Proline-induced hinges in transmembrane helices: possible roles in ion channel gating

A number of ion channels contain transmembrane (TM) alpha-helices that contain proline-induced molecular hinges. These TM helices include the channel-forming peptide alamethicin (Alm), the S6 helix from voltage-gated potassium (Kv) channels, and the D5 helix from voltage-gated chloride (CLC) channels. For both Alm and KvS6, experimental data implicate hinge-bending motions of the helix in an aspect of channel gating. We have compared the hinge-bending motions of these TM helices in bilayer-like environments by multi-nanosecond MD simulations in an attempt to describe motions of these helices that may underlie possible modes of channel gating. Alm is an alpha-helical channel-forming peptide, which contains a central kink associated with a Gly-x-x-Pro motif in its sequence. Simulations of Alm in a TM orientation for 10 ns in an octane slab indicate that the Gly-x-x-Pro motif acts as a molecular hinge. The S6 helix from Shaker Kv channels contains a Pro-Val-Pro motif. Modeling studies and recent experimental data suggest that the KvS6 helix may be kinked in the vicinity of this motif. Simulations (10 ns) of an isolated KvS6 helix in an octane slab and in a POPC bilayer reveal hinge-bending motions. A pattern-matching approach was used to search for possible hinge-bending motifs in the TM helices of other ion channel proteins. This uncovered a conserved Gly-x-Pro motif in TM helix D5 of CLC channels. MD simulations of a model of hCLC1-D5 spanning an octane slab suggest that this channel also contains a TM helix that undergoes hinge-bending motion. In conclusion, our simulations suggest a model in which hinge-bending motions of TM helices may play a functional role in the gating mechanisms of several different families of ion channels.     

Molecular dynamics investigation of membrane-bound bundles of the channel-forming transmembrane domain of viral protein U from the human immunodeficiency virus HIV-1

Molecular dynamics (MD) simulations have been carried out on bundles of the channel-forming transmembrane (TM) domain of the viral protein U (VPU(1-27) and VPU(6-27)) from the human immunodeficiency virus (HIV-1). Simulations of hexameric and pentameric bundles of VPU(6-27) in an octane/water membrane mimetic system suggested that the pentamer is the preferred oligomer. Accordingly, an unconstrained pentameric helix bundle of VPU(1-27) was then placed in a hydrated palmitoyl-oleyl-3-n-glycero-phosphatidylethanolamine (POPE) lipid bilayer and its structural properties calculated from a 3-ns MD run. Some water molecules, initially inside the channel lumen, were expelled halfway through the simulation and the bundle adopted a conical structure reminiscent of previous MD results obtained for VPU(6-27) in an octane/water system. The pore constriction generated may correspond to a closed state of the channel and underlies the relocation of the W residue toward the pore lumen. The relative positions of the helices with respect to the bilayer and their interactions with the lipids are discussed. The observed structure is stabilized via specific interactions between the VPU helices and the carbonyl oxygen atoms of the lipid molecules, particularly at the Q and S residues.     

Helix-coil transition of PrP106-126: molecular dynamic study.

A set of 34 molecular dynamic (MD) simulations totaling 305 ns of simulation time of the prion protein-derived peptide PrP106-126 was performed with both explicit and implicit solvent models. The objective of these simulations is to investigate the relative stability of the alpha-helical conformation of the peptide and the mechanism for conversion from the helix to a random-coil structure. At neutral pH, the wild-type peptide was found to lose its initial helical structure very fast, within a few nanoseconds (ns) from the beginning of the simulations. The helix breaks up in the middle and then unwinds to the termini. The spontaneous transition into the random coil structure is governed by the hydrophobic interaction between His(111) and Val(122). The A117V mutation, which is linked to GSS disease, was found to destabilize the helix conformation of the peptide significantly, leading to a complete loss of helicity approximately 1 ns faster than in the wild-type. Furthermore, the A117V mutant exhibits a different mechanism for helix-coil conversion, wherein the helix begins to break up at the C-terminus and then gradually to unwind towards the N-terminus. In most simulations, the mutation was found to speed up the conversion through an additional hydrophobic interaction between Met(112) and the mutated residue Val(117), an interaction that did not exist in the wild-type peptide. Finally, the beta-sheet conformation of the wild-type peptide was found to be less stable at acidic pH due to a destabilization of the His(111)-Val(122), since at acidic pH this histidine is protonated and is unlikely to participate in hydrophobic interaction.     

