Molecular dynamics of nicotinic acetylcholine receptor correlating biological functions

The nicotinic acetylcholine receptor (nAChR) that mediates fast intercellular communication in response to neurotransmitters is a paradigm of ligand-gated ion channels. Molecular dynamics (MD) simulations are valuable in understanding membrane protein function at atomic level, providing useful clues for further experimental/theoretical studies. In this brief review, recent progress in MD simulations of the nAChR has been illustrated, mainly focusing on the latest simulation of the whole transmembrane domain of the receptor. On the basis of MD simulations, asymmetrical and asynchronous motions of five subunits were observed both in the ligand binding and transmembrane domains; a closed-to-open conformational shift of the gate was captured in different simulation systems; the contributions from the lipid molecules and other transmembrane segments rather than M2 to the gate switch as well as the conformational change of the whole channel were assessed; the dynamic behavior and related physical/chemical properties of the water molecules and cations within the ion channel were examined; and an experimentally comparable single-channel conductance and ion selectivity were obtained.     

Molecular dynamics simulation links conformation of a pore-flanking region to hyperekplexia-related dysfunction of the inhibitory glycine receptor

Inhibitory glycine receptors mediate rapid synaptic inhibition in mammalian spinal cord and brainstem. The previously identified hyperekplexia mutation GLRA1(P250T), located within the intracellular TM1-2 loop of the GlyR alpha1 subunit, results in altered receptor activation and desensitization. Here, elementary steps of ion channel function of alpha1(250) mutants were resolved and shown to correlate with hydropathy and molar volume of residue alpha1(250). Single-channel recordings and rapid activation kinetic studies using laser pulse photolysis showed reduced conductance but similar open probability of alpha1(P250T) mutant channels. Molecular dynamics simulation of a helix-turn-helix motif representing the intracellular TM1-2 domain revealed alterations in backbone conformation, indicating an increased flexibility in these mutants that paralleled changes in elementary steps of channel function. Thus, the architecture of the TM1-2 loop is a critical determinant of ion channel conductance and receptor desensitization.     

Photoinduced electron transfer and fluorophore motion as a probe of the conformational dynamics of membrane proteins: application to the influenza a M2 proton channel

The structure and function of the influenza A M2 proton channel have been the subject of intensive investigations in recent years because of their critical role in the life cycle of the influenza virus. Using a truncated version of the M2 proton channel (i.e., M2TM) as a model, here we show that fluctuations in the fluorescence intensity of a dye reporter that arise from both fluorescence quenching via the mechanism of photoinduced electron transfer (PET) by an adjacent tryptophan (Trp) residue and local motions of the dye molecule can be used to probe the conformational dynamics of membrane proteins. Specifically, we find that the dynamics of the conformational transition between the N-terminal open and C-terminal open states of the M2TM channel occur on a timescale of about 500 μs and that the binding of either amantadine or rimantadine does not inhibit the pH-induced structural equilibrium of the channel. These results are consistent with the direct occluding mechanism of inhibition which suggests that the antiviral drugs act by sterically occluding the channel pore.     

Molecular dynamics and brownian dynamics investigation of ion permeation and anesthetic halothane effects on a proton-gated ion channel

Bacterial Gloeobacter violaceus pentameric ligand-gated ion channel (GLIC) is activated to cation permeation upon lowering the solution pH. Its function can be modulated by anesthetic halothane. In the present work, we integrate molecular dynamics (MD) and Brownian dynamics (BD) simulations to elucidate the ion conduction, charge selectivity, and halothane modulation mechanisms in GLIC, based on recently resolved X-ray crystal structures of the open-channel GLIC. MD calculations of the potential of mean force (PMF) for a Na(+) revealed two energy barriers in the extracellular domain (R109 and K38) and at the hydrophobic gate of transmembrane domain (I233), respectively. An energy well for Na(+) was near the intracellular entrance: the depth of this energy well was modulated strongly by the protonation state of E222. The energy barrier for Cl(-) was found to be 3-4 times higher than that for Na(+). Ion permeation characteristics were determined through BD simulations using a hybrid MD/continuum electrostatics approach to evaluate the energy profiles governing the ion movement. The resultant channel conductance and a near-zero permeability ratio (P(Cl)/P(Na)) were comparable to experimental data. On the basis of these calculations, we suggest that a ring of five E222 residues may act as an electrostatic gate. In addition, the hydrophobic gate region may play a role in charge selectivity due to a higher dehydration energy barrier for Cl(-) ions. The effect of halothane on the Na(+) PMF was also evaluated. Halothane was found to perturb salt bridges in GLIC that may be crucial for channel gating and open-channel stability, but had no significant impact on the single ion PMF profiles.     

