Explicit Membrane Environment Research Articles

Voltage-dependent structural models of the human Hv1 proton channel from long-timescale molecular dynamics simulations

The voltage-gated Hv1 proton channel is a ubiquitous membrane protein that has roles in a variety of cellular processes, including proton extrusion, pH regulation, production of reactive oxygen species, proliferation of cancer cells, and increased brain damage during ischemic stroke. A crystal structure of an Hv1 construct in a putative closed state has been reported, and structural models for the channel open state have been proposed, but a complete characterization of the Hv1 conformational dynamics under an applied membrane potential has been elusive. We report structural models of the Hv1 voltage-sensing domain (VSD), both in a hyperpolarized state and a depolarized state resulting from voltage-dependent conformational changes during a 10-μs-timescale atomistic molecular dynamics simulation in an explicit membrane environment. In response to a depolarizing membrane potential, the S4 helix undergoes an outward displacement, leading to changes in the VSD internal salt-bridge network, resulting in a reshaping of the permeation pathway and a significant increase in hydrogen bond connectivity throughout the channel. The total gating charge displacement associated with this transition is consistent with experimental estimates. Molecular docking calculations confirm the proposed mechanism for the inhibitory action of 2-guanidinobenzimidazole (2GBI) derived from electrophysiological measurements and mutagenesis. The depolarized structural model is also consistent with the formation of a metal bridge between residues located in the core of the VSD. Taken together, our results suggest that these structural models are representative of the closed and open states of the Hv1 channel.

Ion Selective Pentameric Pore Formation by Ebola Virus Delta Peptide

The Ebola produces the pore forming glycoprotein delta peptide which is implicated in facilitating the release of viral particles through the host cell's membrane. In this work, we investigate the molecular details of the pore forming mechanism of the delta peptide viroporin. Using information from experimental studies, we computationally modelled membrane pores formed by delta peptide oligomers. We performed all-atom molecular dynamics (MD) simulations in an explicit membrane environment to investigate the pore-forming mechanism and stability of the pores. Our results suggest that the delta peptide forms stable pentameric pores that are selective for chloride ion transport. We found that a disulfide bond between cysteine residues in the C-terminal of the peptide is essential for the pore stabilization and ion permeation. Our study provides helpful information on the pore-forming mechanism of filovirus delta peptides and such structural information can be important in designing and developing molecular modulators that target the delta peptide pore and disrupt the pathology of the Ebola virus.

OR08-4 A Novel Mutation in the Follicle-Stimulating Hormone Receptor (Ile423Thr) in a Woman with Normal Pubertal Development and Primary Amenorrhea: In Vivo, In Vitro, and In Silico Studies

Women with inactivating mutations in the FSHR may exhibit an array of phenotypes, from lack of pubertal development, primary amenorrhea, and complete insensitivity to FSH, to secondary amenorrhea and premature ovarian failure. In contrast to the LHCGR, only 19 inactivating mutations in the FSHR have been described to date. We report herein the case of a 22 year old women who presented with normal pubertal development but primary amenorrhea, associated with elevated serum FSH and LH levels (27-30 IU/L), low serum estradiol (E2) (21-40 pg/ml) and normal serum AMH (2.2-2.5 ng/ml) levels. After administration of recombinant FSH (total dose 1425 IU/9 days) serum E2 levels rose from 20 to 65 pg/ml with a parallel increase in number (from 5 to 17 follicles) and size (from 3 to 10 mm diameter) of follicles, and ovarian volumes (from 4 to 10 cm3). Sequencing of the FSHR revealed a new homozygous mutation in codon 423, where ATC was changed to ACC, generating the Ile423Thr substitution at the junction of the transmembrane domain (TM) 2 and the exo-loop (EL) 1 of the FSHR. The mother and two half-brothers were heterozygous for the same mutation. The molecular physiopathogenesis of the novel Ile423Thr mutation was analyzed by a series of in silico and in vitro biochemical studies in HEK293 cells transiently expressing either the wild-type (Wt) FSHR or the Ile423Thr mutant cloned into the pSG5 vector, and the results were compared with those obtained in cells transfected with the expression-deficient Asp408Tyr mutant, which leads to a more severe form of hypergonadotropic hypogonadism (1). Compared with the Wt FSHR, the plasma membrane expression of the mature (~80 kDa) form of the Ile423Thr mutant was slightly reduced, while that of the Asp408Tyr mutant was remarkably low, as disclosed by immunoblotting. A dissociation in the responses to FSH-stimulated signaling between the mutant receptors was found: compared with the Wt FSHR, FSH-stimulated cAMP production and ERK phosphorylation were reduced by 67% and 27% in the Ile423Thr mutant and by 52% and 43% in the Asp408Tyr FSHR, respectively. In silico molecular dynamics simulations of the FSHRs in an explicit membrane environment unveiled important differences in the behavior of both mutants vs the Wt receptor, particularly in the interhelical interactions of Thr423 with TM7 and EL2, and Tyr408 with TMs 5 and 7. Concurrently, these data suggest that conformational differences during the inactive (particularly during trafficking of the receptor proteins from the endoplasmic reticulum to the plasma membrane) and active (agonist-occupied receptor) states, account for the distinct expression levels and differential signaling exhibited by the Ile423Thr and Asp408Tyr mutant FSHRs. (1) Bramble MS, et al. A novel follicle-stimulating hormone receptor mutation causing primary ovarian failure: a fertility application of whole exome sequencing. Hum. Reprod. 2016; 31;905-914.