Structure and dynamics of K channel pore-lining helices: a comparative simulation study

Isolated pore-lining helices derived from three types of K-channel have been analyzed in terms of their structural and dynamic features in nanosecond molecular dynamics (MD) simulations while spanning a lipid bilayer. The helices were 1) M1 and M2 from the bacterial channel KcsA (Streptomyces lividans), 2) S5 and S6 from the voltage-gated (Kv) channel Shaker (Drosophila melanogaster), and 3) M1 and M2 from the inward rectifier channel Kir6.2 (human). In the case of the Kv and Kir channels, for which x-ray structures are not known, both short and long models of each helix were considered. Each helix was incorporated into a lipid bilayer containing 127 palmitoyloleoylphosphatidylcholine molecules, which was solvated with approximately 4000 water molecules, yielding approximately 20, 000 atoms in each system. Nanosecond MD simulations were used to aid the definition of optimal lengths for the helix models from Kv and Kir. Thus the study corresponds to a total simulation time of 10 ns. The inner pore-lining helices (M2 in KcsA and Kir, S6 in Shaker) appear to be slightly more flexible than the outer pore-lining helices. In particular, the Pro-Val-Pro motif of S6 results in flexibility about a molecular hinge, as was suggested by previous in vacuo simulations (, Biopolymers. 39:503-515). Such flexibility may be related to gating in the corresponding intact channel protein molecules. Analysis of H-bonds revealed interactions with both water and lipid molecules in the water/bilayer interfacial region. Such H-bonding interactions may lock the helices in place in the bilayer during the folding of the channel protein (as is implicit in the two-stage model of membrane protein folding). Aromatic residues at the extremities of the helices underwent complex motions on both short (<10 ps) and long (>100 ps) time scales.     

Influence of proline residues in transmembrane helix packing

Integral membrane proteins often contain proline residues in their alpha-helical transmembrane (TM) fragments, which may strongly influence their folding and association. Pro-scanning mutagenesis of the helical domain of glycophorin A (GpA) showed that replacement of the residues located at the center abrogates helix packing while substitution of the residues forming the ending helical turns allows dimer formation. Synthetic TM peptides revealed that a point mutation of one of the residues of the dimerization motif (L75P) located at the N-terminal helical turn of the GpA TM fragment, adopts a secondary structure and oligomeric state similar to the wild-type sequence in detergents. In addition, both glycosylation mapping in biological membranes and molecular dynamics showed that the presence of a proline residue at the lipid/water interface has as an effect the extension of the helical end. Thus, helix packing can be an important factor that determines appearance of proline in TM helices. Membrane proteins might accumulate proline residues at the two ends of their TM segments in order to modulate the exposition of key amino acid residues at the interface for molecular recognition events while allowing stable association and native folding.     

Surface binding of alamethicin stabilizes its helical structure: molecular dynamics simulations.

Alamethicin is an amphipathic alpha-helical peptide that forms ion channels. An early event in channel formation is believed to be the binding of alamethicin to the surface of a lipid bilayer. Molecular dynamics simulations are used to compare the structural and dynamic properties of alamethicin in water and alamethicin bound to the surface of a phosphatidylcholine bilayer. The bilayer surface simulation corresponded to a loosely bound alamethicin molecule that interacted with lipid headgroups but did not penetrate the hydrophobic core of the bilayer. Both simulations started with the peptide molecule in an alpha-helical conformation and lasted 2 ns. In water, the helix started to unfold after approximately 300 ps and by the end of the simulation only the N-terminal region of the peptide remained alpha-helical and the molecule had collapsed into a more compact form. At the surface of the bilayer, loss of helicity was restricted to the C-terminal third of the molecule and the rod-shaped structure of the peptide was retained. In the surface simulation about 10% of the peptide/water H-bonds were replaced by peptide/lipid H-bonds. These simulations suggest that some degree of stabilization of an amphipathic alpha-helix occurs at a bilayer surface even without interactions between hydrophobic side chains and the acyl chain core of the bilayer.     