Blocking of the nicotinic acetylcholine receptor ion channel by chlorpromazine, a noncompetitive inhibitor: A molecular dynamics simulation study

A large series of pharmacological agents, distinct from the typical competitive antagonists, block in a noncompetitive manner the permeability response of the nicotinic acetylcholine receptor (nAChR) to the neurotransmitter acetylcholine. Taking the neuroleptic chlorpromazine (CPZ) as an example of such agents, the blocking mechanism of noncompetitive inhibitors to the ion channel pore of the nAChR has been explored at the atomic level using both conventional and steered molecular dynamics (MD) simulations. Repeated steered MD simulations have permitted calculation of the free energy (approximately 36 kJ/mol) of CPZ binding and identification of the optimal site in the region of the serine and leucine rings, at approximately 4 A from the pore entrance. Coulomb and the Lennard-Jones interactions between CPZ and the ion channel as well as the conformational fluctuations of CPZ were examined to assess the contribution of each to the binding of CPZ to the nAChR. The MD simulations disclose a dynamic interaction of CPZ binding to the nAChR ionic channel. The cationic ammonium head of CPZ forms strong hydrogen bonds with Glu262 (alpha), Asp268 (beta), Glu272 (beta), Ser276 (beta), Glu280 (delta), Gln271 (gamma), Glu275 (gamma), and Asn279 (gamma) nAChR residues. Finally, the conventional MD simulation of CPZ at its identified binding site demonstrates that the binding of CPZ not only blocks ion transport through the channel but also markedly inhibits the conformational transitions of the channel, necessary for nAChR to carry out its biological function.     

Local chain dynamics of a model polycarbonate near glass transition temperature: A molecular dynamics simulation

Constant pressure constant temperature molecular dynamics method is employed to investigate the atomistic scale dynamics of a model Bisphenol A polycarbonate in the vicinity of its glass transition temperature. First, the glass transition temperature and the thermal expansion coefficients of the polymer are predicted by performing simulations at different temperatures. To explore the significance of different modes of motion, various types of time correlation functions are utilized in analyzing the trajectories. In these nanosecond scale simulations, the motion of the chain segments is found to be highly localized with little reorientation of the vectors representing these segments. Detailed analysis of trajectories and the correlation functions of the backbone dihedrals and side methyl groups indicates that they exhibit numerous conformational transitions. The activation energies of the conformational transitions obtained from the simulation are generally larger than the potential barriers for the rotations of these dihedrals, however, both show the same trend. We also have estimated the phenylene ring flip activation energy as 12.6 kcal/mol and the flip frequency as 0.77 MHz at 300 K. These values fall either fall within the range determined by various NMR spectroscopy experiments or slightly out of the range. The study shows that the conformational transitions between the adjacent dihedrals are strongly correlated. Three basic cooperative modes are identified from the simulation. They are: a positive synchronous rotation of two phenylene rings, a negative synchronous rotation of two phenylene rings, and a carbonate group rotation. Above the glass transition temperature, the large scale cooperative motions become much more significant.     

NMR detection of pH-dependent histidine-water proton exchange reveals the conduction mechanism of a transmembrane proton channel

