Computational study of the transmembrane domain of the acetylcholine receptor

The nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel protein whose transmembrane domain (TM-domain) is believed to be responsible for channel gating via a hydrophobic effect. In this work, we perform molecular dynamics and Brownian dynamics simulations to investigate the effect of transmembrane potential on the conformation and water occupancy of TM-domain, and the resulting ion permeation events. The results show that the behavior of the hydrophobic gate is voltage-dependent. Large hyperpolarized membrane potential can change the conformation of TM-domain and water occupancy in this region, which may enable ion conduction. An electrostatic gating mechanism is also proposed from our simulations, which seems to play a role in addition to the well-known hydrophobic effect.     

Homology modeling and molecular dynamics simulations of transmembrane domain structure of human neuronal nicotinic acetylcholine receptor

A three-dimensional model of the transmembrane domain of a neuronal-type nicotinic acetylcholine receptor (nAChR), (alpha4)2(beta2)3, was constructed from a homology structure of the muscle-type nAChR recently determined by cryo-electron microscopy. The neuronal channel model was embedded in a fully hydrated DMPC lipid bilayer, and molecular-dynamics simulations were performed for 5 ns. A comparative analysis of the neuronal- versus muscle-type nAChR models revealed many conserved pore-lining residues, but an important difference was found near the periplasmic mouth of the pore. A flickering salt-bridge of alpha4-E266 with its adjacent beta2-K260 was observed in the neuronal-type channel during the course of the molecular-dynamics simulations. The narrowest region, with a pore radius of approximately 2 A formed by the salt-bridges, does not seem to be the restriction site for a continuous water passage. Instead, two hydrophobic rings, formed by alpha4-V259, alpha4-L263, and the homologous residues in the beta2-subunits, act as the gates for water flow, even though the region has a slightly larger pore radius. The model offers new insight into the water transport across the (alpha4)2(beta2)3 nAChR channel, and may lead to a better understanding of the structures, dynamics, and functions of this family of ion channels.     

Structure and dynamics of the gammaM4 transmembrane domain of the acetylcholine receptor in lipid bilayers: insights into receptor assembly and function

A 28-mer peptide (gammaM4) corresponding to the fourth transmembrane segment of the nicotinic acetylcholine receptor (AChR) gamma-subunit, with a single tryptophan residue (Trp6), was reconstituted into lipid bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), loaded with either high or low amounts of cholesterol, i.e., in the conjugated liquid-ordered and liquid-disordered phases, respectively, at room temperature. By making use of the Trp intrinsic fluorescence, both steady-state and time-resolved fluorescence techniques were employed, namely, red-edge excitation shift effect, decay-associated spectra (DAS), and time-resolved anisotropy. The results obtained here, together with previous studies on the same reconstituted peptide, indicate that: (i) Trp6 is strongly anchored in the bilayer with a defined transverse location; (ii) the modifications in the measured DAS are related to the complex result of a self-quenching process on the decay parameters; (iii) the wobbling movement of the indole moiety of Trp6 is fast but severely restricted in amplitude; and, (iv) in the liquid-ordered phase, the bilayer properties and the tilt angle of the peptide enhance peptide-peptide interactions, with the formation of peptide rich patches and possibly some anti-parallel helix-helix aggregates, showing different dynamics from that of the peptide in the liquid-disordered phase where the peptide is randomly distributed.     

Tryptophan scanning mutagenesis of the gammaM4 transmembrane domain of the acetylcholine receptor from Torpedo californica

