A high resolution structure of the putative hinge region in M2 channel-lining segments of the nicotinic acetylcholine receptor

Transmembrane peptide helices play key roles in signal transduction across cell membranes, yet little is known about their high-resolution structure or the role membrane composition plays in their association, structure, dynamics and ultimately their performance. Using magic angle spinning (MAS) homonuclear dipolar recoupling experiments, the backbone structure at positions L10, L11, and A12 of the M2 ion channel peptide was determined in two lipid systems. Their measurements are in agreement with M2 forming transmembrane helices, but the torsion angles vary considerably from common alpha-helical values. These measurements show remarkable agreement with a previous computational model of M2 peptides forming a pore domain in which their helices are kinked near the central leucine, L11 [R. Sankararamakrishnan, C. Adcock, M.S.P. Sansom, The pore domain of the nicotinic acetylcholine receptor: Molecular modeling, pore dimensions, and electrostatics, Biophys. J. 71 (1996) 1659-1671]. The generation of high resolution data for transmembrane helices is of critical importance in refining structures for membrane protein and developing models of helix packing interactions.     

Assembly of the M2 Tetramer Is Strongly Modulated by Lipid Chain Length

The influenza virus matrix protein 2 (M2) assembles into a tetramer in the host membrane during viral uncoating and maturation. It has been used as a model system to understand the relative contributions of protein-lipid and protein-protein interactions to membrane protein structure and association. Here we investigate the effect of lipid chain length on the association of the M2 transmembrane domain into tetramers using Förster resonance energy transfer. We observe that the interactions between the M2 helices are much stronger in 1,2-dilauroyl-sn-glycero-3-phosphocholine than in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayers. Thus, lipid chain length and bilayer thickness not only modulate peptide interactions, but could also be a major determinant of the association of transmembrane helices into functional membrane protein oligomers.     

A structural model of the acetylcholine receptor channel based on partition energy and helix packing calculations.

A structural model of the transmembrane portion of the acetylcholine receptor was developed from sequences of all its subunits by using transfer energy calculations to locate transmembrane alpha-helices and to calculate which helical side chains should be in contact with water inside the channel, with portions of other transmembrane helices, or with lipid hydrocarbon chains. "Knobs-into-holes" side chain packing calculations were used with other factors to stack the transmembrane alpha-helices together. In the model each subunit has the following structures in order along the sequence from the NH2 terminus: a large extracellular domain of undetermined structure, a short apolar alpha-helix that lies on the extracellular lipid surface of the membrane; three apolar transmembrane alpha-helices (I, II, and III), a cytoplasmic domain of undetermined structure, an amphipathic transmembrane alpha-helix (L) that forms the channel lining, a short extracellular alpha-helix, another apolar transmembrane alpha-helix (IV), and a small cytoplasmic domain formed by the COOH-terminal end of the chain. Three concentric layers form the pore. A bundle of five amphipathic L helices forms the channel lining. This bundle is surrounded by a bundle of 10 alternating II and III helices. Helices I and IV cover portions of the outer surface of the bundle formed by helices II and III. Positions of disulfide bridges are predicted and a mechanism for opening and closing conformational changes is proposed that requires tilting transmembrane helices and possibly a thiol-disulfide interchange reaction.     

Viral ion channels: molecular modeling and simulation

In a number of membrane-bound viruses, ion channels are formed by integral membrane proteins. These channel proteins include M2 from influenza A, NB from influenza B, and, possibly, Vpu from HIV-1. M2 is important in facilitating uncoating of the influenza A viral genome and is the target of amantadine, an anti-influenza drug. The biological roles of NB and Vpu are less certain. In all cases, the protein contains a single transmembrane alpha-helix close to its N-terminus. Channels can be formed by homo-oligomerization of these proteins, yielding bundles of transmembrane helices that span the membrane and surround a central ion-permeable pore. Molecular modeling may be used to integrate and interpret available experimental data concerning the structure of such transmembrane pores. This has proved successful for the M2 channel domain, where two independently derived models are in agreement with one another, and with solid-state nuclear magnetic resonance (NMR) data. Simulations based on channel models may yield insights into possible ion conduction and selectivity mechanisms.     

