Solid-State <sup>19</sup>F NMR Spectroscopy Reveals That Trp<sub>41</sub> Participates in the Gating Mechanism of the M2 Proton Channel of Influenza A Virus

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The M2 protein of influenza virus A is a proton-selective ion channel activated by pH. Structure determination by solid-state and solution NMR and X-ray crystallography has contributed significantly to our understanding, but channel activation may involve conformations not captured by these studies. Indeed, solidstate NMR data demonstrate that the M2 protein possesses significant conformational heterogeneity. Here, we report molecular dynamics (MD) simulations of the M2 transmembrane domain (TMD) in the absence and presence of the antiviral drug amantadine. The ensembles of MD conformations for both apo and bound forms reproduced the NMR data well. The TMD helix was found to kink around Gly-34, where water molecules penetrated deeply into the backbone. The amantadine-bound form exhibited a single peak Ϸ10°in the distribution of helix-kink angle, but the apo form exhibited 2 peaks, Ϸ0°and 40°. Conformations of the apo form with small and large kink angles had narrow and wide pores, respectively, around the primary gate formed by His-37 and Trp-41. We propose a structural model for channel activation, in which the small-kink conformations dominate before proton uptake by His-37 from the exterior, and proton uptake makes the large-kink conformations more favorable, thereby priming His-37 for proton release to the interior.solid-state NMR ͉ MD simulations ͉ helix kink ͉ histidine tetrad ͉ amantadine T he M2 protein of influenza virus A is a proton-selective ion channel activated by pH. Its function, proton conductance, is essential for effective replication of the virus. The transmembrane domain (TMD) of this homotetrameric protein contains a single helix (residues Ser-22 to Leu-46) from each subunit. Within the TMD, residues His-37 and Trp-41 constitute the primary gate that is critical for proton conductance and selectivity and pH activation (1-3). On the N-terminal (i.e., virus exterior) side, disulfide bonds involving Cys-17 and Cys-19 provide stabilization (4). On the C-terminal side, an amphiphilic helix may also be involved in pH activation (5). The antiviral drug amantadine and its derivatives inhibit the channel function by putatively binding to the TMD (6-8). The structures of the M2 TMD in the apo form and the amantadine-bound form were first determined by solid-state NMR (9, 10). In addition, extensive experimental (1, 2, 11-16) and computational (17-20) studies have been performed to model the structure of the M2 TMD and understand the molecular mechanisms of conductance and selectivity of the proton channel. Recently, 2 new structures were solved by X-ray crystallography (21) and solution NMR spectroscopy (22). Those 2 studies have generated controversies, especially about the mechanism of drug inhibition. Additional structural results have recently been published that add to the controversies surrounding inhibitor binding (23,24) and the closed-to open-state conformational transition (25).Channel activation may involve conformations not captured by the X-ray and NMR structures. Indeed, solid-state NMR...

Section: Conformational Ensemble Of Apo Form At Low Phsupporting

confidence: 86%

Section: Discussionmentioning

confidence: 99%

Conformational heterogeneity of the M2 proton channel and a structural model for channel activation

Cross

Zhou

2009

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100

136

“…M2 is one of the smallest bona fide channel/transporter proteins (96 residues), capable of pHdependent activation and highly selective conduction of protons vs. other ions (20)(21)(22)(23)(24)(25). A narrow pore leads to the highly conserved His37 and Trp41 residues (16,17,(26)(27)(28)(29), which are respectively responsible for proton selectivity (30) and asymmetry in the magnitude of conductance when the proton gradient is reversed (31). Thus the control of proton diffusion across the membrane relies on the ability of the imidazole moieties of His37 to accept and store protons from water molecules in the pore.…”

mentioning

confidence: 99%

Structure and mechanism of proton transport through the transmembrane tetrameric M2 protein bundle of the influenza A virus