The periodicity of structural and functional effects induced by tryptophan scanning mutagenesis has been successfully used to define function and secondary structure of various transmembrane domains of the acetylcholine receptor of Torpedo californica. We expand the tryptophan scanning of the AchR of T. californica to the gammaM4 transmembrane domain (gammaTM4) by introducing tryptophan, at residues 451-462, along the gammaTM4. Wild type (WT) and mutant AChR were expressed in Xenopus laevis oocytes. Using [(125)I]alpha-bungarotoxin binding assays and voltage clamp, we determined that the nAChR expression, EC(50), and Hill coefficient values for WT are 1.8 +/- 0.4 fmol, 30.3 +/- 1.1 microM, and 1.8 +/- 0.3, respectively. Mutations L456W, F459W, and G462W induce a significant increase in nAChR expression (2.8 +/- 0.5, 3.6 +/- 0.6, and 3.0 +/- 0.5 fmol, respectively) when compared with WT. These data suggest that these residues are important for AChR oligomerization. Mutations A455W, L456W, F459W, and G462W result in a significant decrease in EC(50) (19.5 +/- 1.7, 11.4 +/- 0.7, 16.4 +/- 3.8, and 19.1 +/- 2.6 microM, respectively), thus suggesting a gain in function when compared with WT. In contrast, mutation L458W induced an increase in EC(50) (42.8 +/- 6.8 microM) or loss in function when compared with WT. The Hill coefficient values were the same for WT and all of the mutations studied. The periodicity in function (EC(50) and macroscopic peak current) and nAChR expression reveals an average of 3.3 and 3.0 amino acids respectively, thus suggesting a helical secondary structure for the gammaTM4.     

Scanning mutagenesis of transmembrane domain 3 of the m1 muscarinic acetylcholine receptor

Scanning mutagenesis of transmembrane domain 3 of the M1 muscarinic acetylcholine receptor has revealed a highly-differentiated α-helical structure. Lipid-facing residues are distinguished from a patch of residues which selectively stabilise the ground state of the receptor, and from a band of amino acids extending the full length of the helix, which contribute to the active agonist-receptor-G protein complex. The most important residues are strongly conserved in the GPCR superfamily.     

Functional analysis of transmembrane domain 2 of the M1 muscarinic acetylcholine receptor

Ala substitution scanning mutagenesis has been used to probe the functional role of amino acids in transmembrane (TM) domain 2 of the M1 muscarinic acetylcholine receptor, and of the highly conserved Asn43 in TM1. The mutation of Asn43, Asn61, and Leu64 caused an enhanced ACh affinity phenotype. Interpreted using a rhodopsin-based homology model, these results suggest the presence of a network of specific contacts between this group of residues and Pro415 and Tyr418 in the highly conserved NPXXY motif in TM7 that exhibit a similar mutagenic phenotype. These contacts may be rearranged or broken when ACh binds. D71A, like N414A, was devoid of signaling activity. We suggest that formation of a direct hydrogen bond between the highly conserved side chains of Asp71 and Asn414 may be a critical feature stabilizing the activated state of the M1 receptor. Mutation of Leu67, Ala70, and Ile74 also reduced the signaling efficacy of the ACh-receptor complex. The side chains of these residues are modeled as an extended surface that may help to orient and insulate the proposed hydrogen bond between Asp71 and Asn414. Mutation of Leu72, Gly75, and Met79 in the outer half of TM2 primarily reduced the expression of functional receptor binding sites. These residues may mediate contacts with TM1 and TM7 that are preserved throughout the receptor activation cycle. Thermal inactivation measurements confirmed that a reduction in structural stability followed the mutation of Met79 as well as Asp71.     

Structure of the first transmembrane domain of the neuronal acetylcholine receptor beta2 subunit

The recent cryoelectron microscopy structure of the Torpedo nicotinic acetylcholine receptor (nAChR) at 4-Å resolution shows long helices for all transmembrane (TM) domains. This is in disagreement with several previous reports that the first TM domain of nAChR and other Cys-loop receptors are not entirely helical. In this study, we determined the structure and backbone dynamics of an extended segment encompassing the first TM domain (TM1e) of nAChR β₂ subunit in dodecylphosphocholine micelles, using solution-state NMR and circular dichroism (CD) spectroscopy. Both CD and NMR results show less helicity in TM1e than in Torpedo nAChR structure (Protein Data Bank: 2BG9). The helical ending residues at the C-terminus are the same in the TM1e NMR structure and the Torpedo nAChR structure, but the helical starting residue (I-217) in TM1e is seven residues closer to the C-terminus. Interestingly, the helical starting residue is two residues before the highly conserved P-219, in accordance with the hypothesis that proline causes helical distortions at three residues preceding it. The NMR relaxation measurements show a dynamics pattern consistent with TM1e structure. The substantial nonhelical content adds greater flexibilities to TM1e, thereby implicating a different molecular basis for nAChR function compared to a longer and more rigid helical TM1.     