Structure, Dynamics, and Ion Conductance of the Phospholamban Pentamer

A 52-residue membrane protein, phospholamban (PLN) is an inhibitor of an adenosine-5′-triphosphate-driven calcium pump, the Ca²⁺-ATPase. Although the inhibition of Ca²⁺-ATPase involves PLN monomers, in a lipid bilayer membrane, PLN monomers form stable pentamers of unknown biological function. The recent NMR structure of a PLN pentamer depicts cytoplasmic helices extending normal to the bilayer in what is known as the bellflower conformation. The structure shows transmembrane helices forming a hydrophobic pore 4 Å in diameter, which is reminiscent of earlier reports of possible ion conductance through PLN pentamers. However, recent FRET measurements suggested an alternative structure for the PLN pentamer, known as the pinwheel model, which features a narrower transmembrane pore and cytoplasmic helices that lie against the bilayer. Here, we report on structural dynamics and conductance properties of the PLN pentamers from all-atom (AA) and coarse-grained (CG) molecular dynamics simulations. Our AA simulations of the bellflower model demonstrate that in a lipid bilayer membrane or a detergent micelle, the cytoplasmic helices undergo large structural fluctuations, whereas the transmembrane pore shrinks and becomes asymmetric. Similar asymmetry of the transmembrane region was observed in the AA simulations of the pinwheel model; the cytoplasmic helices remained in contact with the bilayer. Using the CG approach, structural dynamics of both models were investigated on a microsecond timescale. The cytoplasmic helices of the CG bellflower model were observed to fall against the bilayer, whereas in the CG pinwheel model the conformation of the cytoplasmic helices remained stable. Using steered molecular dynamics simulations, we investigated the feasibility of ion conductance through the pore of the bellflower model. The resulting approximate potentials of mean force indicate that the PLN pentamer is unlikely to function as an ion channel.     

Reversible transition between α-helix and β-sheet conformation of a transmembrane domain

Despite the important functions of protein transmembrane domains, their structure and dynamics are often scarcely known. The SNARE proteins VAMP/synaptobrevin and syntaxin 1 are implicated in membrane fusion. Using different spectroscopic approaches we observed a marked sensitivity of their transmembrane domain structure in regard to the lipid/peptide ratio. In the dilute condition, peptides corresponding to the complete transmembrane domain fold into an α-helix inserted at ∼ 35° to the normal of the membranes, an observation in line with molecular simulations. Upon an increase in the peptide/lipid ratio, the peptides readily exhibited transition to β-sheet structure. Moreover, the insertion angle of these β-sheets increased to 54° and was accompanied by a derangement of lipid acyl chains. For both proteins the transition from α-helix to β-sheet was reversible under certain conditions by increasing the peptide/lipid ratio. This phenomenon was observed in different model systems including multibilayers and small unilamellar vesicles. In addition, differences in peptide structure and transitions were observed when using distinct lipids (DMPC, DPPC or DOPC) thus indicating parameters influencing transmembrane domain structure and conversion from helices to sheets. The putative functional consequences of this unprecedented dynamic behavior of a transmembrane domain are discussed.     

Parallel helix bundles and ion channels: molecular modeling via simulated annealing and restrained molecular dynamics.

A parallel bundle of transmembrane (TM) alpha-helices surrounding a central pore is present in several classes of ion channel, including the nicotinic acetylcholine receptor (nAChR). We have modeled bundles of hydrophobic and of amphipathic helices using simulated annealing via restrained molecular dynamics. Bundles of Ala20 helices, with N = 4, 5, or 6 helices/bundle were generated. For all three N values the helices formed left-handed coiled coils, with pitches ranging from 160 A (N = 4) to 240 A (N = 6). Pore radius profiles revealed constrictions at residues 3, 6, 10, 13, and 17. A left-handed coiled coil and a similar pattern of pore constrictions were observed for N = 5 bundles of Leu20. In contrast, N = 5 bundles of Ile20 formed right-handed coiled coils, reflecting loosened packing of helices containing beta-branched side chains. Bundles formed by each of two classes of amphipathic helices were examined: (a) M2a, M2b, and M2c derived from sequences of M2 helices of nAChR; and (b) (LSSLLSL)3, a synthetic channel-forming peptide. Both classes of amphipathic helix formed left-handed coiled coils. For (LSSLLSL)3 the pitch of the coil increased as N increased from 4 to 6. The M2c N = 5 helix bundle is discussed in the context of possible models of the pore domain of nAChR.     

