clear binding modes of Ryd in the RyR cavity and offer structural mechanisms explaining functional data collected on RyR blockade.

Computational membrane biophysics: From ion channel interactions with drugs to cellular function

The rapid development of experimental and computational techniques has changed fundamentally our understanding of cellular-membrane transport. The advent of powerful computers and refined force-fields for proteins, ions, and lipids has expanded the applicability of Molecular Dynamics (MD) simulations. A myriad of cellular responses is modulated through the binding of endogenous and exogenous ligands (e.g. neurotransmitters and drugs, respectively) to ion channels. Deciphering the thermodynamics and kinetics of the ligand binding processes to these membrane proteins is at the heart of modern drug development. The ever-increasing computational power has already provided insightful data on the thermodynamics and kinetics of drug-target interactions, free energies of solvation, and partitioning into lipid bilayers for drugs. This review aims to provide a brief summary about modeling approaches to map out crucial binding pathways with intermediate conformations and free-energy surfaces for drug-ion channel binding mechanisms that are responsible for multiple effects on cellular functions. We will discuss post-processing analysis of simulation-generated data, which are then transformed to kinetic models to better understand the molecular underpinning of the experimental observables under the influence of drugs or mutations in ion channels. This review highlights crucial mathematical frameworks and perspectives on bridging different well-established computational techniques to connect the dynamics and timescales from all-atom MD and free energy simulations of ion channels to the physiology of action potentials in cellular models. This article is part of a Special Issue entitled: Biophysics in Canada, edited by Lewis Kay, John Baenziger, Albert Berghuis and Peter Tieleman.

Molecular Mechanism of Conductance Enhancement in Narrow Cation-Selective Membrane Channels

Membrane proteins known as ryanodine receptors (RyRs) display large conductance of ∼1 nS and nearly ideal charge selectivity. Both properties are inversely correlated in other large-conductance but nonselective biological nanopores (i.e., α-hemolysin) used as industrial biosensors. Although recent cryo-electron microscopy structures of RyR2 show similarities to K+- and Na+-selective channels, it remains unclear whether similar ion conduction mechanisms occur in RyR2. Here, we combine microseconds of all-atom molecular dynamics (MD) simulations with mutagenesis and electrophysiology experiments to investigate large K+ conductance and charge selectivity (cation vs anion) in an open-state structure of RyR2. Our results show that a water-mediated knock-on mechanism enhances the cation permeation. The polar Q4863 ring may function as a confinement zone amplifying charge selectivity, while the cytoplasmic vestibule can contribute to the efficiency of the cation attraction. We also provide direct evidence that the rings of acidic residues at the channel vestibules are critical for both conductance and charge discrimination in RyRs.

Understanding the Molecular Mechanism of Cation Permeation in the Cardiac Ryanodine Receptor (RyR2) Channel using Computational Electrophysiology

582-Pos Board B352 Strained Collagen Resists Bacterial Collagenase Degradation Karanvir Saini, Manorama Tiwari, Jerome Irianto, Charlotte Pfeifer, Cory Alvey, Dennis E. Discher. Univ of Pennsylvania, Univ of Pennsylvania, Philadelphia, PA, USA. Background: Collagen, a triple-helical, self-organizing protein, is the predominant structural protein in mammals, found in tendon, bone, ligament, cartilage, intervertebral disc, skin, blood vessel, and cornea. Tendons have 7080% dry weight as collagen, and function as dynamic structures which respond to the magnitude, direction, frequency, and duration of physiologic as well as pathologic mechanical loads via complex interactions between cellular pathways and the highly specialized extracellular matrix. This extracellular matrix of tendon is hierarchical structure having collagen fibrils oriented along longitudinal axis of tendon. The theory suggests that the mechanisms which drive the preferential accumulation of collagen of loaded tissue are not only cell-driven but operate at the molecular level. The concept reduces control of matrix morphology in degrading chemical environment via mechanical strain. Methodology: The investigation was carried out in an environmentally-controlled microbioreactor in which fascicle extracted from mice tail tendon was subjected to strain gradients using three-point bending. The unstrained fascicle was labelled with dye and stripes were created on it via photobleaching. The deformation of fascicle lead to change in the displacement between stripes which resulted in quantification of strain magnitude. Thereafter, deformed fascicle was exposed to bacterial collagenase (BC) and its degradation was tracked using Second Harmonic Generation (SHG) signal produced by collagen fibrils present in it. It was found that mechanical strain significantly increased degradation time of Collagen fibrils present in the regions of fascicle having high tensile strain compared to the ones having low strain magnitudes. Conclusions/Significance:Our study demonstrates for the first time that applied mechanical strain preferentially preserves collagen fibrils forming tendon tissue in the presence of a pathologicallyimportant BC. Our results have the potential to extend our understanding of phenomena like development, adaptation, remodeling and disease of many collagen matrices.