M2 Helix Research Articles

Dynamic Properties of Na + Ions in Models of Ion Channels: A Molecular Dynamics Study

We present simulation results for the effective diffusion coefficients of a sodium ion in a series of model ion channels of different diameters and hydrophobicities, including models of alamethicin, a leucine-serine peptide, and the M2 helix bundle of the nicotinic acetylcholine receptor. The diffusion coefficient, which in the simulations has a value of 0.15(2) Å 2 ps −1 in bulk water, is found to be reduced to as little as 0.02(1) Å 2 ps −1 in the narrower channels, and to about 0.10(5) Å 2 ps −1 in wider channels such as the nicotinic acetylcholine receptor. It is anticipated that this work will be useful in connection with calculations of channel conductivity using such techniques as the Poisson-Nernst-Planck equation, Eyring rate theory, or Brownian dynamics.

Lipid properties and the orientation of aromatic residues in OmpF, influenza M2, and alamethicin systems: molecular dynamics simulations.

Molecular dynamics simulations allow a direct study of the structure and dynamics of membrane proteins and lipids. We describe the behavior of aromatic residues and lipid properties in POPE and POPC bilayer models with the Escherichia coli OmpF trimer, single alamethicin and Influenza M2 helices, 4-helix M2 bundles, and two alamethicin 6-helix channel models. The total simulation time is over 24 ns, of systems containing solvent, protein, and between 104 and 318 lipids. Various types of adjustment between lipids and proteins occur, depending on the size of the protein and the degree of hydrophobic mismatch between lipid and protein. Single helices cause little measurable effect on nearby lipids whereas the 4-helix bundles, 6-helix channel models, and OmpF cause a significant lowering of order parameters in nearby lipid chains, an increased difference between odd and even chain dihedrals in the magnitude of the trans dihedral fractions and dihedral transition rates, and in most cases a decreased gauche population and a decrease in bilayer thickness. An increased tilt of the lipid chains near the proteins can account for most of the observed decrease in order parameters. The orientation of tryptophans and tyrosines on the outside of the proteins is determined by packing at the protein exterior and non-specific hydrogen bonding with lipids and solvent. The tyrosines in the broad bands that delimit the hydrophobic exterior of OmpF show little change in orientation over one nanosecond. Their rings are oriented predominantly perpendicular to the bilayer plane, with the hydroxyl group pointing toward the lipid-water interface. Phenylalanines in OmpF, alamethicin, and Influenza M2 are more mobile and assume a variety of orientations.

Two models of the influenza A M2 channel domain: verification by comparison

The influenza M2 protein is a simple membrane protein, containing a single transmembrane helix. It is representative of a very large family of single-transmembrane helix proteins. The functional protein is a tetramer, with the four transmembrane helices forming a proton-permeable channel across the bilayer. Two independently derived models of the M2 channel domain are compared, in order to assess the success of applying molecular modelling approaches to simple membrane proteins. The Calpha RSMD between the two models is 1.7 A. Both models are composed of a left-handed bundle of helices, with the helices tilted roughly 15 degrees relative to the (presumed) bilayer normal. The two models have similar pore radius profiles, with a pore cavity lined by the Ser31 and Gly34 residues and a pore constriction formed by the ring of His37 residues. Independent studies of M2 have converged on the same structural model for the channel domain. This model is in agreement with solid state NMR data. In particular, both model and NMR data indicate that the M2 helices are tilted relative to the bilayer normal and form a left-handed bundle. Such convergence suggests that, at least for simple membrane proteins, restraints-directed modelling might yield plausible models worthy of further computational and experimental investigation.

Mutational analysis of trans-membrane helices M3, M4, M5 and M7 of the fast-twitch Ca2+-ATPase.