The structure of the M2 channel-lining segment from the nicotinic acetylcholine receptor

The structures of functional peptides corresponding to the predicted channel-lining M2 segment of the nicotinic acetylcholine (AChR) were determined using solution NMR experiments on micelle samples, and solid-state NMR experiments on bilayer samples. The AChR M2 peptide forms a straight transmembrane α-helix, with no kinks. M2 inserts in the lipid bilayer at an angle of 12° relative to the bilayer normal, with a rotation about the helix long axis such that the polar residues face the N-terminus of the peptide, which is assigned to be intracellular. A molecular model of the AChR channel pore, constructed from the solid-state NMR 3-D structure of the AChR M2 helix in the membrane assuming a pentameric organization, results in a funnel-like architecture for the channel with the wide opening on the N-terminal intracellular side. A central narrow pore has a diameter ranging from about 3.0 Å at its narrowest, to 8.6 Å at its widest. Nonpolar residues are predominantly on the exterior of the bundle, while polar residues line the pore. This arrangement is in fair agreement with evidence collected from permeation, mutagenesis, affinity labeling and cysteine accessibility measurements. A pentameric M2 helical bundle may, therefore, represent the structural blueprint for the inner bundle that lines the channel of the nicotinic AChR.     

Deuterium/hydrogen exchange factors measured by solution nuclear magnetic resonance spectroscopy as indicators of the structure and topology of membrane proteins

Deuterium/hydrogen exchange factors (chi) were measured for the backbone amide sites of the membrane-bound forms of the 50-residue fd coat protein and the 23-residue magainin2 peptide in lipid micelles by solution nuclear magnetic resonance spectroscopy. By combining kinetic and thermodynamic effects, deuterium/hydrogen exchange factors overcome the principal limitations encountered in the measurements of kinetic protection factors and thermodynamic fractionation factors for membrane proteins. The magnitudes of the exchange factors can be correlated with the structure and topology of membrane-associated polypeptides. In fd coat protein, residues in the transmembrane helix have exchange factors that are substantially smaller than those in the amphipathic surface helix or the loop connecting the two helices. For the amphipathic helical peptide, magainin2, the exchange factors of residues exposed to the solvent are appreciably larger than those that face the hydrocarbon portion of membrane bilayers. These examples demonstrate that deuterium/hydrogen exchange factors can be measured by solution NMR spectroscopy and used to identify residues in transmembrane helices as well as to determine the polarity of amphipathic helices in membrane proteins.     

Gating Transition of Pentameric Ligand-Gated Ion Channels

Pentameric ligand-gated ion channels are an important family of membrane proteins and play key roles in physiological processes, including signal transduction at chemical synapses. Here, we study the conformational changes associated with the opening and closing of the channel pore. Based on recent crystal structures of two prokaryotic members of the family in open and closed states, respectively, mixed elastic network models are constructed for the transmembrane domain. To explore the conformational changes in the gating transition, a coarse-grained transition path is computed that smoothly connects the closed and open conformations of the channel. We find that the conformational transition involves no major rotations of the transmembrane helices, and is instead characterized by a concerted tilting of helices M2 and M3. In addition, helix M2 changes its bending state, which results in an early closure of the pore during the open-to-closed transition.     

Transmembrane helix uniformity examined by spectral mapping of torsion angles

The environment and unique balance of molecular forces within lipid bilayers has a profound impact upon the structure, dynamics, and function of membrane proteins. We describe the biophysical foundations for the remarkable uniformity of many transmembrane helices that result from the molecular interactions within lipid bilayers. In fact, the characteristic uniformity of transmembrane helices leads to unique spectroscopic opportunities allowing for phi,psi torsion angles to be mapped directly onto solid state nuclear magnetic resonance (NMR) PISEMA spectra. Results from spectral simulations, the solid state NMR-derived structure of the influenza A M2 proton channel transmembrane domain, and high-resolution crystal structures of 27 integral membrane proteins demonstrate that transmembrane helices tend to be more uniform than previously thought. The results are discussed through the definition of a preferred range of backbone varphi,psi torsion angles for transmembrane alpha helices and are presented with respect to improving biophysical characterizations of integral membrane proteins.     