A transmembrane peptide from the human EGF receptor: behaviour of the cytoplasmic juxtamembrane domain in lipid bilayers

Solid state (2)H NMR spectroscopy was employed to study peptides related to the transmembrane domain of the human epidermal growth factor receptor, for insight into the interaction of its cytoplasmic juxtamembrane domain with the membrane surface. Since such receptors have clusters of (+)charged amino acids in this region, the effect of (-)charged phosphatidylserine at the concentration found naturally in the cytoplasmic leaflet (15 mol%) was considered. Each peptide contained 34 amino acids, which included the hydrophobic 23 amino acid stretch thought to span the membrane and a ten amino acid segment beyond the 'cytoplasmic' surface. Non-perturbing deuterium probe nuclei were located within alanine side chains in intramembranous and extramembranous portions. (2)H NMR spectra were recorded at 35 degrees C and 65 degrees C in fluid lipid bilayers consisting of (zwitterionic) 1-palmitoyl-2-oleoylphosphatidylcholine, with and without 15 mol% (anionic) phosphatidylserine. The cationic extramembranous portion of the receptor backbone was found to be highly rotationally mobile on a time scale of 10(-4)-10(-5) s in both types of membrane - as was the alpha-helical intramembranous portion. Deuterium nuclei in alanine side chains (-CD(3)) detected modest changes in peptide backbone orientation and/or dynamics related to the presence of 1-stearoyl-2-oleoylphosphatidylserine: in the case of the extramembranous portion of the peptide these seemed related to lipid charge. Temperature effects on the peptide backbone external to the membrane were qualitatively different from effects on the helical transmembrane domain - likely reflecting the different physical constraints on these peptide regions and the greater flexibility of the extramembranous domain. Effects related to lipid charge could be detected in the spectrum of CD(3) groups on the internally mobile side chain of Val(650), six residues beyond the membrane surface.     

Conformation-sensitive steroid and fatty acid sites in the transmembrane domain of the nicotinic acetylcholine receptor

The mechanism by which some hydrophobic molecules such as steroids and free fatty acids (FFA) act as noncompetitive inhibitors of the nicotinic acetylcholine receptor (AChR) is still not known. In the present work, we employ F?rster resonance energy transfer (FRET) between the intrinsic fluorescence of membrane-bound Torpedo californica AChR and the fluorescent probe Laurdan using the decrease in FRET efficiency (E) caused by steroids and FFA to identify potential sites of these hydrophobic molecules. Structurally different steroids produced similar changes (DeltaE) in FRET, and competition studies between them demonstrate that they occupy the same site(s). They also share their binding site(s) with FFA. Furthermore, the FRET conditions define the location of the sites at the lipid-protein interface. Endogenous production of FFA by controlled phospholipase A2 enzymatic digestion of membrane phospholipids yielded DeltaE values similar to those obtained by addition of exogenous ligand. This finding, together with the preservation of the sites in membranes subjected to controlled proteolysis of the extracellular AChR moiety with membrane-impermeable proteinase K, further refines the topology of the sites at the AChR transmembrane domain. Agonist-induced desensitization resulted in the masking of the sites observed in the absence of agonist, thus demonstrating the conformational sensitivity of FFA and steroid sites in the AChR.     

Cholesterol modulates the organization of the gammaM4 transmembrane domain of the muscle nicotinic acetylcholine receptor

A 28-mer gammaM4 peptide, obtained by solid-state synthesis and corresponding to the fourth transmembrane segment of the nicotinic acetylcholine receptor gamma-subunit, possesses a single tryptophan residue (Trp453), making it an excellent model for studying peptide-lipid interactions in membranes by fluorescence spectroscopy. The gammaM4 peptide was reconstituted with synthetic lipids (vesicles of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, i.e., POPC) rich and poor in cholesterol and analyzed using steady-state and time-resolved fluorescence techniques. The decrease in gammaM4 intrinsic fluorescence lifetime observed upon incorporation into a cholesterol-rich lo phase could be rationalized on the basis of a dynamic self-quenching owing to the formation of peptide-rich patches in the membrane. This agrees with the low F?rster type resonance energy transfer efficiency from the Trp453 residue to the fluorescent cholesterol analog, dehydroergosterol, in the lo phase. In the absence of cholesterol the gammaM4 nicotinic acetylcholine receptor peptide is randomly distributed in the POPC bilayer with its hydrophobic moiety matching the membrane thickness, whereas in the presence of cholesterol the increase in the membrane thickness and variation of the material properties favor the formation of peptide-enriched patches, i.e., interhelix interaction energy is essential for obtaining a stabilized structure. Thus, the presence of a cholesterol-rich, ordered POPC phase drives the organization of peptide-enriched patches, in which the gammaM4 peptide occupies approximately 30% of the patch area.     