Molecular dynamics simulation of the follicle-stimulating hormone receptor. Understanding the conformational dynamics of receptor variants at positions N680 and D408 from in silico analysis.

Follicle-stimulating hormone receptor (FSHR) is a G-protein coupled receptor (GPCR) and a prototype of the glycoprotein hormone receptors subfamily of GPCRs. Structural data of the FSHR ectodomain in complex with follicle-stimulating hormone suggests a “pull and lift” activation mechanism that triggers a conformational change on the seven α-helix transmembrane domain (TMD). To analyze the conformational changes of the FSHR TMD resulting from sequence variants associated with reproductive impairment in humans, we set up a computational approach combining helix modeling and molecular simulation methods to generate conformational ensembles of the receptor at room (300 K) and physiological (310 K) temperatures. We examined the receptor dynamics in an explicit membrane environment of polyunsaturated phospholipids and solvent water molecules. The analysis of the conformational dynamics of the functional (N680 and S680) and dysfunctional (mutations at D408) variants of the FSHR allowed us to validate the FSHR-TMD model. Functional variants display a concerted motion of flexible intracellular regions at TMD helices 5 and 6. Disruption of side chain interactions and conformational dynamics were detected upon mutation at D408 when replaced with alanine, arginine, or tyrosine. Dynamical network analysis confirmed that TMD helices 2 and 5 may share communication pathways in the functional FSHR variants, whereas no connectivity was detected in the dysfunctional mutants, indicating that the global dynamics of the FSHR was sensitive to mutations at amino acid residue 408, a key position apparently linked to misfolding and variable cell surface plasma membrane expression of FSHRs with distinct mutations at this position.

What Can and Cannot Be Learned from Molecular Dynamics Simulations of Bacterial Proton-Coupled Oligopeptide Transporter GkPOT?

We have performed an extensive set of all-atom molecular dynamics (MD) simulations of a bacterial proton-coupled oligopeptide transporter (POT) in an explicit membrane environment. We have characterized both the local and global conformational dynamics of the transporter upon the proton and/or substrate binding, within a statistical framework. Our results reveal a clearly distinct behavior for local conformational dynamics in the absence and presence of the proton at the putative proton binding residue E310. Particularly, we find that the substrate binding conformation is drastically different in the two conditions, where the substrate binds to the protein in a lateral/vertical manner, in the presence/absence of the proton. We do not observe any statistically significant distinctive behavior in terms of the global conformational changes in different simulation conditions, within the time scales of our simulations. Our extensive simulations and analyses call into question the implicit assumption of many MD studies that local conformational changes observed in short simulations could provide clues to the global conformational changes that occur on much longer time scales. The linear regression analysis of quantities associated with the global conformational fluctuations, however, provides an indication of a mechanism involving the concerted motion of the transmembrane helices, consistent with the rocker-switch mechanism.

Communication over the network of binary switches regulates the activation of A2A adenosine receptor.