Exploring conformational states and helical packings in the P2X receptor transmembrane domain by molecular dynamics simulation

The P2X receptor is a trimeric transmembrane protein that acts as an ATP-gated ion channel. Its transmembrane domain (TMD) contains only six helices and three of them, the M2 helices, line the ion conduction pathway. Here, using molecular dynamics simulation, I identify four conformational states of the TMD that are associated with four types of packing between M2 helices. Packing in the extracellular half of the M2 helix produces closed conformations, while packing in the intracellular half produces both open and closed conformations. State transition is observed and supports a mechanism where iris-like twisting of the M2 helices switches the location of helical packing between the extracellular and the intracellular halves of the helices. In addition, this twisting motion alters the position and orientation of residue side-chains relative to the pore and therefore influences the pore geometry and possibly ion permeation. Helical packing, on the other hand, may restrict the twisting motion and generate discrete conformational states.     

The structure of the M2 channel-lining segment from the nicotinic acetylcholine receptor

The structures of functional peptides corresponding to the predicted channel-lining M2 segment of the nicotinic acetylcholine (AChR) were determined using solution NMR experiments on micelle samples, and solid-state NMR experiments on bilayer samples. The AChR M2 peptide forms a straight transmembrane alpha-helix, with no kinks. M2 inserts in the lipid bilayer at an angle of 12 degrees relative to the bilayer normal, with a rotation about the helix long axis such that the polar residues face the N-terminus of the peptide, which is assigned to be intracellular. A molecular model of the AChR channel pore, constructed from the solid-state NMR 3-D structure of the AChR M2 helix in the membrane assuming a pentameric organization, results in a funnel-like architecture for the channel with the wide opening on the N-terminal intracellular side. A central narrow pore has a diameter ranging from about 3.0 A at its narrowest, to 8.6 A at its widest. Nonpolar residues are predominantly on the exterior of the bundle, while polar residues line the pore. This arrangement is in fair agreement with evidence collected from permeation, mutagenesis, affinity labeling and cysteine accessibility measurements. A pentameric M2 helical bundle may, therefore, represent the structural blueprint for the inner bundle that lines the channel of the nicotinic AChR.     

Gating mechanisms in Cys-loop receptors

The Cys-loop receptor superfamily of ligand-gated ion channels has a prominent role in neuronal signalling. These receptors are pentamers, each subunit containing ten β-strands in the extracellular domain and four α-helical transmembrane domains (M1–M4). The M2 domain of each subunit lines the intrinsic ion channel pore and residues within the extracellular domain form ligand binding sites. Ligand binding initiates a conformational change that opens the ion-selective pore. The coupling between ligand binding in the extracellular domain and opening of the intrinsic ion channel pore located in the membrane is not fully understood. Several loop structures, such as loop 2, the Cys-loop, the pre-M1 region and the M2–M3 loop have been implicated in receptor activation. The current “conformational change wave” hypothesis suggests that binding of a ligand initiates a rotation of the β-sheets around an axis that passes through the Cys-loop. Due to this rotation, the Cys-loop and loop 2 are displaced. Movement of the M2–M3 loop then twists the M2 domain leading to a separation of the helices and opening of the pore. The publication of a crystal structure of an acetylcholine binding protein and the refined structure of the Torpedo marmorata acetylcholine receptor have improved the understanding of the mechanisms and structures involved in coupling ligand binding to channel gating. In this review, the most recent findings on some of these loop structures will be reported and discussed in view of their role in the gating mechanism.     