Interactions of alpha-helices with lipid bilayers: a review of simulation studies

Membrane proteins, of which the majority seem to contain one or more alpha-helix, constitute approx. 30% of most genomes. A complete understanding of the nature of helix/bilayer interactions is necessary for an understanding of the structural principles underlying membrane proteins. This review describes computer simulation studies of helix/bilayer interactions. Key experimental studies of the interactions of alpha-helices and lipid bilayers are briefly reviewed. Surface associated helices are found in some membrane-bound enzymes (e.g. prostaglandin synthase), and as stages in the mechanisms of antimicrobial peptides and of pore-forming bacterial toxins. Transmembrane alpha-helices are found in most integral membrane proteins, and also in channels formed by amphipathic peptides or by bacterial toxins. Mean field simulations, in which the lipid bilayer is approximated as a hydrophobic continuum, have been used in studies of membrane-active peptides (e.g. alamethicin, melittin, magainin and dermaseptin) and of simple membrane proteins (e.g. phage Pf1 coat protein). All atom molecular dynamics simulations of fully solvated bilayers with transmembrane helices have been applied to: the constituent helices of bacteriorhodopsin; peptide-16 (a simple model TM helix); and a number of pore-lining helices from ion channels. Surface associated helices (e.g. melittin and dermaseptin) have been simulated, as have alpha-helical bundles such as bacteriorhodopsin and alamethicin. From comparison of the results from the two classes of simulation, it emerges that a major theoretical challenge is to exploit the results of all atom simulations in order to improve the mean field approach.     

A prokaryotic glutamate receptor: homology modelling and molecular dynamics simulations of GluR0

GluR0 is a prokaryotic homologue of mammalian glutamate receptors that forms glutamate-activated, potassium-selective ion channels. The topology of its transmembrane (TM) domain is similar to that of simple potassium channels such as KcsA. Two plausible alignments of the sequence of the TM domain of GluR0 with KcsA are possible, differing in the region of the P helix. We have constructed homology models based on both alignments and evaluated them using 6 ns duration molecular dynamics simulations in a membrane-mimetic environment. One model, in which an insertion in GluR0 relative to KcsA is located in the loop between the M1 and P helices, is preferred on the basis of lower structural drift and maintenance of the P helix conformation during simulation. This model also exhibits inter-subunit salt bridges that help to stabilise the TM domain tetramer. During the simulation, concerted K(+) ion-water movement along the selectivity filter is observed, as is the case in simulations of KcsA. K(+) ion exit from the central cavity is associated with opening of the hydrophobic gate formed by the C-termini of the M2 helices. In the intact receptor the opening of this gate will be controlled by interactions with the extramembranous ligand-binding domains.     

Molecular dynamics simulations predict a tilted orientation for the helical region of dynorphin A(1-17) in dimyristoylphosphatidylcholine bilayers