Dynamics and functions of G-protein coupled receptors (GPCRs) are accurately regulated by the type of ligands that bind to the orthosteric or allosteric binding sites. To glean the structural and dynamical origin of ligand-dependent modulation of GPCR activity, we performed total ~ 5 μsec molecular dynamics simulations of A2A adenosine receptor (A2AAR) in its apo, antagonist-bound, and agonist-bound forms in an explicit water and membrane environment, and examined the corresponding dynamics and correlation between the 10 key structural motifs that serve as the allosteric hotspots in intramolecular signaling network. We dubbed these 10 structural motifs “binary switches” as they display molecular interactions that switch between two distinct states. By projecting the receptor dynamics on these binary switches that yield 210 microstates, we show that (i) the receptors in apo, antagonist-bound, and agonist-bound states explore vastly different conformational space; (ii) among the three receptor states the apo state explores the broadest range of microstates; (iii) in the presence of the agonist, the active conformation is maintained through coherent couplings among the binary switches; and (iv) to be most specific, our analysis shows that W246, located deep inside the binding cleft, can serve as both an agonist sensor and actuator of ensuing intramolecular signaling for the receptor activation. Finally, our analysis of multiple trajectories generated by inserting an agonist to the apo state underscores that the transition of the receptor from inactive to active form requires the disruption of ionic-lock in the DRY motif.

Genomics-Aided Structural Modeling of an Antiparallel Homodimeric Fluoride Channel

‘Flucs’ are small membrane proteins widespread in bacteria, single-celled eukaryotes, and plants. Only recently characterized, Flucs act as fluoride-specific ion channels, forming antiparallel dimers of four-helix transmembrane bundles. Little else is known about this protein family. To gain insight into the structure and function of the Flucs, we use direct-coupling analysis (DCA) and ab initio molecular modeling to generate all-atom models of the E. coli Fluc homodimer EC2. DCA uses large multiple sequence alignments to infer the interdependencies between residue positions in protein families and is robust at predicting protein contacts from sequence alone. Taking into account simple geometric considerations and strong experimental evidence for an antiparallel homodimer, we are able to parse the inter- and intra-monomeric contacts predicted by DCA. These contacts are used to bias a conformational search performed by a custom Rosetta fold-and-dock protocol. Final refined models are further relaxed with all-atom molecular dynamics simulations in an explicit membrane environment. Possible mechanisms for fluoride selectivity and permeation are discussed in light of the model. This study demonstrates the utility of DCA in the ab initio modeling of oligomers, suggests a novel sequence-based approach to identify dual-topology proteins, and provides a strong foundation for more directed experimental characterizations of the Fluc protein family.

Studying Conformational Changes of Mhp1 using Unbiased All-Atom Molecular Simulations

Mhp1 is a secondary active transporter from nucleobase/cation symporter-1 (NCS1) family. We have developed the implicit membrane-explicit membrane (IM-EX) hybrid simulation method to study conformational changes in secondary active transporters (JMB. 404:506). This novel approach starts with IM simulations and certain confirmations from the implicit simulation are placed in an explicit membrane environment to get the final conformation. Studying conformational changes of Mhp1 is an important test to our IM-EX method. Three different conformations of Mhp1 are known, i.e., inward-open, outward-occluded and outward-open. For Mhp1, the implicit simulation is applied by using SGLD-fp method (JCP. 135: 204101). The main advantage of SGLD-fp method is to overcome the energy barriers in a reasonable amount of simulation time. Then, certain conformations that show structural change during the implicit simulation are simulated with MD in an explicit membrane. Crystal structures of different conformations were used as initial conformation for SGLD-fp simulations. Glu289 was protonated based on its pKa value. The 20-ns simulation for inward-open conformation showed opening in extracellular gate but no closure for intracellular gate. In case of outward-occluded conformation, the result showed opening of EC gate while IC gate is still closed. The final structure agrees with the outward-open conformation, which means SGLD-fp method can predict conformational changes in a short time. For the outward-open conformation, simulations with SGLD-fp maintained this conformation. Protonation of other residues, choosing different initial seeds and effect of sodium and substrate are currently being studied to see how these factors affect transition between states.