Subunit-selective role of the M3 transmembrane domain of the nicotinic acetylcholine receptor in channel gating

The nicotinic acetylcholine receptor (AChR) can be either hetero-pentameric, composed of alpha and non-alpha subunits, or homo-pentameric, composed of alpha7 subunits. To explore the subunit-selective contributions of transmembrane domains to channel gating we analyzed single-channel activity of chimeric muscle AChRs. We exchanged M3 between alpha1 and epsilon or alpha7 subunits. The replacement of M3 in alpha1 by epsilonM3 significantly alters activation properties. Channel activity appears as bursts of openings whose durations are 20-fold longer than those of wild-type AChRs. In contrast, 7-fold briefer openings are observed in AChRs containing the reverse epsilon chimeric subunit. The duration of the open state decreases with the increase in the number of alpha1M3 segments, indicating additive contributions of M3 of all subunits to channel closing. Each alpha1M3 segment decreases the energy barrier of the closing process by approximately 0.8 kcal/mol. Partial chimeric subunits show that small stretches of the M3 segment contribute additively to the open duration. The replacement of alpha1 sequence by alpha7 in M3 leads to 3-fold briefer openings whereas in M1 it leads to 10-fold prolonged openings, revealing that the subunit-selective role is unique to each transmembrane segment.     

Subunit-selective role of the M3 transmembrane domain of the nicotinic acetylcholine receptor in channel gating

The nicotinic acetylcholine receptor (AChR) can be either hetero-pentameric, composed of α and non-α subunits, or homo-pentameric, composed of α7 subunits. To explore the subunit-selective contributions of transmembrane domains to channel gating we analyzed single-channel activity of chimeric muscle AChRs. We exchanged M3 between α1 and ? or α7 subunits. The replacement of M3 in α1 by ?M3 significantly alters activation properties. Channel activity appears as bursts of openings whose durations are 20-fold longer than those of wild-type AChRs. In contrast, 7-fold briefer openings are observed in AChRs containing the reverse ? chimeric subunit. The duration of the open state decreases with the increase in the number of α1M3 segments, indicating additive contributions of M3 of all subunits to channel closing. Each α1M3 segment decreases the energy barrier of the closing process by ∼ 0.8 kcal/mol. Partial chimeric subunits show that small stretches of the M3 segment contribute additively to the open duration. The replacement of α1 sequence by α7 in M3 leads to 3-fold briefer openings whereas in M1 it leads to 10-fold prolonged openings, revealing that the subunit-selective role is unique to each transmembrane segment.     

NMR structure and dynamics of a designed water-soluble transmembrane domain of nicotinic acetylcholine receptor

The nicotinic acetylcholine receptor (nAChR) is an important therapeutic target for a wide range of pathophysiological conditions, for which rational drug designs often require receptor structures at atomic resolution. Recent proof-of-concept studies demonstrated a water-solubilization approach to structure determination of membrane proteins by NMR (Slovic et al., PNAS, 101: 1828-1833, 2004; Ma et al., PNAS, 105: 16537-42, 2008). We report here the computational design and experimental characterization of WSA, a water-soluble protein with ~83% sequence identity to the transmembrane (TM) domain of the nAChR α1 subunit. Although the design was based on a low-resolution structural template, the resulting high-resolution NMR structure agrees remarkably well with the recent crystal structure of the TM domains of the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel (GLIC), demonstrating the robustness and general applicability of the approach. NMR T(2) dispersion measurements showed that the TM2 domain of the designed protein was dynamic, undergoing conformational exchange on the NMR timescale. Photoaffinity labeling with isoflurane and propofol photolabels identified a common binding site in the immediate proximity of the anesthetic binding site found in the crystal structure of the anesthetic-GLIC complex. Our results illustrate the usefulness of high-resolution NMR analyses of water-solubilized channel proteins for the discovery of potential drug binding sites.     