The structural properties of the endogenous opioid peptide dynorphin A(1-17) (DynA), a potential analgesic, were studied with molecular dynamics simulations in dimyristoylphosphatidylcholine bilayers. Starting with the known NMR structure of the peptide in dodecylphosphocholine micelles, the N-terminal helical segment of DynA (encompassing residues 1-10) was initially inserted in the bilayer in a perpendicular orientation with respect to the membrane plane. Parallel simulations were carried out from two starting structures, systems A and B, that differ by 4 A in the vertical positioning of the peptide helix. The complex consisted of approximately 26,400 atoms (dynorphin + 86 lipids + approximately 5300 waters). After >2 ns of simulation, which included >1 ns of equilibration, the orientation of the helical segment of DynA had undergone a transition from parallel to tilted with respect to the bilayer normal in both the A and B systems. When the helix axis achieved a approximately 50 degrees angle with the bilayer normal, it remained stable for the next 1 ns of simulation. The two simulations with different starting points converged to the same final structure, with the helix inserted in the bilayer throughout the simulations. Analysis shows that the tilted orientation adopted by the N-terminal helix is due to specific interactions of residues in the DynA sequence with phospholipid headgroups, water, and the hydrocarbon chains. Key elements are the "snorkel model"-type interactions of arginine side chains, the stabilization of the N-terminal hydrophobic sequence in the lipid environment, and the specific interactions of the first residue, Tyr. Water penetration within the bilayer is facilitated by the immersed DynA, but it is not uniform around the surface of the helix. Many water molecules surround the arginine side chains, while water penetration near the helical surface formed by hydrophobic residues is negligible. A mechanism of receptor interaction is proposed for DynA, involving the tilted orientation observed from these simulations of the peptide in the lipid bilayer.     

An N-terminal fragment of barnase has residual helical structure similar to that in a refolding intermediate.

A fragment of barnase comprising amino acids 1 to 36 (B(1-36)) that encompasses the region containing the two large helices (residues 6-18 and 26-34) of the native protein has been obtained by cleavage of the barnase mutant Val36----Met with cyanogen bromide. The circular dichroism (c.d.) spectrum of B(1-36) in the far ultraviolet indicates that the fragment is only weakly structured in water at neutral pH. The two-dimensional 1H nuclear magnetic resonance spectrum of B(1-36) shows, however, that a fraction of the population does have helical structure, spanning amino acid residues 8 to 18. B(1-36) becomes more helical in 35% trifluoroethanol. This is indicated by the c.d. spectrum and the increase from 6.6 to 7.0 in the pKa of His18, which is known to interact with the dipole of helix 6-18 in native barnase. The helical region of B(1-36) in 35% trifluoroethanol extends to residue 6. It is calculated from extrapolation of a trifluoroethanol titration of the ellipticity at 222 nm that B(1-36) exhibits in water approximately 6% of helical structure, calculated for a 36 residue alpha-helical peptide. This corresponds to approximately 20% of that expected for an 11-residue alpha-helical region. In trifluoroethanol, c.d. measurements indicate that approximately 30% of the 36-residue peptide is helical. It has been shown from extensive studies of the refolding of barnase that there is a folding intermediate that contains residues 8 to 18 in a helical conformation and that residue 6 is mainly unfolded. The experiments on the conformation of B(1-36) show that a small, but significant fraction, of its population in water adopts the conformation of the major alpha-helix during the barnase folding pathway, in the absence of tertiary interactions. Thus, in the folding of native barnase, secondary structure formation can precede the docking of the major alpha-helix onto the beta-sheet.     

Molecular dynamics of individual alpha-helices of bacteriorhodopsin in dimyristol phosphatidylocholine. I. Structure and dynamics.

Understanding the role of the lipid bilayer in membrane protein structure and dynamics is needed for tertiary structure determination methods. However, the molecular details are not well understood. Molecular dynamics computer calculations can provide insight into these molecular details of protein:lipid interactions. This paper reports on 10 simulations of individual alpha-helices in explicit lipid bilayers. The 10 helices were selected from the bacteriorhodopsin structure as representative alpha-helical membrane folding components. The bilayer is constructed of dimyristoyl phosphatidylcholine molecules. The only major difference between simulations is the primary sequence of the alpha-helix. The results show dramatic differences in motional behavior between alpha-helices. For example, helix A has much smaller root-mean-squared deviations than does helix D. This can be understood in terms of the presence of aromatic residues at the interface for helix A that are not present in helix D. Additional motions are possible for the helices that contain proline side chains relative to other amino acids. The results thus provide insight into the types of motion and the average structures possible for helices within the bilayer setting and demonstrate the strength of molecular simulations in providing molecular details that are not directly visualized in experiments.     

