Identification of the functional core of the influenza A virus A/M2 proton-selective ion channel

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144

The M2 proton channel from influenza A virus transmits protons across membranes via a narrow aqueous pore lined by water and a proton sensor, His37. Near the center of the membrane, a water cluster is stabilized by the carbonyl of Gly34 and His37, the properties of which are modulated by protonation of His37. At low pH (5-6), where M2 conducts protons, this region undergoes exchange processes on the microsecond to second timescale. Here, we use 2D IR to examine the instantaneous conformational distribution and hydration of G34, and the evolution of the ensemble on the femtosecond to picosecond timescale. The channel water is strongly pH dependent as gauged by 2D IR which allows recording of the vibrational frequency autocorrelation function of a 13 C ¼ 18 O Gly34 probe. At pH 8, where entry and exit of protons within the channel are very slow, the carbonyl groups appear to adopt a single conformation/environment. The high-pH conformer does not exhibit spectral dynamics near the Gly34, and water in the channel must form a relatively rigid ice-like structure. By contrast, two vibrational forms of G34 are seen at pH 6.2, neither of which is identical to the high-pH form. In at least one of these low-pH forms, the probe is immersed in a very mobile, bulk-like aqueous environment having a correlation time ca. 1.3 ps at pH 6.2. Thus, protonation of His37 at low pH causes liquid-like water molecules to flow into the neighborhood of the Gly34.M2 channel influenza | transmembrane protein | water dynamics | infrared | two-dimensional

Section: Discussionmentioning

confidence: 99%

Tidal surge in the M2 proton channel, sensed by 2D IR spectroscopy

Ghosh

Qiu

DeGrado

et al. 2011

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144

“…A similarly short sequence was shown to associate into tetramers that faithfully reproduce the salient electrophysiological and pharmacological features of the full-length protein (33). The present structure reveals a proton conduction path composed of alternating layers of sidechains and well-ordered water clusters.…”

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confidence: 86%

“…An M2 peptide (residues , 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 are bound to the His residues in the tetrad (34) with surprisingly high affinity (pK a ¼ 8.2), stabilized via low-barrier H-bonds.…”

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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

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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 ...

“…the His37 tetrad) that reside in the middle of the AM2 transmembrane domain (AM2/TM) (Fig. 1A), which has been experimentally shown (15) to account for the proton permeation behavior of the full AM2 protein. The "shutter" mechanism (16,17) suggests that the His37 tetrad works as a gate.…”

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confidence: 99%

Multiscale simulation reveals a multifaceted mechanism of proton permeation through the influenza A M2 proton channel

Liang

Swanson

et al. 2014

139

The influenza A virus M2 channel (AM2) is crucial in the viral life cycle. Despite many previous experimental and computational studies, the mechanism of the activating process in which proton permeation acidifies the virion to release the viral RNA and core proteins is not well understood. Herein the AM2 proton permeation process has been systematically characterized using multiscale computer simulations, including quantum, classical, and reactive molecular dynamics methods. We report, to our knowledge, the first complete free-energy profiles for proton transport through the entire AM2 transmembrane domain at various pH values, including explicit treatment of excess proton charge delocalization and shuttling through the His37 tetrad. The free-energy profiles reveal that the excess proton must overcome a large freeenergy barrier to diffuse to the His37 tetrad, where it is stabilized in a deep minimum reflecting the delocalization of the excess charge among the histidines and the cost of shuttling the proton past them. At lower pH values the His37 tetrad has a larger total charge that increases the channel width, hydration, and solvent dynamics, in agreement with recent 2D-IR spectroscopic studies. The proton transport barrier becomes smaller, despite the increased charge repulsion, due to backbone expansion and the more dynamic pore water molecules. The calculated conductances are in quantitative agreement with recent experimental measurements. In addition, the free-energy profiles and conductances for proton transport in several mutants provide insights for explaining our findings and those of previous experimental mutagenesis studies.T he influenza type A virus is a highly pathogenic RNA virus that causes flu in birds and mammals (1). The influenza A M2 (AM2) protein (2) contains a homotetramer channel that transports protons across the viral membrane and acidifies the virion interior, enabling the dissociation of the viral matrix proteins, which is a crucial step in viral replication (3). The protein has been the target of antiviral drugs amantadine and rimantadine (4, 5). Much effort has been devoted to discovering the structure and proton transport (PT) mechanism of the AM2 channel, resulting in many crystal structures available in the protein data bank (6-14). Based on the crystal structures and electrophysiology experiments, several PT models have been suggested. These mechanisms can be divided into two main categories, delineated by the role of the four histidine residues (a.k.a. the His37 tetrad) that reside in the middle of the AM2 transmembrane domain (AM2/TM) (Fig. 1A), which has been experimentally shown (15) to account for the proton permeation behavior of the full AM2 protein. The "shutter" mechanism (16,17) suggests that the His37 tetrad works as a gate. At low pH the gate opens due to the electrostatic repulsion between the biprotonated, positively charged histidine residues. The excess proton is then transferred through continuous water wire via the Grotthuss mechanism, without changing the pro...