Acharya

Carnevale

Fiorin

et al. 2010

250

520

The M2 proton channel from influenza A virus is an essential protein that mediates transport of protons across the viral envelope. This protein has a single transmembrane helix, which tetramerizes into the active channel. At the heart of the conduction mechanism is the exchange of protons between the His37 imidazole moieties of M2 and waters confined to the M2 bundle interior. Protons are conducted as the total charge of the four His37 side chains passes through 2 þ and 3 þ with a pK a near 6. A 1.65 Å resolution X-ray structure of the transmembrane protein (residues 25-46), crystallized at pH 6.5, reveals a pore that is lined by alternating layers of sidechains and well-ordered water clusters, which offer a pathway for proton conduction. The His37 residues form a box-like structure, bounded on either side by water clusters with wellordered oxygen atoms at close distance. The conformation of the protein, which is intermediate between structures previously solved at higher and lower pH, suggests a mechanism by which conformational changes might facilitate asymmetric diffusion through the channel in the presence of a proton gradient. Moreover, protons diffusing through the channel need not be localized to a single His37 imidazole, but instead may be delocalized over the entire His-box and associated water clusters. Thus, the new crystal structure provides a possible unification of the discrete site versus continuum conduction models.ion channels | M2 proton channel | membrane proteins | water clusters | histidine protonation W ater molecules confined at interfaces or in cavities behave differently from those in the bulk. Studies of water clusters have shed light not only on their fundamental properties (1-4) but also on the mechanism employed by various nano-bio systems to fine-tune water and proton transport (5-9). A relevant example from biology is the M2 protein of the influenza A virus (10, 11), which is the target of the influenza drugs amantadine and rimantadine (12-17). Tetrameric M2 (18) transports protons across the viral envelope to acidify the virion interior and trigger uncoating of the viral RNA prior to fusion of the viral envelope with the endosomal bilayer (19). M2 is one of the smallest bona fide channel/transporter proteins (96 residues), capable of pHdependent activation and highly selective conduction of protons vs. other ions (20)(21)(22)(23)(24)(25). A narrow pore leads to the highly conserved His37 and Trp41 residues (16,17,(26)(27)(28)(29), which are respectively responsible for proton selectivity (30) and asymmetry in the magnitude of conductance when the proton gradient is reversed (31). Thus the control of proton diffusion across the membrane relies on the ability of the imidazole moieties of His37 to accept and store protons from water molecules in the pore.An M2 peptide (residues 22-46), slightly longer than the transmembrane domain (32), associates into a functional four-helix bundle (33). Solid state 15 N nuclear magnetic resonance (ssNMR) experiments indicate that the first protons ...

“…A 25-residue TM segment: residues 22-46 (called M2-TM hereafter) spans the hydrophobic region of the membrane, and includes a few hydrophilic residues on either end. M2-TM forms tetrameric bundles (with the four chains referred to as A-D hereafter) and binds adamantane-containing drugs as the full M2 protein does, both in micelles (2) and in lipid bilayers (3)(4)(5)(6).…”

mentioning

confidence: 99%

Molecular dynamics calculations suggest a conduction mechanism for the M2 proton channel from influenza A virus

Khurana

Peraro²,

DeVane³

et al. 2009

116

166

ion channel ͉ transporter ͉ pH activated ͉ His gate ͉ simulations T he M2 protein from the influenza A virus is commonly described as a pH-activated proton channel based on its function of transferring protons into a virus. After endocytosis of the virus, the low pH in the endosome activates the channel. The transfer of protons to the viral interior via the M2 protein permits the uncoating of the viral RNA and fusion of the viral envelope with the endosomal bilayer, an important step in the life cycle of the virus (1). The M2 protein is a homotetramer, with each monomer consisting of a 24-residue N-terminal extracellular domain, a transmembrane (TM) domain of 19 residues, and a 54-residue cytoplasmic domain. A 25-residue TM segment: residues 22-46 (called M2-TM hereafter) spans the hydrophobic region of the membrane, and includes a few hydrophilic residues on either end. M2-TM forms tetrameric bundles (with the four chains referred to as A-D hereafter) and binds adamantane-containing drugs as the full M2 protein does, both in micelles (2) and in lipid bilayers (3-6).Experimentally based model structures of M2-TM have been available for some time (4-9) and have been computationally investigated by several authors (10 -18); however, highresolution structures have only recently been determined. Schnell and Chou have reported an NMR structural ensemble for the peptide, spanning residues 18-60 in detergent micelles at high pH (19). Stouffer et al., however, have used x-ray diffraction to solve the structures of M2-TM: crystallized under neutral and low pH conditions (20). Importantly, solid-state NMR studies of the helix-spanning region of the channel (in bilayers that most closely resemble a native membrane) have provided highresolution measurements of individual helical distances and orientations: these data are in agreement with the crystallographic and solution NMR structures (8, 9).The flux of protons through the M2 protein increases as the pH is lowered, which is a result of the increase in the permeant ion concentration and a ''gating'' process that involves His-37 and Trp-41 residues (which line the pore of M2-TM near the C terminus) (21). The x-ray crystal structure of the channel, solved at low pH in the presence of the channel-blocking drug amantadine, is closed near the N terminus but open in the vicinity of His-37 and Trp-41. The NMR structure, however, is completely occluded at the C terminus, and has only a very small opening near the N terminus. Surprisingly, the crystallographic structure determined for M2-TM (crystallized near neutral pH and in the absence of channel-blocking drugs) has hybrid characteristics: The N-terminal half of the structure possesses fourfold rotational symmetry, with the channel pore very constricted near Val-27. In contrast, the C-terminal half of the structure exhibits some asymmetry; one of the four helices (chain D) shows a slight bend near the middle of the TM region centered on Gly-34. This bend allows aromatic interactions between Trp-41 on chain D and His-37 on chain ...