Structural Modeling of Toxin Interactions with the Human Voltage-Gated Sodium Channel Pore

Voltage-gated sodium (Nav) channel subtypes are targets for the development of novel analgesics. We are constraining structural models of human Nav subtypes, as the lack of high-resolution structures of channels makes rational drug design challenging. The human Nav1.4 channel is associated with paralysis and human Nav1.7 channel plays an important role in pain signaling. We used the bacterial Nav channel NavRh (pdb id: 4DXW) as a template to generate homology/de novo models of human Nav1.4 and Nav1.7 channel pore-forming domains. Due to significant sequence differences between human and bacterial Nav channels in the P2 helix region of the selectivity filter, we predicted the structure of these regions de novo using Rosetta loop modeling methods. We simulated interactions of human Nav1.4 and Nav1.7 channels with pore blocking toxins - tetrodotoxin, saxitoxin, and μ-conotoxin KIIIA - using Rosetta methods and molecular dynamics in an all-atom explicit membrane environment. Virtual alanine scans of key residues forming toxin receptor sites within human Nav1.4 and Nav1.7 channel pore-forming domains showed good correlation with available experimental data. High-resolution structural models of the human Nav channels provide critical starting points for design of novel analgesics.

Conformational Transition Pathway of GlpT Transporter, Characterized by Nonequilibrium Molecular Dynamics Simulations

Glycerol-3-phosphate transporter (GlpT) is a member of major facilitator superfamily (MFS), the largest family of secondary transporters. Similar to other transporters, GlpT is believed to function using an “alternating-access” mechanism during which it undergoes large conformational changes. The only available structure of GlpT is in the apo inward-facing (IF) state. In our earlier work using equilibrium molecular dynamics (MD) simulations, the binding site of GlpT was characterized and the initial substrate-induced closing on the cytoplasmic side was observed. However, an atomic model of the outward-facing (OF) state of GlpT and the IF-to-OF transition pathway remain unknown. Here we have used a novel approach based on nonequilibrium MD simulations to drive the transition of GlpT from its available IF state to its unknown OF state in an explicit membrane environment. Based on the interhelical symmetries, a set of non-conventional collective variables were defined for the transmembrane helices to specifically reflect the conformational changes involved during the IF-to-OF transition. The system was then steered along these collective variables in a nonequilibrium scheme. The obtained IF-to-OF transition pathway was further optimized adaptively by minimizing the nonequilibrium work, which is a good way to produce more realistic transition pathways. Thus, the protocol of inducing the transition was optimized after each simulation based on the resulted trajectory. Nonequilibrium work relations were used to analyze the trajectories and extract information about the OF state structure, the IF-to-OF transition mechanism, the intermediate/occluded states, and the characteristics of the OF-state binding site, providing insight about the general mechanism of MFS transporters and more generally introducing a novel approach to study the IF-to-OF transitions in membrane transporters.

Exploring a Model of a Chemokine Receptor/Ligand Complex in an Explicit Membrane Environment by Molecular Dynamics Simulation: The Human CCR1 Receptor

The seven transmembrane helices G-protein-coupled receptors (GPCRs) form one of the largest superfamilies of signaling proteins found in humans. Homology modeling, molecular docking, and molecular dynamics (MD) simulation were carried out to construct a reliable model for CCR1 as one of the GPCRs and to explore the structural features and the binding mechanism of BX471 as one of the most potent CCR1 inhibitors. In this study, BX471 has been docked into the active site of the CCR1 protein. After docking, one 20 ns MD simulation was performed on the CCR1-ligand complex to explore effects of the presence of lipid membrane in the vicinity of the CCR1-ligand complex. At the end of the MD simulation, a change in the position and orientation of the ligand in the binding site was observed. This important observation indicated that the application of MD simulation after docking of ligands is useful. Explorative runs of molecular dynamics simulation on the receptor-ligand complex revealed that except for Phe85, Phe112, Tyr113, and Ile259, the rest of the residues in the active site determined by docking are changed. The results obtained are in good agreement with most of the experimental data reported by others. Our results show that molecular modeling and rational drug design for chemokine targets is a possible approach.