Mutational analysis of trans-membrane helices M3, M4, M5 and M7 of the Ca2+-ATPase revealed a novel phenotypic variant, M4 [Y295A (the one-letter symbols are used for amino acid residues throughout)], displaying an increased affinity for Pi and decreased affinity for MgATP, while retaining the ability to translocate Ca2+ ions across the endoplasmic reticulum membrane. The properties of this mutant suggest that the E1-E2 equilibrium is shifted towards E2, and indicate a key role for this aromatic residue (Y295) at the end of trans-membrane helix M4. A mutant containing three amino acid residue substitutions at the end of the seventh trans-membrane helix, M7 (F834A, F835A, T837F), showed a complete loss of ATPase activity and a reduced ability to phosphorylate with Pi, although MgATP-initiated phosphorylation was unaffected. The observation that single mutations in this cluster of residues had no effect on Ca2+ transport suggests that correct anchoring of the helix at the lipid-water interface by these aromatic residues is important in the functioning of the ATPase. Mutation of polar residues in helix M3 did not affect inhibition of the ATPase by thapsigargin, thapsivillosin A or t-butyl hydroquinone, suggesting that hydrogen-bonding partners for the essential -OH groups on these inhibitors lie elsewhere in the ATPase.

Electrostatics and the Ion Selectivity of Ligand-Gated Channels

The nicotinic acetylcholine receptor (nAChR) is a cation-selective ion channel that opens in response to acetylcholine binding. The related glycine receptor (GlyR) is anion selective. The pore-lining domain of each protein may be modeled as a bundle of five parallel M2 helices. Models of the pore-lining domains of homopentameric nAChR and GlyR have been used in continuum electrostatics calculations to probe the origins of ion selectivity. Calculated pK A values suggest that “rings” of acidic or basic side chains at the mouths of the nAChR or GlyR M2 helix bundles, respectively, may not be fully ionized. In particular, for the nAChR the ring of glutamate side chains at the extracellular mouth of the pore is predicted to be largely protonated at neutral pH, whereas those glutamate side chains in the intracellular and intermediate rings (at the opposite mouth of the pore) are predicted to be fully ionized. Inclusion of the other domains of each protein represented as an irregular cylindrical tube in which the M2 bundles are embedded suggests that both the M2 helices and the extramembrane domains play significant roles in determining ion selectivity.

Modelling and Simulation of Ion Channels: Applications to the Nicotinic Acetylcholine Receptor

Molecular dynamics simulations with experimentally derived restraints have been used to develop atomic models of M2 helix bundles forming the pore-lining domains of the nicotinic acetylcholine receptor and related ligand-gated ion channels. M2 helix bundles have been used in microscopic simulations of the dynamics and energetics of water and ions within an ion channel. Translational and rotational motion of water are restricted within the pore, and water dipoles are aligned relative to the pore axis by the surrounding helix dipoles. Potential energy profiles for translation of a Na+ion along the pore suggest that the protein and water components of the interaction energy exert an opposing effect on the ion, resulting in a relatively flat profile which favors cation permeation. Empirical conductance calculations based on a pore radius profile suggest that the M2 helix model is consistent with a single channel conductance of ca. 50 pS. Continuum electrostatics calculations indicate that a ring of glutamate residues at the cytoplasmic mouth of the α7 nicotinic receptor M2 helix bundle may not be fully ionized. A simplified model of the remainder of the channel protein when added to the M2 helix bundle plays a significant role in enhancing the ion selectivity of the channel.

Kinked-Helices Model of the Nicotinic Acetylcholine Receptor Ion Channel and Its Complexes with Blockers: Simulation by the Monte Carlo Minimization Method

A model of the nicotinic acetylcholine receptor ion channel was elaborated based on the data from electron microscopy, affinity labeling, cysteine scanning, mutagenesis studies, and channel blockade. A restrained Monte Carlo minimization method was used for the calculations. Five identical M2 segments (the sequence EKMTLSISVL 10LALTVFLLVI 20V) were arranged in five-helix bundles with various geometrical profiles of the pore. For each bundle, energy profiles for chlorpromazine, QX-222, pentamethonium, and other blocking drugs pulled through the pore were calculated. An optimal model obtained allows all of the blockers free access to the pore, but retards them at the rings of residues known to contribute to the corresponding binding sites. In this model, M2 helices are necessarily kinked. They come into contact with each other at the cytoplasmic end but diverge at the synaptic end, where N-termini of M1 segments may contribute to the pore. The kinks disengage α-helical H-bonds between Ala 12 and Ser 8. The uncoupled lone electron pairs of Ser 8 carbonyl oxygens protrude into the pore, forming a hydrophilic ring that may be important for the permeation of cations. A split network of H-bonds provides a flexibility to the chains Val 9-Ala 12, the numerous conformations of which form only two or three intrasegment H-bonds. The cross-ectional dimensions of the interface between the flexible chains vary essentially at the level of Leu 11. We suggest that conformational transitions in the chains Val 9-Ala 12 are responsible for the channel gating, whereas rotations of more stable α-helical parts of M2 segments may be necessary to transfer the channel in the desensitized state.