Characterization of the interacting domain of the HIV-1 fusion peptide with the transmembrane domain of the T-cell receptor

HIV infection is initiated by the fusion of the viral membrane with the target T-cell membrane. The HIV envelope glycoprotein, gp41, contains a fusion peptide (FP) in the N terminus that functions together with other gp41 domains to fuse the virion with the host cell membrane. We recently reported that FP co-localizes with CD4 and T-cell receptor (TCR) molecules, co-precipitates with TCR, and inhibits antigen-specific T-cell proliferation and pro-inflammatory cytokine secretion. Molecular dynamic simulation implicated an interaction between an alpha-helical transmembrane domain (TM) of the TCRalpha chain (designated CP) and the beta-sheet 5-13 region of the 16 N-terminal amino acids of FP (FP(1-16)). To correlate between the theoretical prediction and experimental data, we synthesized a series of mutants derived from the interacting motif GALFLGFLG stretch (FP(5-13)) and investigated them structurally and functionally. The data reveal a direct correlation between the beta-sheet structure of FP(5-13) and its mutants and their ability to interact with CP and induce immunosuppressive activity; the phenylalanines play an important role. Furthermore, studies with fluorescently labeled peptides revealed that this interaction leads to penetration of the N terminus of FP and its active analogues into the hydrophobic core of the membrane. A detailed understanding of the molecular interactions mediating the immunosuppressive activity of the FP(5-13) motif should facilitate evaluating its contribution to HIV pathology and its exploitation as an immunotherapeutic tool.     

The transmembrane domain of Neu in a lipid bilayer: molecular dynamics simulations

The results of full-atom molecular dynamics simulations of the transmembrane domains (TMDs) of both native, and Glu⁶⁶⁴-mutant (either protonated or unprotonated) Neu in an explicit fully hydrated dimyristoylphosphatidylcholine (DMPC) lipid bilayer are presented. For the native TMD peptide, a 10.05 ns trajectory was collected, while for the mutant TMD peptides 5.05 ns trajectories were collected for each. The peptides in all three simulations display stable predominantly -helical hydrogen bonding throughout the trajectories. The only significant exception occurs near the C-terminal end of the native and unprotonated mutant TMDs just outside the level of the lipid headgroups, where -helical hydrogen bonding develops, introducing a kink in the backbone structure. However, there is no indication of the formation of a bulge within the hydrophobic region of either native or mutant peptides. Over the course of the simulation of the mutant peptide, it is found that a significant number of water molecules penetrate the hydrophobic region of the surrounding lipid molecules, effectively hydrating Glu⁶⁶⁴. If the energy cost of such water penetration is significant enough, this may be a factor in the enhanced dimerization affinity of Glu⁶⁶⁴-mutant Neu.     

Investigating the mechanism of peptide aggregation: insights from mixed monte carlo-molecular dynamics simulations

The early stages of peptide aggregation are currently not accessible by experimental techniques at atomic resolution. In this article, we address this problem through the application of a mixed simulation scheme in which a preliminary coarse-grained Monte Carlo analysis of the free-energy landscape is used to identify representative conformations of the aggregates and subsequent all-atom molecular dynamics simulations are used to analyze in detail possible pathways for the stabilization of oligomers. This protocol was applied to systems consisting of multiple copies of the model peptide GNNQQNY, whose detailed structures in the aggregated state have been recently solved in another study. The analysis of the various trajectories provides dynamical and structural insight into the details of aggregation. In particular, the simulations suggest a hierarchical mechanism characterized by the initial formation of stable parallel β-sheet dimers and identify the formation of the polar zipper motif as a fundamental feature for the stabilization of initial oligomers. Simulation results are consistent with experimentally derived observations and provide an atomically detailed view of the putative initial stages of fibril formation.     