Tetrahydrofolate Recognition by the Mitochondrial Folate Transporter

A mitochondrial carrier family (MCF) of transport proteins facilitates the transfer of charged small molecules across the inner mitochondrial membrane. The human genome has ∼50 genes corresponding to members of this family. All MCF proteins contain three repeats of a characteristic and conserved PX(D/E)XX(K/R) motif thought to be central to the mechanism of these transporters. The mammalian mitochondrial folate transporter (MFT) is one of a few MCF members, known as the P(I/L)W subfamily, that have evolved a tryptophan residue in place of the (D/E) in the second conserved motif; the function of this substitution (Trp-142) is unclear. Molecular dynamics simulations of the MFT in its explicit membrane environment identified this tryptophan, as well as several other residues lining the transport cavity, to be involved in a series of sequential interactions that coordinated the movement of the tetrahydrofolate substrate within the transport cavity. We probed the function of these residues by mutagenesis. The mutation of every residue identified by molecular dynamics to interact with tetrahydrofolate during simulated transit into the aqueous channel severely impaired folate transport. Mutation of the subfamily-defining tryptophan residue in the MFT to match the MCF consensus at this position (W142D) was incompatible with cell survival. These studies indicate that MFT Trp-142, as well as other residues lining the transporter interior, coordinate tetrahydrofolate descent and positioning of the substrate in the transporter basin. Overall, we identified residues in the walls and at the base of the transport cavity that are involved in substrate recognition by the MFT.

Modeling KCNQ1 Channel and KCNE1 Interactions

KCNQ1 is the pore-forming component of the slow delayed rectifier (Iks) channel in human heart. Although KCNQ1 can function as a voltage-gated (Kv) channel on its own, it requires KCNE1 to modulate its functions. To understand the mechanism of how KCNE1 modulate KCNQ1, molecular simulations are employed to model the interactions between KCNQ1 and KCNE1. A homology model of KCNQ1 was constructed based on the crystal structure of Kv1.2-Kv2.1 paddle chimera channel (PDB entry: 2R9R) template. For KCNE1, we used a NMR structure as an initial conformation, and manually adjusted the orientations of its N- and C- terminal domains to avoid their folding into membrane. Then 100ns molecular dynamics (MD) simulations were conducted to sample possible conformations of KCNE1 in POPC lipid bilayers. The cluster analysis was conducted on KCNE1 tansmembrane domain (KCNE1-TMD) from 40ns to 100ns simulation period. Five representative structures of KCNE1-TMD were selected, and used for docking simulations to KCNQ1. We performed 2 dimensional Brownian Dynamics (2D-BD) protein-docking simulations on each representative structure of KCNE1-TMD and KCNQ1 homology model. After selecting compact complexes by using a distance-based filter to remove loose contact complexes, a series of experimental restraints were applied to select one final KCNQ1/KCNE1-TMD complex structure. The N- and C- terminal domains of KCNE1 were reconstructed to generate the whole complex of KCNQ1/KCNE1, and followed by 100ns MD simulations in explicit membrane environment. For comparison, we also conducted 100ns MD simulations on KCNQ1 alone. Based on the two simulation studies, we analyzed the dynamics of the KCNQ1 channel with and without KCNE1 present through a systematic examination of hydrogen bond network patterns and ion permeation pathway of the channel. The results could provide us helpful insights of the molecular mechanism of how KCNE1 modulating KCNQ1 channel.

Molecular Dynamics Simulations of PIP 2-Driven Kir Channel Activation

Inwardly rectifying K+ (Kir) channels are gated by the signaling phospholipid phosphatidylinositol-bisphosphate (PIP2). The molecular mechanism of how PIP2 interacts with Kir channels and induces its structural transition from the closed to the open state remains unclear. We used computational approaches, molecular docking and molecular dynamics (MD) simulations, to model the PIP2-driven Kir channel activation based on crystal structures of a Kir3.1 chimera (BacKir3.1, PDB entry: 2QKS). The BacKir3.1 serves as a valid structural and functional model of Kir3 channel behavior (Nishida et al., 2007; Leal-Pinto et al., 2010). BacKir3.1 was crystallized in two conformers, dilated and constricted forms, which differ mainly in the conformations of the cytosolic G-loop gate. We built four channel systems including dilated and constricted BacKir3.1 channels with and without PIP2 present in the explicit POPC membrane environment, and performed 100ns MD simulations, respectively. We monitored the interactions between PIP2 and the channel during the simulations that showed C-alpha RMSD stability after 50ns. Several key residues in the Slide helix and the B-loop were identified to form hydrogen bonds and/or salt bridges with PIP2. Average radii of the ion permeation pathway along the channel were calculated during the 95-100ns simulation interval. Both systems in which PIP2 was present became wider in the G-loop regions along the ion permeation pathway compared to their counterpart systems in the absence of PIP2. In the “dilated + PIP2” system, the MD simulations identified in one subunit of the channel an outward rotation on the residue Phe181, that is located in the helix bundle-crossing (HBC) gate of TM2. The relationship of the channel-PIP2 interacting residues and their effects on the conformations of the G-loop and HBC gates are actively pursued through a systematic examination of hydrogen bond network patterns and principal component analysis.