Site-directed Disulfide Mapping of Helices M4 and M6 in the Ca2+ Binding Domain of SERCA1a, the Ca2+ ATPase of Fast Twitch Skeletal Muscle Sarcoplasmic Reticulum

In an attempt to define the spatial relationships among SERCA1a transmembrane helices M4, M5, M6, and M8, involved in Ca2+ binding, all six cysteine residues were removed from predicted transmembrane sequences by substitution with Ser or Ala. The cysteine-depleted protein retained 44% of wild type Ca2+ transport activity. Pairs of cysteine residues were then reintroduced to determine whether their juxtaposition would result in the formation of disulfide cross-links between transmembrane helices. In initial studies designed to map the juxtaposition of Ca2+ binding residues, Cys was substituted for Glu309 or Gly310 in transmembrane sequence M4, in combination with the substitution of Cys for Glu771 in M5; for Asn796, Thr799, or Asp800 in M6; or for Glu908 in M8. These double mutants all retained the capacity to form a phosphoenzyme intermediate from Pi (but not from ATP in the presence of Ca2+), and in all but mutants E309C/N796C and G310C/N796C, phosphoenzyme formation was insensitive to 100 microM Ca2+. These results support the view that both Glu309 and Asn796 contribute to Ca2+ binding site II, which is not required for conversion of E2, the substrate for Pi phosphorylation, to E1. Cross-linking in mutants E309C/N796C and G310C/D800C established reference points for the orientation of M4 and M6 relative to each other and provided the basis for the prediction of potential additional cross-links. Strong links were formed with the pairs T317C/A804C and T317C/L807C near the cytoplasmic ends of the two helices and with A305C/L792C and A305C/L793C near the lumenal ends. These combined results support the conclusion that M4 and M6 form a right-handed coiled-coil structure that forms part of the pathway of Ca2+ translocation. In addition to providing a possible explanation for the mutation sensitivity of several pairs of residues in these helices, the proposed association of M4 and M6 supports a new model for the orientation of the two Ca2+ binding sites among transmembrane helices M4, M5, and M6.

ATPase gene transfer and mutational analysis of the cation translocation mechanism.

The peptide segment interposed between cation binding and phosphorylation domains retains a high degree of homology in all cation transport ATPases. Mutational analysis and chimeric replacements of Ca2+ ATPase components with corresponding Na+,K(+)-ATPase components indicate that this segment is utilized by various cation ATPases as a common structural device for a long-range functional linkage of enzyme phosphorylation and cation transport. Vectorial displacement of bound cation is rendered possible by a transmembrane channel formed by four clustered helices (M4, M5, M6, and M8). Originating from the four helices, the oxygen functions of Glu309, Glu771, Thr799, Asp800, and Glu908 form a duplex Ca2+ binding site in the middle of the channel, while Lys297 seals the luminal end of the channel with its positively charged side chain. The perturbation triggered by enzyme phosphorylation is apparently transmitted through the linkage segment to produce rotational displacement of the M4 helix with minimal change of secondary structure. The cation binding site is thereby disrupted and the Lys297 side chain removed, permitting Ca2+ to dissociate in exchange for H+ and to flow through the luminal end of the channel.