The functional topography of transmembrane domain 3 of the M1 muscarinic acetylcholine receptor, revealed by scanning mutagenesis

Alanine-scanning mutagenesis has been applied to residues 100-121 in transmembrane domain 3 of the M1 muscarinic acetylcholine receptor. This study complements a previous investigation of the triad Asp122-Arg123-Tyr124 (Lu, Z-L., Curtis, C. A., Jones, P. G., Pavia, J., and Hulme, E. C. (1997) Mol. Pharmacol. 51, 234-241). The results demonstrate the alpha-helical secondary structure of the domain and suggest its orientation with respect to the other transmembrane domains. The C-terminal part of the helix appears to be largely buried within the receptor structure. On its surface, there is a patch of three residues, Val113, Leu116, and Ser120, which may form intramolecular contacts that help to stabilize the inactive ground state of the receptor. Mutagenic disruption of these increased agonist affinity and signaling efficacy. In two cases (L116A and S120A), this led to constitutive activation of the receptor. Parallel to the helix axis and spanning the whole transmembrane region, a distinct strip of residues on one face of transmembrane domain 3 forms intermolecular (acetylcholine-receptor, receptor-G protein) or intrareceptor bonds that contribute to the activated state. The binding of acetylcholine may destabilize the first set of contacts while favoring the formation of the second.     

Fluorescence and molecular dynamics studies of the acetylcholine receptor gammaM4 transmembrane peptide in reconstituted systems

A combination of fluorescence spectroscopy and molecular dynamics (MD) is applied to assess the conformational dynamics of a peptide making up the outermost ring of the nicotinic acetylcholine receptor (AChR) transmembrane region and the effect of membrane thickness and cholesterol on the hydrophobic matching of this peptide. The fluorescence studies exploit the intrinsic fluorescence of the only tryptophan residue in a synthetic peptide corresponding to the fourth transmembrane domain of the AChR gamma subunit (gammaM4-Trp(6)) reconstituted in lipid bilayers of varying thickness, and combine this information with quenching studies using depth-sensitive phosphatidylcholine spin-labeled probes and acrylamide, polarization of fluorescence, and generalized polarization of Laurdan. A direct correlation was found between bilayer width and the depth of insertion of Trp(6). We further extend our recent MD study of the conformational dynamics of the AChR channel to focus on the crosstalk between M4 and the lipid-belt region. The isolated gammaM4 peptide is shown to possess considerable orientational flexibility while maintaining a linear alpha-helical structure, and to vary its tilt depending on bilayer width and cholesterol (Chol) content. MD studies also show that gammaM4 also establishes contacts with the other TM peptides on its inner face, stabilizing a shorter TM length that is still highly sensitive to the lipid environment. In the native membrane the topology of the M4 ring is likely to exhibit a similar behavior, dynamically modifying its tilt to match the hydrophobic thickness of the bilayer.     

NMR structure and dynamics of the second transmembrane domain of the neuronal acetylcholine receptor beta 2 subunit

Structure and backbone dynamics of a selectively [(15)N]Leu-labeled 28-residue segment of the extended second transmembrane domain (TM2e) of the human neuronal nicotinic acetylcholine receptor (nAChR) beta(2) subunit were studied by (1)H and (15)N solution-state NMR in dodecylphosphocholine micelles. The TM2e structure was determined on the basis of the nuclear Overhauser effects (NOEs) and the hydrogen bond restraints, which were inferred from the presence of H(alpha)(i)-H(N)(i+3), H(alpha)(i)-H(beta)(i+3), and H(alpha)(i)-H(N)(i+4) NOE connectivity and from the slow amide hydrogen exchange with D(2)O. The TM2e structure of the nAChR beta(2) subunit contains a helical region between T4 and K22. Backbone dynamics were calculated using the model-free approach based on the (15)N relaxation rate constants, R(1) and R(2), and on the (15)N-[(1)H] NOE. The data acquired at 9.4 and 14.1 T and calculations using different dynamic models demonstrated no conformational exchange and internal motions on the nanosecond time scale. The global tumbling time of TM2e in micelles was 14.4 +/- 0.2 ns; the NOE values were greater than 0.63 at 9.4 T, and the order parameter, S(2), was 0.83-0.96 for all (15)N-labeled leucine residues, suggesting a restricted internal motion. This is the first report of NMR structure and backbone dynamics of the second transmembrane domain of the human nAChR beta(2) subunit in a membrane-mimetic environment, providing the basis for subsequent studies of subunit interactions in the transmembrane domain complex of the neuronal nAChR.