The acid-activated proton channel formed by the influenza M2 protein is important for the life cycle of the virus. A single histidine, His37, in the M2 transmembrane domain (M2TM) is responsible for pH activation and proton selectivity of the channel. Recent studies suggested three models for how His37 mediates proton transport: a shuttle mechanism involving His37 protonation and deprotonation, a H-bonded imidazole-imidazolium dimer model, and a transporter model involving large protein conformational changes in synchrony with proton conduction. Using magic-angle-spinning (MAS) solid-state NMR spectroscopy, we examined the proton exchange and backbone conformational dynamics of M2TM in a virus-envelope-mimetic membrane. At physiological temperature and pH, (15)N NMR spectra show fast exchange of the imidazole (15)N between protonated and unprotonated states. To quantify the proton exchange rates, we measured the (15)N T(2) relaxation times and simulated them for chemical-shift exchange and fluctuating N-H dipolar fields under (1)H decoupling and MAS. The exchange rate is 4.5 × 10(5) s(-1) for Nδ1 and 1.0 × 10(5) s(-1) for Nε2, which are approximately synchronized with the recently reported imidazole reorientation. Binding of the antiviral drug amantadine suppressed both proton exchange and ring motion, thus interfering with the proton transfer mechanism. By measuring the relative concentrations of neutral and cationic His as a function of pH, we determined the four pK(a) values of the His37 tetrad in the viral membrane. Fitting the proton current curve using the charge-state populations from these pK(a)'s, we obtained the relative conductance of the five charge states, which showed that the +3 channel has the highest time-averaged unitary conductance. At physiologically relevant pH, 2D correlation spectra indicated that the neutral and cationic histidines do not have close contacts, ruling out the H-bonded dimer model. Moreover, a narrowly distributed nonideal helical structure coexists with a broadly distributed ideal helical conformation without interchange on the sub-10 ms time scale, thus excluding the transporter model in the viral membrane. These data support the shuttle mechanism of proton conduction, whose essential steps involve His-water proton exchange facilitated by imidazole ring reorientations.     

Anesthetics as chemical tools to study the structure and function of nicotinic acetylcholine receptors

The nicotinic acetylcholine receptor (AChR) is the archetype of the Cys-loop ligand-gated ion channel receptor superfamily. Noncompetitive antagonists inhibit the AChR without interacting directly with agonist sites. Among noncompetitive antagonists, general and local anesthetics have been used for decades to study the structure and function of muscle- as well as neuronal-type AChRs. In this review, we address and update all information regarding the characterization of binding sites and the mechanism of action for n-alkanols, barbiturates, inhalational and dissociative general anesthetics, as well as for tertiary and quaternary local anesthetics. The experimental evidence outlined in this review suggest that: (1) several neuronal-type AChRs might be targets for the pharmacological action of distinct anesthetics; (2) the molecular components of a specific anesthetic locus on a certain receptor type are different from the structural determinants of the site for the same anesthetic on a different receptor type; (3) there are unique binding sites for distinct anesthetics in the same receptor; (4) the affinity of a specific anesthetic depends on the AChR conformational state; (5) anesthetics may inhibit AChRs by different mechanisms including open-channel-blocking, augmenting the desensitization process, and/or inactivating the opening of resting receptors; and (6) some anesthetics may potentiate AChR activity.     

Conformational analysis of six- and twelve-membered ring compounds by molecular dynamics

A molecular dynamics (MD)-based conformational analysis has been performed on a number of cycloalkanes in order to demonstrate the reliability and generality of MD as a tool for conformational analysis. MD simulations on cyclohexane and a series of methyl-substituted cyclohexanes were performed at temperatures between 400 and 1200 K. Depending on the simulation temperature, different types of interconversions (twist-boat–twist-boat, twist- boat–chair and chair–chair) could be observed, and the MD simulations demonstrated the expected correlation between simulation temperature and ring inversion barriers. A series of methyl-substituted 1,3- dioxanes were investigated at 1000 K, and the number of chair–chair interconversions could be quantitatively correlated to the experimentally determined ring inversion barrier. Similarly, the distribution of sampled minimum-energy conformations correlated with the energy-derived Boltzmann distribution. The macrocyclic ring system cyclododecane was subjected to an MD simulation at 1000 K and 71 different conformations could be sampled. These conformations were compared with the results of previously reported conformational analyses using stochastic search methods, and the MD method provided 19 out of the 20 most stable conformations found in the MM2 force field. Finally, the general performance of the MD method for conformational analysis is discussed.     