Structure of the 1-36 amino-terminal fragment of human phospholamban by nuclear magnetic resonance and modeling of the phospholamban pentamer

The structure of a 36-amino-acid-long amino-terminal fragment of phospholamban (phospholamban[1-36]) in aqueous solution containing 30% trifluoroethanol was determined by nuclear magnetic resonance. The peptide, which comprises the cytoplasmic domain and six residues of the transmembrane domain of phospholamban, assumes a conformation characterized by two alpha-helices connected by a turn. The residues of the turn are Ile18, Glu19, Met20, and Pro21, which are adjacent to the two phosphorylation sites Ser16 and Thr17. The proline is in a trans conformation. The helix comprising amino acids 22-36 is well determined (the root mean square deviation for the backbone atoms, calculated for a family of 18 nuclear magnetic resonance structures is 0.57 A). Recently, two molecular models of the transmembrane domain of phospholamban were proposed in which a symmetric homopentamer is composed of a left-handed coiled coil of alpha-helices. The two models differ by the relative orientation of the helices. The model proposed by,Simmerman et al. (H.K. Simmerman, Y.M. Kobayashi, J.M. Autry, and L.R. Jones, 1996, J. Biol. Chem. 271:5941-5946), in which the coiled coil is stabilized by a leucine-isoleucine zipper, is similar to the transmembrane pentamer structure of the cartilage oligomeric membrane protein determined recently by x-ray (V. Malashkevich, R. Kammerer, V Efimov, T. Schulthess, and J. Engel, 1996, Science 274:761-765). In the model proposed by Adams et al. (P.D. Adams, I.T. Arkin, D.M. Engelman, and A.T. Brunger, 1995, Nature Struct. Biol. 2:154-162), the helices in the coiled coil have a different relative orientation, i.e., are rotated clockwise by approximately 50 degrees. It was possible to overlap and connect the structure of phospholamban[1-36] derived in the present study to the two transmembrane pentamer models proposed. In this way two models of the whole phospholamban in its pentameric form were generated. When our structure was connected to the leucine-isoleucine zipper model, the inner side of the cytoplasmic domain of the pentamer (where the helices face one another) was lined by polar residues (Gln23, Gln26, and Asn30), whereas the five Arg25 side chains were on the outer side. On the contrary, when our structure was connected to the other transmembrane model, in the inner side of the cytoplasmic domain of the pentamer, the five Arg25 residues formed a highly charged cluster.     

alpha-helical structural elements within the voltage-sensing domains of a K(+) channel

Voltage-gated K(+) channels are tetramers with each subunit containing six (S1-S6) putative membrane spanning segments. The fifth through sixth transmembrane segments (S5-S6) from each of four subunits assemble to form a central pore domain. A growing body of evidence suggests that the first four segments (S1-S4) comprise a domain-like voltage-sensing structure. While the topology of this region is reasonably well defined, the secondary and tertiary structures of these transmembrane segments are not. To explore the secondary structure of the voltage-sensing domains, we used alanine-scanning mutagenesis through the region encompassing the first four transmembrane segments in the drk1 voltage-gated K(+) channel. We examined the mutation-induced perturbation in gating free energy for periodicity characteristic of alpha-helices. Our results are consistent with at least portions of S1, S2, S3, and S4 adopting alpha-helical secondary structure. In addition, both the S1-S2 and S3-S4 linkers exhibited substantial helical character. The distribution of gating perturbations for S1 and S2 suggest that these two helices interact primarily with two environments. In contrast, the distribution of perturbations for S3 and S4 were more complex, suggesting that the latter two helices make more extensive protein contacts, possibly interfacing directly with the shell of the pore domain.     

Structural models of the MscL gating mechanism.

Three-dimensional structural models of the mechanosensitive channel of large conductance, MscL, from the bacteria Mycobacterium tuberculosis and Escherichia coli were developed for closed, intermediate, and open conformations. The modeling began with the crystal structure of M. tuberculosis MscL, a homopentamer with two transmembrane alpha-helices, M1 and M2, per subunit. The first 12 N-terminal residues, not resolved in the crystal structure, were modeled as an amphipathic alpha-helix, called S1. A bundle of five parallel S1 helices are postulated to form a cytoplasmic gate. As membrane tension induces expansion, the tilts of M1 and M2 are postulated to increase as they move away from the axis of the pore. Substantial expansion is postulated to occur before the increased stress in the S1 to M1 linkers pulls the S1 bundle apart. During the opening transition, the S1 helices and C-terminus amphipathic alpha-helices, S3, are postulated to dock parallel to the membrane surface on the perimeter of the complex. The proposed gating mechanism reveals critical spatial relationships between the expandable transmembrane barrel formed by M1 and M2, the gate formed by S1 helices, and "strings" that link S1s to M1s. These models are consistent with numerous experimental results and modeling criteria.     