NMR-Based Simulation Studies of Pf1 Coat Protein in Explicit Membranes

As time- and ensemble-averaged measures, NMR observables contain information about both protein structure and dynamics. This work represents a computational study to extract such information for membrane proteins from orientation-dependent NMR observables: solid-state NMR chemical shift anisotropy and dipolar coupling, and solution NMR residual dipolar coupling. We have performed NMR-restrained molecular dynamics simulations to refine the structure of the membrane-bound form of Pf1 coat protein in explicit lipid bilayers using the recently measured chemical shift anisotropy, dipolar coupling, and residual dipolar coupling data. From the simulations, we have characterized detailed protein-lipid interactions and explored the dynamics. All simulations are stable and the NMR restraints are well satisfied. The C-terminal transmembrane (TM) domain of Pf1 finds its optimal position in the membrane quickly (within 6 ns), illustrating efficient solvation of TM domains in explicit bilayer environments. Such rapid convergence also leads to well-converged interaction patterns between the TM helix and the membrane, which clearly show the interactions of interfacial membrane-anchoring residues with the lipids. For the N-terminal periplasmic helix of Pf1, we identify a stable, albeit dynamic, helix orientation parallel to the membrane surface that satisfies the amphiphatic nature of the helix in an explicit lipid bilayer. Such detailed information cannot be obtained solely from NMR observables. Therefore, the present simulations illustrate the usefulness of NMR-restrained MD refinement of membrane protein structure in explicit membranes.     

NMR-Based Simulation Studies of Pf1 Coat Protein in Explicit Membranes

As time- and ensemble-averaged measures, NMR observables contain information about both protein structure and dynamics. This work represents a computational study to extract such information for membrane proteins from orientation-dependent NMR observables: solid-state NMR chemical shift anisotropy and dipolar coupling, and solution NMR residual dipolar coupling. We have performed NMR-restrained molecular dynamics simulations to refine the structure of the membrane-bound form of Pf1 coat protein in explicit lipid bilayers using the recently measured chemical shift anisotropy, dipolar coupling, and residual dipolar coupling data. From the simulations, we have characterized detailed protein-lipid interactions and explored the dynamics. All simulations are stable and the NMR restraints are well satisfied. The C-terminal transmembrane (TM) domain of Pf1 finds its optimal position in the membrane quickly (within 6 ns), illustrating efficient solvation of TM domains in explicit bilayer environments. Such rapid convergence also leads to well-converged interaction patterns between the TM helix and the membrane, which clearly show the interactions of interfacial membrane-anchoring residues with the lipids. For the N-terminal periplasmic helix of Pf1, we identify a stable, albeit dynamic, helix orientation parallel to the membrane surface that satisfies the amphiphatic nature of the helix in an explicit lipid bilayer. Such detailed information cannot be obtained solely from NMR observables. Therefore, the present simulations illustrate the usefulness of NMR-restrained MD refinement of membrane protein structure in explicit membranes.     

Proline-induced distortions of transmembrane helices

Proline residues in the transmembrane (TM) alpha-helices of integral membrane proteins have long been suspected to play a key role for helix packing and signal transduction by inducing regions of helix distortion and/or dynamic flexibility (hinges). In this study we try to characterise the effect of proline on the geometric properties of TM alpha-helices. We have examined 199 transmembrane alpha-helices from polytopic membrane proteins of known structure. After examining the location of proline residues within the amino acid sequences of TM helices, we estimated the helix axes either side of a hinge and hence identified a hinge residue. This enabled us to calculate helix kink and swivel angles. The results of this analysis show that proline residues occur with a significant concentration in the centre of sequences of TM alpha-helices. In this location, they may induce formation of molecular hinges, located on average about four residues N-terminal to the proline residue. A superposition of proline-containing TM helices structures shows that the distortion induced is anisotropic and favours certain relative orientations (defined by helix kink and swivel angles) of the two helix segments.     