Computational analysis of ligand recognition mechanisms by prostaglandin E2 (subtype 2) and D2 receptors

The eight members of the prostanoid receptor family belong to the class A G protein-coupled receptors. We investigated the evolutionary relationship of the eight members by a molecular phylogenetic analysis and found that prostaglandin E₂ receptor subtype 2 (EP₂) and prostaglandin D₂ receptor (DP) were closely related. The structures of the ligands for the two receptors are similar to each other but are distinguished by the exchanged locations of the carbonyl oxygen and the hydroxy group in the cyclopentane ring. The ligand recognition mechanisms of the receptors were examined by an integrated approach using several computational methods, such as amino acid sequence comparison, homology modeling, docking simulation, and molecular dynamics simulation. The results revealed the similar location of the ligand between the two receptors. The common carboxy group of the ligands interacts with the Arg residue on the seventh transmembrane (TM) helix, which is invariant among the prostanoid receptors. EP₂ uses a Ser on TM1 to recognize the carbonyl oxygen in the cyclopentane ring of the ligand. The Ser is specifically conserved within EP₂. On the other hand, DP uses a Lys on TM2 to recognize the hydroxy group of the ?? chain of the ligand. The Lys is also specifically conserved within DP. The interaction network between the D(E)RY motif and TM6 was found in EP₂. However, DP lacked this network, due to the mutation in the D(E)RY motif. Based on these observations and the previously published mutational studies on the motif, the possibility of another activation mechanism that does not involve the interaction between the D(E)RY motif and TM6 will be discussed.     

Modeling anesthetic binding sites within the glycine alpha one receptor based on prokaryotic ion channel templates: the problem with TM4

Ligand-gated ion channels (LGICs) significantly modulate anesthetic effects. Their exact molecular structure remains unknown. This has led to ambiguity regarding the proper amino acid alignment within their 3D structure and, in turn, the location of any anesthetic binding sites. Current controversies suggest that such a site could be located in either an intra- or intersubunit locale within the transmembrane domain of the protein. Here, we built a model of the glycine alpha one receptor (GlyRa1) based on the open-state structures of two new high-resolution ion channel templates from the prokaryote, Gloebacter violaceus (GLIC). Sequence scoring suggests reasonable homology between GlyRa1 and GLIC. Three of the residues notable for modulating anesthetic action are on transmembrane segments 1-3 (TM1-3): (ILE229, SER 267, and ALA 288). They line an intersubunit interface, in contrast to previous models. However, residues from the fourth transmembrane domain (TM4) that are known to modulate a variety of anesthetic effects are quite distant from this putative anesthetic binding site. While this model can account for a large proportion of the physicochemical data regarding such proteins, it cannot readily account for the alterations on anesthetic effects that are due to mutations within TM4.     

Single molecule conformational dynamics of adenylate kinase: energy landscape, structural correlations, and transition state ensembles

We developed a coarse grained two-well model to study the single molecule protein conformational dynamics in microscopic detail at the residue level, overcoming the often encountered computational bottleneck. In particular, we explored the underlying conformational energy landscape of adenylate kinase, a crucial protein for signal transduction in the cell, and identified two major kinetic pathways for the conformational switch between open and closed states through either the intermediate state or the transient state. Based on the parameters fitted to the room-temperature experimental data, we predicted open and closed kinetic rates at the whole temperature ranges from 10 to 50 degrees C, which agree well with the experimental turnover numbers. After uncovering the underlying mechanism for conformational dynamics and exploring the structural correlations, we found the crucial dynamical interplay between the nucleoside monophosphate binding domain (NMP) and the ATP-binding domain (LID) in controlling the conformational switch. The key residues and contacts responsible for the conformational transitions are identified by following the time evolution of the two-dimensional spatial contact maps and characterizing the transition state as well as intermediate structure ensembles through phi value analysis. Our model provides a general framework to study the conformational dynamics of biomolecules and can be applied to many other systems.     

Dimer asymmetry in superoxide dismutase studied by molecular dynamics simulation

Summary Molecular dynamics (MD) simulations of 100 ps have been carried out to study the active-site behaviour of the Cu,Zn superoxide dismutase dimer (SOD) in water. The active site of each subunit was monitored during the whole simulation by calculating the distances between functional residues and the catalytic copper. The results indicate that charge orientation is maintained at each active site but the solvent accessibility varies. Analysis of the MD simulation, carried out by using the atomic displacement covariance matrix, has shown a different intra-subunit correlation pattern for the two monomers and the presence of inter-subunit correlations. The MD simulation presented here indicates an asymmetry in the two active sites and different dynamic behaviour of the two SOD subunits.     