Folding simulations of gramicidin A into the β-helix conformations: Simulated annealing molecular dynamics study

Gramicidin A is a linear hydrophobic 15-residue peptide which consists of alternating D- and L-amino acids and forms a unique tertiary structure, called the beta(6.3)-helix, to act as a cation-selective ion channel in the natural conditions. In order to investigate the intrinsic ability of the gramicidin A monomer to form secondary structures, we performed the folding simulation of gramicidin A using a simulated annealing molecular dynamics (MD) method in vacuum mimicking the low-dielectric, homogeneous membrane environment. The initial conformation was a fully extended one. From the 200 different MD runs, we obtained a right-handed beta(4.4)-helix as the lowest-potential-energy structure, and left-handed beta(4.4)-helix, right-handed and left-handed beta(6.3)-helix as local-minimum energy states. These results are in accord with those of the experiments of gramicidin A in homogeneous organic solvent. Our simulations showed a slight right-hand sense in the lower-energy conformations and a quite beta-sheet-forming tendency throughout almost the entire sequence. In order to examine the stability of the obtained right-handed beta(6.3)-helix and beta(4.4)-helix structures in more realistic membrane environment, we have also performed all-atom MD simulations in explicit water, ion, and lipid molecules, starting from these beta-helix structures. The results suggested that beta(6.3)-helix is more stable than beta(4.4)-helix in the inhomogeneous, explicit membrane environment, where the pore water and the hydrogen bonds between Trp side-chains and lipid-head groups have a role to further stabilize the beta(6.3)-helix conformation.

Tilt and Azimuthal Angles of a Transmembrane Peptide: A Comparison between Molecular Dynamics Calculations and Solid-State NMR Data of Sarcolipin in Lipid Membranes

We report molecular dynamics simulations in the explicit membrane environment of a small membrane-embedded protein, sarcolipin, which regulates the sarcoplasmic reticulum Ca-ATPase activity in both cardiac and skeletal muscle. In its monomeric form, we found that sarcolipin adopts a helical conformation, with a computed average tilt angle of 28 ± 6° and azymuthal angles of 66 ± 22°, in reasonable accord with angles determined experimentally (23 ± 2° and 50 ± 4°, respectively) using solid-state NMR with separated-local-field experiments. The effects of time and spatial averaging on both 15N chemical shift anisotropy and 1H/ 15N dipolar couplings have been analyzed using short-time averages of fast amide out-of-plane motions and following principal component dynamic trajectories. We found that it is possible to reproduce the regular oscillatory patterns observed for the anisotropic NMR parameters (i.e., PISA wheels) employing average amide vectors. This work highlights the role of molecular dynamics simulations as a tool for the analysis and interpretation of solid-state NMR data.

Backbone Conformation and Dynamics of the Lipid-Modified Membrane Anchor of Human N-Ras Investigated by Solid-State NMR and Molecular Dynamics Simulations

Many proteins involved in signal transduction are anchored to membranes by covalently attached lipid modifications. In this study we investigated the conformation and dynamics of the backbone and side chains of the N-Ras membrane anchoring domain. Experimental solid-state NMR studies involved doubly lipid-modified uniformly 13C and 15N labeled heptapeptides representing the C-terminus of N-Ras, which were incorporated into DMPC bilayers. A structural model of the peptide was calculated on the basis of isotropic chemical shifts, explicit torsion angle measurements, and nuclear Overhauser effects determined by solid-state NMR. The amplitude of molecular motions was assessed by 1H-13C order parameter measurements using separated local field NMR. For determination of the correlation times of the motions, T1 and T2 relaxation times were measured and analyzed using a generalized relaxation approach. To further understand the dynamics of Ras, molecular dynamics simulations of the molecule in lipid bilayers were conducted. In generating starting conditions for the simulation, special attention was paid to the backbone conformation since transitions between conformations were found to be rare events in a previous simulation of 100 ns length on this system [1]. Therefore, the experimentally determined conformation of the peptide backbone was equilibrated using a replica exchange technique in an explicit membrane environment. This enabled us to identify different conformers and to assess their relative probability. The resulting distribution of conformations was used subsequently for a long conventional MD simulation that was analyzed with regard to the experimental data. The combined simulations and experimental approach enabled a detailed model of the dynamics of the peptide to be obtained.[1] Vogel, A. Tan, K.-T. Waldmann, H. Feller, S.E. Brown, M.F. Huster, D. Biophys. J. 2007, 93, 2697-2712.