Conformational and sequence signatures in beta helix proteins

beta helix proteins are characterized by a repetitive fold, in which the repeating unit is a beta-helical coil formed by three strand segments linked by three loop segments. Using a data set of left- and right-handed beta helix proteins, we have examined conformational features at equivalent positions in successive coils. This has provided insights into the conformational rules that the proteins employ to fold into beta helices. Left-handed beta helices attain their equilateral prism fold by incorporating "corners" with the conformational sequence P(II)-P(II)-alpha(L)-P(II), which imposes sequence restrictions, resulting in the first and third P(II) residues often being G and a small, uncharged residue (V, A, S, T, C), respectively. Right-handed beta helices feature mid-sized loops (4, 5, or 6 residues) of conserved conformation, but not of conserved sequence; they also display an alpha-helical residue at the C-terminal end of L2 loops. Backbone conformational parameters (phi,psi) that permit the formation of continuous, loopless beta helices (Perutz nanotubes) have also been investigated.     

Arginine in Membranes: The Connection Between Molecular Dynamics Simulations and Translocon-Mediated Insertion Experiments

Several laboratories have carried out molecular dynamics (MD) simulations of arginine interactions with lipid bilayers and found that the energetic cost of placing arginine in lipid bilayers is an order of magnitude greater than observed in molecular biology experiments in which Arg-containing transmembrane helices are inserted across the endoplasmic reticulum membrane by the Sec61 translocon. We attempt here to reconcile the results of the two approaches. We first present MD simulations of guanidinium groups alone in lipid bilayers, and then, to mimic the molecular biology experiments, we present simulations of hydrophobic helices containing single Arg residues at different positions along the helix. We discuss the simulation results in the context of molecular biology results and show that the energetic discrepancy is reduced, but not eliminated, by considering free energy differences between Arg at the interface and at the center of the model helices. The reduction occurs because Arg snorkeling to the interface prevents Arg from residing in the bilayer center where the energetic cost of desolvation is highest. We then show that the problem with MD simulations is that they measure water-to-bilayer free energies, whereas the molecular biology experiments measure the energetics of partitioning from translocon to bilayer, which raises the fundamental question of the relationship between water-to-bilayer and water-to-translocon partitioning. We present two thermodynamic scenarios as a foundation for reconciliation of the simulation and molecular biology results. The simplest scenario is that translocon-to-bilayer partitioning is independent of water-to-bilayer partitioning; there is no thermodynamic cycle connecting the two paths.     

Conformational dynamics of helix S6 from Shaker potassium channel: simulation studies

Prolines in transmembrane (TM) alpha-helices are believed to play an important structural and/or functional role in membrane proteins. At a structural level a proline residue distorts alpha-helical structure due to the loss of at least one stabilizing backbone hydrogen bond, and introduces flexibility in the helix that may result in substantial kink and swivel motions about the effective "hinge." At a functional level, for example in Kv channels, it is believed that proline-induced molecular hinges may have a direct role in gating, i.e., the conformational change linked to opening/closing the channel to movement of ions. In this article we study the conformational dynamics of the S6 TM helix from of the Kv channel Shaker, which possesses the motif PVP--a motif that is conserved in Kv channels. We perform multiple molecular dynamics simulations of single S6 helices in a membrane-mimetic environment in order to effectively map the kink-swivel conformational space of the protein, exploiting the ability of multiple simulations to achieve greater sampling. We show that the presence of proline locally perturbs the helix, disrupting local dihedral angles and producing local twist and unwinding in the region of the hinge--an effect that is relaxed with distance from the PVP motif. We furthermore show that motions about the hinge are highly anisotropic, reflecting a preferred region of kink-swivel conformation space that may have implications for the gating process.     