Revealing time bunching effect in single-molecule enzyme conformational dynamics

In this perspective, we focus our discussion on how the single-molecule spectroscopy and statistical analysis are able to reveal enzyme hidden properties, taking the study of T4 lysozyme as an example. Protein conformational fluctuations and dynamics play a crucial role in biomolecular functions, such as in enzymatic reactions. Single-molecule spectroscopy is a powerful approach to analyze protein conformational dynamics under physiological conditions, providing dynamic perspectives on a molecular-level understanding of protein structure-function mechanisms. Using single-molecule fluorescence spectroscopy, we have probed T4 lysozyme conformational motions under the hydrolysis reaction of a polysaccharide of E. coli B cell walls by monitoring the fluorescence resonant energy transfer (FRET) between a donor-acceptor probe pair tethered to T4 lysozyme domains involving open-close hinge-bending motions. Based on the single-molecule spectroscopic results, molecular dynamics simulation, a random walk model analysis, and a novel 2D statistical correlation analysis, we have revealed a time bunching effect in protein conformational motion dynamics that is critical to enzymatic functions. Bunching effect implies that conformational motion times tend to bunch in a finite and narrow time window. We show that convoluted multiple Poisson rate processes give rise to the bunching effect in the enzymatic reaction dynamics. Evidently, the bunching effect is likely common in protein conformational dynamics involving in conformation-gated protein functions. In this perspective, we will also discuss a new approach of 2D regional correlation analysis capable of analyzing fluctuation dynamics of complex multiple correlated and anti-correlated fluctuations under a non-correlated noise background. Using this new method, we are able to map out any defined segments along the fluctuation trajectories and determine whether they are correlated, anti-correlated, or non-correlated; after which, a cross correlation analysis can be applied for each specific segment to obtain a detailed fluctuation dynamics analysis.     

Multidimensional umbrella sampling and replica‐exchange molecular dynamics simulations for structure prediction of transmembrane helix dimers

Structural information of a transmembrane (TM) helix dimer is useful in understanding molecular mechanisms of important biological phenomena such as signal transduction across the cell membrane. Here, we describe an umbrella sampling (US) scheme for predicting the structure of a TM helix dimer in implicit membrane using the interhelical crossing angle and the TM–TM relative rotation angles as the reaction coordinates. This scheme conducts an efficient conformational search on TM–TM contact interfaces, and its robustness is tested by predicting the structures of glycophorin A (GpA) and receptor tyrosine kinase EphA1 (EphA1) TM dimers. The nuclear magnetic resonance (NMR) structures of both proteins correspond to the global free‐energy minimum states in their free‐energy landscapes. In addition, using the landscape of GpA as a reference, we also examine the protocols of temperature replica‐exchange molecular dynamics (REMD) simulations for structure prediction of TM helix dimers in implicit membrane. A wide temperature range in REMD simulations, for example, 250–1000 K, is required to efficiently obtain a free‐energy landscape consistent with the US simulations. The interhelical crossing angle and the TM–TM relative rotation angles can be used as reaction coordinates in multidimensional US and be good measures for conformational sampling of REMD simulations. © 2013 Wiley Periodicals, Inc.     

Alamethicin channels in a membrane: molecular dynamics simulations

Alamethicin (Alm) is a 20 residue peptide which forms a kinked alpha-helix in membrane and membrane-mimetic environments. Ion channels formed by intramembraneous aggregates of Alm are thought to be formed by bundles of approximately parallel Alm helices surrounding a central bilayer pore. Different channel conductance levels correspond to different numbers of helices per bundle, ranging from N = 5 to N > 8. Calculation of the predicted pKA values of the ring of Glu18 sidechains at the C-terminal mouth of the pore suggests that at neutral pH most or all of these sidechains will remain protonated. Nanosecond molecular dynamics (MD) simulations of N = 5, 6, 7 and 8 bundles of Alm helices in a POPC bilayer have been run, corresponding to a total simulation time of 4 ns. These simulations explore the stability and conformational dynamics of these helix bundle channels when embedded in a full phospholipid bilayer in an aqueous environment. The structural and dynamic properties of water in these model channels are examined. As in earlier in vacuo simulations (J. Breed, R. Sankararamakrishnan, I. D. Kerr and M. S. P. Sansom, Biophys. J., 1996, 70, 1643) the dipole moments of water molecules within the pores are aligned antiparallel to the helix dipoles. This helps to contribute to the stability of the helix bundles.     