Molecular dynamics study of water and Na+ ions in models of the pore region of the nicotinic acetylcholine receptor

The nicotinic acetylcholine receptor (nAChR) is an integral membrane protein that forms ligand-gated and cation-selective channels. The central pore is lined by a bundle of five approximately parallel M2 helices, one from each subunit. Candidate model structures of the solvated pore region of a homopentameric (alpha7)5 nAChR channel in the open state, and in two possible forms of the closed state, have been studied using molecular dynamics simulations with restraining potentials. It is found that the mobility of the water is substantially lower within the pore than in bulk, and the water molecules become aligned with the M2 helix dipoles. Hydrogen-bonding patterns in the pore, especially around pore-lining charged and hydrophilic residues, and around exposed regions of the helix backbone, have been determined. Initial studies of systems containing both water and sodium ions together within the pore region have also been conducted. A sodium ion has been introduced into the solvated models at various points along the pore axis and its energy profile evaluated. It is found that the ion causes only a local perturbation of the water structure. The results of these calculations have been used to examine the effectiveness of the central ring of leucines as a component of a gate in the closed-channel model.

Synthesis of a radioactive azido derivative of thapsigargin and photolabeling of the sarcoplasmic reticulum ATPase.

A thapsigargin C8-derivative (ZTG) was synthesized by acylating debutanoylthapsigargin with 4-azido[carboxyl-14C]benzoic acid. ZTG retains the inhibitory activity of thapsigargin (TG) with respect to the Ca2+ ATPase of sarcoplasmic reticulum (SR). Covalent ATPase labeling was obtained by photoactivation of the ZTG azido moiety under conditions optimized to reduce nonspecific association of ZTG with SR vesicles and to approximate a matching ZTG:ATPase stoichiometry. Specific photolabeling of the Ca2+ ATPase with ZTG was obtained with 30% efficiency and was competitively inhibited by TG. Analysis of the labeled protein and its proteolytic fragments demonstrates that the ZTG label is associated covalently with the membrane-bound portion of tryptic subfragment A1, which spans the sequence between Leu253 and Arg324 and includes segments of S3 and S4 in the stalk, the M3 and M4 transmembrane helices, and the intervening lumenal loop. This finding is in agreement with previous spectroscopic observations and mutational analysis.

Molecular dynamics study of water and Na+ ions in models of the pore region of the nicotinic acetylcholine receptor.

The nicotinic acetylcholine receptor (nAChR) is an integral membrane protein that forms ligand-gated and cation-selective channels. The central pore is lined by a bundle of five approximately parallel M2 helices, one from each subunit. Candidate model structures of the solvated pore region of a homopentameric (alpha7)5 nAChR channel in the open state, and in two possible forms of the closed state, have been studied using molecular dynamics simulations with restraining potentials. It is found that the mobility of the water is substantially lower within the pore than in bulk, and the water molecules become aligned with the M2 helix dipoles. Hydrogen-bonding patterns in the pore, especially around pore-lining charged and hydrophilic residues, and around exposed regions of the helix backbone, have been determined. Initial studies of systems containing both water and sodium ions together within the pore region have also been conducted. A sodium ion has been introduced into the solvated models at various points along the pore axis and its energy profile evaluated. It is found that the ion causes only a local perturbation of the water structure. The results of these calculations have been used to examine the effectiveness of the central ring of leucines as a component of a gate in the closed-channel model.

The Influenza A Virus M2 Channel: A Molecular Modeling and Simulation Study

The M2 protein of influenza virus forms ion channels activated by low pH which are proton permeable and play a key role in the life cycle of the virus. M2 is a 97-residue integral membrane protein containing a single transmembrane (TM) helix. M2 is present as disulfide-linked homotetramers. The TM domain of M2 has been modeled as a bundle of four parallel M2 helices. The helix bundle forms a left-handed supercoil surrounding a central pore. Residue H37 has been implicated in the mechanism of low-pH activation of the channel. Models generated with H37 in a fully deprotonated state exhibit a pore occluded by a ring of H37 side chains oriented toward the lumen of the pore. Models with H37 in a fully protonated state no longer exhibit such occlusion of the pore, as the H37 side chains adopt a more interfacial location. Extended molecular dynamics simulations with water molecules within and at the mouths of the pores support this distinction between the H37-deprotonated and H37-protonated models. These simulations suggest that only in the H37-protonated model is there a continuous column of water extending the entire length of the central pore. A mechanism for activation of M2 by low pH is presented in which the H37-deprotonated model corresponds to the “closed” form of the channel, while the H37-protonated model corresponds to the “open” form. A switch from the closed to the open form of the channel occurs if H37 is protonated midway through a simulation. The open channel is suggested to contain a wire of H-bonded water molecules which enables proton permeability.

