Influence of Cholesterol and its Stereoisomers on Members of the Serotonin Receptor Family

Despite the ubiquity of cholesterol within the cell membrane, the mechanism by which it influences embedded proteins remains elusive. Numerous GPCRs exhibit dramatic responses to membrane cholesterol, with regard to the ligand-binding affinity and functional properties, including the 5-HT receptor family. Here, we use over 25 μs of unbiased atomistic MD simulations to identify cholesterol interaction sites in the 5-HT1B and 5-HT2B receptors and evaluate their impact on receptor structure. Susceptibility to membrane cholesterol is shown to be subtype-dependent and determined by the quality of interactions between the extracellular loops. Charged residues are essential to maintain the arrangement of the extracellular surface in 5-HT2B; in the absence of such interactions, the extracellular surface of the 5-HT1B is malleable, populating a number of distinct conformations. Elevated-cholesterol density near transmembrane helix 4 is considered to be conducive to the conformation of extracellular loop 2. Occupation of this site is also shown to be stereospecific, illustrated by differential behaviour of nat-cholesterol isomers, ent- and epi-cholesterol. In simulations containing the endogenous agonist, serotonin, cholesterol binding at transmembrane helix 4 biases bound serotonin molecules towards an unexpected binding mode in the extended binding pocket. The results highlight the capability of membrane cholesterol to influence the mobility of the extracellular surface in the 5-HT1 receptor family and manipulate the architecture of the extracellular ligand-binding pocket

The Use of Multiscale Molecular Simulations in Understanding a Relationship between the Structure and Function of Biological Systems of the Brain: The Application to Monoamine Oxidase Enzymes

Computational techniques provide accurate descriptions of the structure and dynamics of biological systems, contributing to their understanding at an atomic level.Classical MD simulations are a precious computational tool for the processes where no chemical reactions take place.QM calculations provide valuable information about the enzyme activity, being able to distinguish among several mechanistic pathways, provided a carefully selected cluster model of the enzyme is considered.Multiscale QM/MM simulation is the method of choice for the computational treatment of enzyme reactions offering quantitative agreement with experimentally determined reaction parameters.Molecular simulation provide insight into the mechanism of both the catalytic activity and inhibition of monoamine oxidases, thus aiding in the rational design of their inhibitors that are all employed and antidepressants and antiparkinsonian drugs. Aging society and therewith associated neurodegenerative and neuropsychiatric diseases, including depression, Alzheimer's disease, obsessive disorders, and Parkinson's disease, urgently require novel drug candidates. Targets include monoamine oxidases A and B (MAOs), acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and various receptors and transporters. For rational drug design it is particularly important to combine experimental synthetic, kinetic, toxicological, and pharmacological information with structural and computational work. This paper describes the application of various modern computational biochemistry methods in order to improve the understanding of a relationship between the structure and function of large biological systems including ion channels, transporters, receptors, and metabolic enzymes. The methods covered stem from classical molecular dynamics simulations to understand the physical basis and the time evolution of the structures, to combined QM, and QM/MM approaches to probe the chemical mechanisms of enzymatic activities and their inhibition. As an illustrative example, the later will focus on the monoamine oxidase family of enzymes, which catalyze the degradation of amine neurotransmitters in various parts of the brain, the imbalance of which is associated with the development and progression of a range of neurodegenerative disorders. Inhibitors that act mainly on MAO A are used in the treatment of depression, due to their ability to raise serotonin concentrations, while MAO B inhibitors decrease dopamine degradation and improve motor control in patients with Parkinson disease. Our results give strong support that both MAO isoforms, A and B, operate through the hydride transfer mechanism. Relevance of MAO catalyzed reactions and MAO inhibition in the context of neurodegeneration will be discussed

Effect of anionic lipids on ion permeation through the KcsA K+-channel

K+-channels are responsible for the efficient and selective conduction of K+ ions across the plasma membrane. The bacterial K+-channel KcsA has historically been used to characterize various aspects of K+ conduction via computational means. The energetic barriers associated with ion translocation across the KcsA selectivity filter have been computed in various studies, leading to the proposal of two alternate mechanisms of conduction, involving or neglecting the presence of water molecules in between the permeating ions. Here, the potential of mean force of K+ permeation is evaluated for KcsA in lipid bilayers containing anionic lipids, which is known to increase the open probability of the channel. In addition, the effect of the protonation/deprotonation of residue E71, which directly interacts with the selectivity filter sequence, is assessed. Both conduction mechanisms are considered throughout. The results obtained provide novel insights into the molecular functioning of K+-channels including the inactivation process

Early Steps in C-Type Inactivation of the hERG Potassium Channel

Fast C-type inactivation confers distinctive functional properties to the hERG potassium channel, and its association to inherited and acquired cardiac arrythmias makes the study of the inactivation mechanism of hERG at the atomic detail of paramount importance. At present, two models have been proposed to describe C-type inactivation in K+-channels. Experimental data and computational work on the bacterial KcsA channel support the hypothesis that C-type inactivation results from a closure of the selectivity filter that sterically impedes ion conduction. Alternatively, recent experimental structures of a mutated Shaker channel revealed a widening of the extracellular portion of the selectivity filter, which might diminish conductance by interfering with the mechanism of ion permeation. Here, we performed molecular dynamics simulations of the wild-type hERG, a non-inactivating mutant (hERG-N629D), and a mutant that inactivates faster than the wild-type channel (hERG-F627Y) to find out which and if any of the two reported C-type inactivation mechanisms applies to hERG. Closure events of the selectivity filter were not observed in any of the simulated trajectories but instead, the extracellular section of the selectivity filter deviated from the canonical conductive structure of potassium channels. The degree of widening of the potassium binding sites at the extracellular entrance of the channel was directly related to the degree of inactivation with hERG-F627Y > wild-type hERG > hERG-N629D. These findings support the hypothesis that C-type inactivation in hERG entails a widening of the extracellular entrance of the channel rather than a closure of the selectivity filter