Role of the N-Terminal Transmembrane Helix Contacts in the Activation of FGFR3

Fibroblast growth factor receptor 3 (FGFR3) is a member of receptor tyrosine kinases, which is involved in skeletal cell growth, differentiation, and migration. FGFR3 transduces biochemical signals from the extracellular ligand-binding domain to the intracellular kinase domain through the conformational changes of the transmembrane (TM) helix dimer. Here, we apply generalized replica exchange with solute tempering method to wild type (WT) and G380R mutant (G380R) of FGFR3. The dimer interface in G380R is different from WT and the simulation results are in good agreement with the solid-state nuclear magnetic resonance (NMR) spectroscopy. TM helices in G380R are extended more than WT, and thereby, G375 in G380R contacts near the N-termini of the TM helix dimer. Considering that both G380R and G375C show the constitutive activation, the formation of the N-terminal contacts of the TM helices can be generally important for the activation mechanism. © 2019 Wiley Periodicals, Inc.     

Molecular dynamics simulation of a hydrated diphytanol phosphatidylcholine lipid bilayer containing an alpha-helical bundle of four transmembrane domains of the influenza A virus M2 protein

An alpha-helical bundle composed of four transmembrane portions of the M2 protein from the Influenza A virus has been studied in a hydrated diphytanol phosphatidylcholine bilayer using molecular dynamics (MD) calculations. Experimentally, the sequence utilized is known to aggregate as a four-helix bundle and act as a pH-gated proton-selective ion channel, which is blocked by the drug amantadine hydrochloride. In the presented simulation, the ion channel was initially set up as a parallel four-helix bundle. The all-atom simulation consisted of almost 16,000 atoms, described classically, using a forcefield from the CHARMM22 database. Bilayers with and without the bundle were shown to be stable throughout the nanosecond timescale of the MD simulation. Structural and dynamical properties of the bilayer both with and without the transmembrane protein are reported.     

Membrane-dependent effects of a cytoplasmic helix on the structure and drug binding of the influenza virus M2 protein

The influenza A M2 protein forms a proton channel for virus infection and also mediates virus assembly and budding. The minimum protein length that encodes both functions contains the transmembrane (TM) domain (roughly residues 22-46) for the amantadine-sensitive proton-channel activity and an amphipathic cytoplasmic helix (roughly residues 45-62) for curvature induction and virus budding. However, structural studies involving the TM domain with or without the amphipathic helix differed on the drug-binding site. Here we use solid-state NMR spectroscopy to determine the amantadine binding site in the cytoplasmic-helix-containing M2(21-61). (13)C-(2)H distance measurements of (13)C-labeled protein and (2)H-labeled amantadine showed that in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers, the first equivalent of drug bound S31 inside the M2(21-61) pore, similar to the behavior of M2 transmembrane peptide (M2TM) in DMPC bilayers. The nonspecific surface site of D44 observed in M2TM is disfavored in the longer peptide. Thus, the pharmacologically relevant drug-binding site in the fully functional M2(21-61) is S31 in the TM pore. Interestingly, when M2(21-61) was reconstituted into a virus-mimetic membrane containing 30% cholesterol, no chemical shift perturbation was observed for pore-lining residues, whereas M2TM in the same membrane exhibited drug-induced chemical shift changes. Reduction of the cholesterol level and the use of unsaturated phospholipids shifted the conformational equilibrium of M2TM fully to the bound state but did not rescue drug binding to M2(21-61). These results suggest that the amphipathic helix, together with cholesterol, modulates the ability of the TM helix to bind amantadine. Thus, the M2 protein interacts with the lipid membrane and small-molecule inhibitors in a complex fashion, and a careful examination of the environmental dependence of the protein conformation is required to fully understand the structure-function relation of this protein.