The pore domain of the nicotinic acetylcholine receptor: molecular modeling, pore dimensions, and electrostatics

The pore domain of the nicotinic acetylcholine receptor has been modeled as a bundle of five kinked M2 helices. Models were generated via molecular dynamics simulations incorporating restraints derived from 9-A resolution cryoelectron microscopy data (Unwin, 1993; 1995), and from mutagenesis data that identify channel-lining side chains. Thus, these models conform to current experimental data but will require revision as higher resolution data become available. Models of the open and closed states of a homopentameric alpha 7 pore are compared. The minimum radius of the closed-state model is less than 2 A; the minimum radius of the open-state models is approximately 6 A. It is suggested that the presence of "bound" water molecules within the pore may reduce the effective minimum radii below these values by up to approximately 3 A. Poisson-Boltzmann calculations are used to obtain a first approximation to the potential energy of a monovalent cation as it moves along the pore axis. The differences in electrostatic potential energy profiles between the open-state models of alpha 7 and of a mutant of alpha 7 are consistent with the experimentally observed change in ion selectivity from cationic to anionic. Models of the open state of the heteropentameric Torpedo nicotinic acetylcholine receptor pore domain are also described. Relatively small differences in pore radius and electrostatic potential energy profiles are seen when the Torpedo and alpha 7 models are compared.

The emerging three-dimensional structure of a receptor. The nicotinic acetylcholine receptor.

The nicotinic acetylcholine receptor is the neurotransmitter receptor with the most-characterized protein structure. The amino acid sequences of its five subunits have been elucidated by cDNA cloning and sequencing. Its shape and dimensions (approximately 12.5 nm x 8 nm) were deduced from electron-microscopy studies. Its subunits are arranged around a five-fold axis of pseudosymmetry in the order (clockwise) alpha H gamma alpha L delta beta. Its two agonist/competitive-antagonist-binding sites have been localized by photolabelling studies to a deep gorge between the subunits near the membrane surface. Its ion channel is formed by five membrane-spanning (M2) helices that are contributed by the five subunits. This finding has been generalized as the Helix M2 model for the superfamily of ligand-gated ion channels. The binding site for regulatory non-competitive antagonists has been localized by photolabelling and site-directed-mutagenesis studies within this ion channel. Therefore a three-dimensional image of the nicotinic acetylcholine receptor is emerging, the most prominent feature of which is an active site that combines the agonist/ competitive-antagonist-binding sites, the regulatory site and the ion channel within a relatively narrow space close to and within the bilayer membrane.

Short and Long Range Functions of Amino Acids in the Transmembrane Region of the Sarcoplasmic Reticulum ATPase: A MUTATIONAL STUDY

Mutational analysis of several amino acids in the transmembrane region of the sarcoplasmic reticulum ATPase was performed by expressing wild type ATPase and 32 site-directed mutants in COS-1 cells followed by functional characterization of the microsomal fraction. Four different phenotype characteristics were observed in the mutants: (a) functions similar to those sustained by the wild type ATPase; (b) Ca2+ transport inhibited to a greater extent than ATPase hydrolytic activity; (c) inhibition of transport and hydrolytic activity in the presence of high levels of phosphorylated enzyme intermediate; and (d) total inhibition of ATP utilization by the enzyme while retaining the ability to form phosphoenzyme by utilization of P(i). Analysis of experimental observations and molecular models revealed short and long range functions of several amino acids within the transmembrane region. Short range functions include: (a) direct involvement of five amino acids in Ca2+ binding within a channel formed by clustered transmembrane helices M4, M5, M6, and M8; (b) roles of several amino acids in structural stabilization of the helical cluster for optimal channel function; and (c) a specific role of Lys297 in sealing the distal end of the channel, suggesting that the M4 helix rotates to allow vectorial flux of Ca2+ upon enzyme phosphorylation. Long range functions are related to the influence of several transmembrane amino acids on phosphorylation reactions with ATP or P(i), transmitted to the extramembranous region of the ATPase in the presence or in the absence of Ca2+.