Structural And Dynamic Effects Of Cholesterol At Preferred Sites Of Interaction With Rhodopsin Identified From Microsecond Length Molecular Dynamics Simulations

A key unresolved question about GPCR function is the role of membrane components in receptor stability and activation. In particular, cholesterol is known to affect the function of membrane proteins, but the details of its effect on GPCRs are still elusive. Here, we describe how cholesterol modulates the behavior of TM1-TM2-TM7-helix 8(H8) functional network, that comprises the highly conserved NPxxY(x)5,6F motif, through specific interactions with the receptor. The inferences are based on the analysis of microsecond molecular dynamics (MD) simulations of Rhodopsin in an explicit membrane environment.

Mechanism of Signal Propagation upon Retinal Isomerization: Insights from Molecular Dynamics Simulations of Rhodopsin Restrained by Normal Modes

As one of the best studied members of the pharmaceutically relevant family of G-protein-coupled receptors, rhodopsin serves as a prototype for understanding the mechanism of G-protein-coupled receptor activation. Here, we aim at exploring functionally relevant conformational changes and signal transmission mechanisms involved in its photoactivation brought about through a cis-trans photoisomerization of retinal. For this exploration, we propose a molecular dynamics simulation protocol that utilizes normal modes derived from the anisotropic network model for proteins. Deformations along multiple low-frequency modes of motion are used to efficiently sample collective conformational changes in the presence of explicit membrane and water environment, consistent with interresidue interactions. We identify two highly stable regions in rhodopsin, one clustered near the chromophore, the other near the cytoplasmic ends of transmembrane helices H1, H2, and H7. Due to redistribution of interactions in the neighborhood of retinal upon stabilization of the trans form, local structural rearrangements in the adjoining H3–H6 residues are efficiently propagated to the cytoplasmic end of these particular helices. In the structures obtained by our simulations, all- trans retinal interacts with Cys 167 on H4 and Phe 203 on H5, which were not accessible in the dark state, and exhibits stronger interactions with H5, while some of the contacts made (in the cis form) with H6 are lost.

Pharmacophore-directed Homology Modeling and Molecular Dynamics Simulation of G Protein-coupled Receptor: Study of Possible Binding Modes of 5-HT<sub>2C</sub> Receptor Agonists

A new pharmacophore-based modeling procedure, including homology modeling, pharmacophore study, flexible molecular docking, and long-time molecular dynamics (MD) simulations, was employed to construct the structure of the human 5-HT(2C) receptor and determine the characteristics of binding modes of 5-HT(2C) receptor agonists. An agonist-receptor complex has been constructed based on homology modeling and a pharmacophore hypothesis model based on some high active compounds. Then MD simulations of the ligand-receptor complex in an explicit membrane environment were carried out. The conformation of the 5-HT(2C) receptor during MD simulation was explored, and the stable binding modes of the studied agonist were determined. Flexible molecular docking of several structurally diverse agonists of the human 5-HT(2C) receptor was carried out, and the general binding modes of these agonists were investigated. According to the models presented in this work and the results of Flexi-Dock, the involvement of the amino acid residues Asp134, Ser138, Asn210, Asn331, Tyr358, Ile131, Ser132, Val135,Thr139, Ile189, Val202, Val208, Leu209, Phe214, Val215, Gly218, Ser219, Phe223, Trp324, Phe327, and Phe328 in agonist recognition was studied. The obtained binding modes of the human 5-HT(2C) receptor agonists have good agreement with the site-directed mutagenesis data and other studies.