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

Abstract

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 ...

2D IR spectroscopy reveals the role of water in the binding of channel-blocking drugs to the influenza M2 channel

“…Noting that the MD simulations were performed with pH 6.5 crystal structures, it is possible that the fast exchange dynamics observed herein reflect not merely a reorganization of solvent density around differently solvated backbone amides but also reorganization of the water near Ala30 in response to hopping and translocation of a proton between different sites. Considering the strong affinity for water of a protonated histidine, 60 and the proximity of Gly34 to the histidine tetrad, which allows water molecules to bridge between the glycine amide and the imidazole nitrogen, 28 it can be expected that the water near Gly34 is more ordered compared to that near the Ala30 site. This is reflected in the absence of any measurable solvent exchange at the Gly34 site.…”

“…This tetrad of water molecules has also been observed in X-ray crystallographic measurements. 28 Guided by these previous observations and predictions, a set of ammonium-containing spiro compounds has recently been designed to target the M2 channel with the expectation that drug binding would be significantly stabilized through interactions with the aforementioned carbonyl tetrads and the distinctive water clusters in the channel. 23 While experimental results 23 indeed indicate that this series of spiro compounds has high affinity toward the M2 channel, MD simulations 23 suggest that the spiro compound binds in an amantadine-like mode deeper within the channel, docking itself in the pore through interactions with the water cluster near Gly34 (Figure 1(c)).…”

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

“…However, the water dynamics in the AM2 protein probed using 2D infrared (2D-IR) spectroscopy revealed that the well-ordered "ice-like" pore water dynamics at pH 8.0 change to more mobile and "liquid-like" dynamics (on the timescale of a few picoseconds) at pH 3.2 (23). This result suggests an interesting pH-dependent behavior of the AM2 protein that is highly relevant for understanding its PT mechanism.Although many computational studies have investigated the features of AM2 that may influence its PT in recent years (12,(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40), only a few have explicitly simulated any aspect of the explicit PT process (24,25,29,37,38). This is because it is challenging to accurately model the charge delocalization and Grotthuss shuttling of the hydrated excess proton in a computationally tractable way.…”

“…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)(7)(8)(9)(10)(11)(12)(13)(14). Based on the crystal structures and electrophysiology experiments, several PT models have been suggested.…”

A simple recipe for setting up the flux equations of cyclic and linear reaction schemes of ion transport with a high number of states: The arrow scheme

“…These reactions imply a cyclic reaction scheme and are related to the resetting of the protonation-induced tilting of the helices. 33,34 For further details see. 11…”