Molecular dynamics simulations of water within models of ion channels

The transbilayer pores formed by ion channel proteins contain extended columns of water molecules. The dynamic properties of such waters have been suggested to differ from those of water in its bulk state. Molecular dynamics simulations of ion channel models solvated within and at the mouths of their pores are used to investigate the dynamics and structure of intra-pore water. Three classes of channel model are investigated: a) parallel bundles of hydrophobic (Ala20) alpha-helices; b) eight-stranded hydrophobic (Ala10) antiparallel beta-barrels; and c) parallel bundles of amphipathic alpha-helices (namely, delta-toxin, alamethicin, and nicotinic acetylcholine receptor M2 helix). The self-diffusion coefficients of water molecules within the pores are reduced significantly relative to bulk water in all of the models. Water rotational reorientation rates are also reduced within the pores, particularly in those pores formed by alpha-helix bundles. In the narrowest pore (that of the Ala20 pentameric helix bundle) self-diffusion coefficients and reorientation rates of intra-pore waters are reduced by approximately an order of magnitude relative to bulk solvent. In Ala20 helix bundles the water dipoles orient antiparallel to the helix dipoles. Such dipole/dipole interaction between water and pore may explain how water-filled ion channels may be formed by hydrophobic helices. In the bundles of amphipathic helices the orientation of water dipoles is modulated by the presence of charged side chains. No preferential orientation of water dipoles relative to the pore axis is observed in the hydrophobic beta-barrel models.

Molecular Dynamics Simulations on Solvated M2 Helix Bundles of Nicotinic Receptors

The nicotinic acetylcholine receptor (nAChR) belongs to the family of neurotransmitter-gated ion channels and is an integral protein present in post-synaptic membranes. Photolabeling and mutagenesis studies implicate the M2 segments in forming the water-filled ion channel. On the basis of these studies i t has been suggested that five M2 helices form a bundle of a-helices surrounding a central pore.

Energy transduction and kinetic regulation by the peptide segment connecting phosphorylation and cation binding domains in transport ATPases.

The sarcoplasmic reticulum ATPase segment (Thr316-Leu356) connecting the extramembranous phosphorylation domain to the preceding transmembrane helix M4 (which is an integral component of the Ca2+ binding domain) retains a high degree of sequence homology with other cation transport ATPases. Single, non conservative mutations of homologous residues in this segment produces enzyme inhibition (Zhang et al., 1995). We have now produced single and multiple mutations of non-homologous residues in this segment of the Ca2+ ATPase to match the corresponding residues of the Na+, K+ ATPase. We find that the main characteristics of the ATPase mechanism (i.e., Ca2+ dependent phosphoenzyme formation and thapsigargin sensitivity) are retained even when the entire 41-amino acid (Thr316-Leu356) segment of the Ca2+ ATPase is rendered identical to the corresponding segment of the Na+, K+ ATPase by sequential mutations of the 14 non-homologous amino acids. However, the phosphoenzyme turnover (likely rate limited by the "Ca2.E1-P-->Ca.E2-P transition") is progressively reduced if four or more Ca2+ ATPase residues are mutated to the corresponding residues of the Na+, K+ ATPase. The time course of enzyme inactivation by EGTA (likely rate limited by the "E1 to E2 transition") is also prolonged. Our findings suggest that an analogous peptide segment provides a functional linkage for energy transduction between phosphorylation and cation binding domains in various cation transport ATPase. However, its kinetic influence on rate-limiting conformational transitions is dependent on matching specific structures in each ATPase.

Water-mediated conformational transitions in nicotinic receptor M2 helix bundles: a molecular dynamics study

The ion channel of the nicotinic acetylcholine receptor is a water-filled pore formed by five M2 helix segments, one from each subunit. Molecular dynamics simulations on bundles of five M2α7 helices surrounding a central column of water and with caps of water molecules at either end of the pore have been used to explore the effects of intrapore water on helix packing. Interactions of water molecules with the N-terminal polar sidechains lead to a conformational transition from right- to left-handed supercoils during these simulations. These studies reveal that the pore formed by the bundle of M2 helices is flexible. A structural role is proposed for water molecules in determining the geometry of bundles of isolated pore-forming helices.