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

Abstract

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 ...

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

“…Helix kinking in the apo form has been observed by X-ray crystallography (21), EPR (5), and solid-state NMR (23). A very recent MD study of the apo form with various protontation states of His-37 also showed large kink angles (20).…”

“…Based on MD simulations, Khurana et al (20) proposed a transporter-like mechanism for proton conductance, in which the M2 TMD cycles between outside open/inside closed and outside closed/inside open conformations and His-37 is protonated/deprotonated during each cycle. Here, we put forward a more conventional structural model, by injecting conformational details into the proton relay mechanism proposed by Pinto et al (14).…”

“…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)(12)(13)(14)(15)(16) and computational (17)(18)(19)(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).…”

Exploring the Proton Conductance and Drug Resistance of BM2 Channel through Molecular Dynamics Simulations and Free Energy Calculations at Different pH Conditions

“…or 2+ (such as "bddb", "bedd", etc.) states, their subsystems demonstrated that the His19 gate was open or closed in response to the specific protonation state of each His19 residue; see Figure S3 30 the open gate of the BM2 channel is the consequence of the electrostatic repulsion between the histidine residues being protonated at a low pH condition (see Figures S3 and S4 of the Supporting Information).…”

Viroporins: structure, function and potential as antiviral targets

“…This allows one His of each pair to interact with adjacent Trp41, whereupon a third protonation event induces channel opening via alteration of the helical bundle and opening the Trp41 gate (Chizhmakov et al, 1996;Pielak & Chou, 2010). However, alternative models for M2 gating are also proposed, including a 'shuttle' mechanism of proton conductance, whereby exchange of protons between His37 and water residues are facilitated by imidazole ring reorientations (Hong & DeGrado, 2012;Hu et al, 2010;Khurana et al, 2009;Phongphanphanee et al, 2010). Thus, despite its apparent simplicity compared with cellular ion channels and a wealth of structural information, the fundamental properties of this viroporin paradigm remain a topic of considerable debate.…”