Stability of loops in the structure of lactose permease

Structural analysis of peptide fragments has provided useful information on the secondary structure of integral membrane proteins built from a helical bundle (up to seven transmembrane segments). Comparison of those results to recent X-ray crystallographic results showed agreement between the structures of the fragments and the structures of the intact proteins. Lactose permease of Escherichia coli (lac Y) offers an opportunity to test that hypothesis on a substantially larger integral membrane protein. Lac Y contains a bundle of 12 transmembrane segments connected by 11 loops. Eleven segments, each corresponding to one of the loops in this protein, were studied. Five of these segments form defined structures in solution as determined by multidimensional nuclear magnetic resonance. Four peptides form turns, and one peptide reveals the end of one of the transmembrane helices. These results suggest that some loops in helical bundles are stabilized by short-range interactions, particularly in smaller bundles, and such intrinsically stable loops may contribute to protein stability and influence the pathway of folding. Greater conformational flexibility may be found in large integral membrane proteins.     

Transmembrane interactions in the activation of the Neu receptor tyrosine kinase

The Neu receptor tyrosine kinase is constitutively activated by a single amino acid change in the transmembrane domain of the receptor. The mutation of Val664 to glutamate or glutamine induces receptor dimerization and autophosphorylation of the receptor's intracellular kinase domain. The ability of this single mutation to activate the receptor is sequence-dependent, suggesting that specific helix-helix interactions stabilize the transmembrane dimer. We have determined the local secondary structure and interhelical contacts in the region of position 664 in peptide models of the activated receptor using solid-state rotational resonance and rotational echo double-resonance (REDOR) NMR methods. Intrahelical (13)C rotational resonance distance measurements were made between 1-(13)C-Thr662 and 2-(13)C-Gly665 on peptides corresponding to the wild-type Neu and activated Neu transmembrane sequences containing valine and glutamate at position 664, respectively. We observed similar internuclear distances (4.5 +/- 0.2 A) in both Neu and Neu*, indicating that the region near residue 664 is helical and is not influenced by mutation. Interhelical (15)N...(13)C REDOR measurements between Gln664 side chains on opposing helices were not consistent with hydrogen bonding between the side chain functional groups. However, interhelical rotational resonance measurements between 1-(13)C-Glu664 and 2-(13)C-Gly665 and between 1-(13)C-Gly665 and 2-(13)C-Gly665 demonstrated close contacts (4.3-4.5 A) consistent with the packing of Gly665 in the Neu* dimer interface. These measurements provide structural constraints for modeling the transmembrane dimer and define the rotational orientation of the transmembrane helices in the activated receptor.     

Bilayer mechanical properties regulate the transmembrane helix mobility and enzymatic state of CD39

CD39 can exist in at least two distinct functional states depending on the presence and intact membrane integration of its two transmembrane helices. In native membranes, the transmembrane helices undergo dynamic rotational motions that are required for enzymatic activity and are regulated by substrate binding. In this study, we show that bilayer mechanical properties regulate conversion between the two enzymatic functional states by modulating transmembrane helix dynamics. Alteration of membrane properties by insertion of cone-shaped or inverse cone-shaped amphiphiles or by cholesterol removal switches CD39 to the same enzymatic state that removal or solubilization of the transmembrane domains does. The same membrane alterations increase the propensity of both transmembrane helices to rotate within the packed structure, resulting in a structure with greater mobility but not an altered primary conformation. Membrane alteration also abolishes the ability of the substrate to stabilize the helices in their primary conformation, indicating a loss of coupling between substrate binding and transmembrane helix dynamics. Removal of either transmembrane helix mimics the effect of membrane alteration on the mobility and substrate sensitivity of the remaining helix, suggesting that the ends of the extracellular domain have intrinsic flexibility. We suggest that a mechanical bilayer property, potentially elasticity, regulates CD39 by altering the balance between the stability and flexibility of its transmembrane helices and, in turn, of its active site.