Insights into the mechanisms of K+ permeation in K+-channels from computer simulations

Ion permeation, selectivity, and the behavior of the K+ channel selectivity filter have been studied intensively in the previous two decades. The agreement among multiple approaches used to study ion flux in K+ channels suggests a consensus mechanism of ion permeation across the selectivity that has been put to the test in recent years with the proposal of an alternative way by which ions can cross the selectivity filter of K+ channels via direct Coulomb repulsion between contacting cations. Past experimental work by Zhou and MacKinnon (J. Mol. Biol. 2004, 338, 839) showed that mutation of the site S4 reduces the total occupancy of the selectivity filter to less than two ions on average by lowering the occupancy of the S2-S4 configuration without changing the S1-S3 configuration much, and this reduction of occupancy means that ion configurations different from the ones involved in the canonical mechanism are likely to be involved. At that time, calculations using complicated kinetic networks to relate occupancy to conduction did not provide deeper insight into the conduction mechanism. Here, to help solve this enigma, umbrella sampling simulations have been performed to evaluate the potential of mean force of two KcsA mutant channels where the S4 site is substituted. Our new results provide insights into the significance of threonine in this position, revealing the effect of substitution on the alternate mechanisms of conduction proposed, involving either water or vacant sites

Ion-triggered selectivity in bacterial sodium channels

Since the availability of the first crystal structure of a bacterial Na+ channel in 2011, understanding selectivity across this family of membrane proteins has been the subject of intense research efforts. Initially, free energy calculations based on molecular dynamics simulations revealed that although sodium ions can easily permeate the channel with their first hydration shell almost intact, the selectivity filter is too narrow for efficient conduction of hydrated potassium ions. This steric view of selectivity was subsequently questioned by microsecond atomic trajectories, which proved that the selectivity filter appears to the permeating ions as a highly degenerate, liquid-like environment. Although this liquid-like environment looks optimal for rapid conduction of Na+, it seems incompatible with efficient discrimination between similar ion species, such as Na+ and K+, through steric effects. Here extensive molecular dynamics simulations, combined with Markov state model analyses, reveal that at positive membrane potentials, potassium ions trigger a conformational change of the selectivity toward a nonconductive metastable state. It is this transition of the selectivity filter, and not steric effects, that prevents the outward flux of K+ at positive membrane potentials. This description of selectivity, triggered by the nature of the permeating ions, might have implications on the current understanding of how ion channels, and in particular bacterial Na+ channels, operate at the atomic scale

Location and character of volatile general anesthetics binding sites in the transmembrane domain of TRPV1

It has been proposed that general anesthesia results from direct multisite interactions with multiple and diverse ion channels in the brain. An understanding of the mechanisms by which general anesthetics modulate ion channels is essential to clarify their underlying behavior and their role in reversible immobilization and amnesia. Despite the fact that volatile general anesthetics are drugs that primarily induce insensitivity to pain, they have been reported to sensitize and active the vanilloid-1 receptor, TRPV1, which is known to mediate the response of the nervous system to certain harmful stimuli and which plays a crucial role in the pain pathway. Currently, the mechanism of action of anesthetics is unknown and the precise molecular sites of interaction have not been identified. Here, using ∼2.5 μs of classical molecular dynamics simulations and metadynamics, we explore these enigmas. Binding sites are identified and the strength of the association is further characterized using alchemical free-energy calculations. Anesthetic binding/unbinding proceeds primarily through a membrane-embedded pathway, and subsequently, a complex scenario is established involving multiple binding sites featuring single or multiple occupancy states of two small volatile drugs. One of the five anesthetic binding sites reported was previously identified experimentally, and another one, importantly, is identical to that of capsaicin, one of the chemical stimuli that activate TRPV1. However, in contrast to capsaicin, isoflurane and chloroform binding free-energies render modest to no association compared to capsaicin, suggesting a different activation mechanism. Uncovering chloroform and isoflurane modulatory sites will further our understanding of the TRPV1 molecular machinery and open the possibility of developing site-specific drugs

Stereospecific Interactions of Cholesterol in a Model Cell Membrane:Implications for the Membrane Dipole Potential

Cholesterol is a major constituent of the plasma membrane in higher order eukaryotes. The effect of cholesterol on the structure and organisation of cell membranes has been studied extensively by both experimental and computational means. In recent years, a wealth of data has been accumulated illustrating how subtle differences in the structure of cholesterol equate to considerable changes in the physical properties of the membrane. The effect of cholesterol stereoisomers, in particular, has been established, identifying a direct link with the activity of specific membrane proteins. In this study, we perform extensive molecular dynamics simulations of phospholipid bilayers containing three isomers of cholesterol, the native form (nat-cholesterol), the enantiomer of the native form (ent-cholesterol), and an epimer of cholesterol that differs by the orientation of the polar hydroxyl group (epi-cholesterol). Based on these simulations, an atomic-level description of the stereospecific cholesterol-phospholipid interactions is provided, establishing a potential mechanism for the perturbation of membrane properties, specifically the membrane dipole potential