Alpha-hairpin stability and folding of transmembrane segments

Molecular Dynamics (MD) simulations at low dielectric constant have been carried out for peptides matching the double spanning segments of transmembrane proteins. Different folding dynamics have been observed. The peptides folded into the stable helix-turn-helix conformation-alpha-hairpin-with antiparallel-oriented strands or unstable alpha-hairpin conformation that unfolded later into the straight helical structure. The peptide having flexible residues in the TM helices often misfolded into a tangled structure that can be avoided by restricting the flexibility of these residues. General conclusions can be drawn from the observed folding dynamics. The stability and folding of some double spanning transmembrane fragments are self-assembling. The following and/or neighboring peptide chains of the protein may support the stability of the hairpin structure of other fragments. The stability of the TM helices containing flexible residues could be maintained due to contacts with neighboring TM segments.     

Conformational and dynamics simulation study of antimicrobial peptide hedistin—heterogeneity of its helix–turn–helix motif

Hedistin is an antimicrobial peptide isolated from the coelomocytes of Nereis diversicolor, possessing activity against a large spectrum of bacteria including the methicillin resistant Staphylococcus aureus and Vibrio alginolyticus. The three-dimensional structure of hedistin in both aqueous solution and deuterated dodecylphosphocholine (DPC) micelles was examined using circular dichroism (CD) and nuclear magnetic resonance (NMR) techniques. And, the early events of the antibacterial process of hedistin were simulated using palmitoyl-oleoyl-phophatidylcholine (POPC) lipid bilayers and molecular dynamics (MD) simulation methods. Hedistin lacks secondary structure in aqueous solution, however, in DPC micelles, it features with a heterogeneous helix–turn–helix moiety and exhibits obvious amphipathic nature. The turn region (residues Val9–Thr12) in the moiety is a four-residue hinge, lying in between the first N-terminal α-helix (residues Leu5–Lys8) and the second α-helix (residues Val13–Ala17) regions and causing an ～ 120° angle between the axes of the two helices. The segmental and nonlinear nature of hedistin structure is referred to as the heterogeneity of its helix–turn–helix motif which was found to be corresponding to a kind of discrete dynamics behavior, herein coined as its dynamical heterogeneity, at the early stage (0–50 ns) of the MD simulations. That is, the first helix segment, prior to (at 310 K) or following (at 363 K) the second helix, binds to the lipid head-group region and subsequently permeates into the hydrophobic lipid tail region, and the hinge is the last portion entering the lipid environment. This result implies that hedistin may adopt a “carpet” model action when disrupting bacterial membrane.     

Structure–function relationships of the human bitter taste receptor hTAS2R1: insights from molecular modeling studies

Bitter taste receptors (T2Rs) belong to G-protein-coupled receptors (GPCRs). Despite extensive studies, the precise mechanisms of GPCR activation are still poorly understood. In this study, the models of the human bitter taste receptor hTAS2R1 alone and in complex with various ligands were constructed on the basis of template-based modeling and molecular docking. Then these models were subjected to all-atom molecular dynamics (MD) simulations in explicit lipid bilayers. The binding pocket of hTAS2R1 is mainly formed by transmembrane helix (TM) III, TM V, TM VI, and TM VII. Most of the residues contributing to ligand binding are positionally conserved comparing with other hTAS2Rs. By comparing the final conformations obtained by extensive MD simulations, we identified the changes in the transmembrane helices and the intra- and extracellular loops, which were expected to initiate the activation of the receptor. The intracellular loop II (ICL2) and TM III were found to play prominent roles in the process of activation. We proposed that a set of interactions between the aromatic Phe115 in the middle of ICL2 and three residues (Tyr103, Lys106, and Val107) at the cytoplasmic end of TM III may serve as a conformational switch of hTAS2R1 activation. All of the residues involved in the switch are highly conserved among T2Rs. This indicates that the control switch we proposed may be universal in T2Rs. Besides, our results also suggest that the formation of a short helical segment in ICL2 may be necessary for the activation of hTAS2R1.