M2 amphipathic helices facilitate pH-dependent conformational transition in influenza A virus

Abstract

The matrix-2 (M2) protein from influenza A virus is a tetrameric, integral transmembrane (TM) protein that plays a vital role in viral replication by proton flux into the virus. The His37 tetrad is a pH sensor in the center of the M2 TM helix that activates the channel in response to the low endosomal pH. M2 consists of different regions that are believed to be involved in membrane targeting, packaging, nucleocapsid binding, and proton transport. Although M2 has been the target of many experimental and theoretical studies that have led to significant insights into its structure and function under differing conditions, the main mechanism of proton transport, its conformational dynamics, and the role of the amphipathic helices (AHs) on proton conductance remain elusive. To this end, we have applied explicit solvent constant pH molecular dynamics using the multisite λ-dynamics approach (CpHMD^MSλD) to investigate the buried ionizable residues comprehensively and to elucidate their effect on the conformational transition. Our model recapitulates the pH-dependent conformational transition of M2 from closed to open state when the AH domain is included in the M2 construct, revealing the role of the amphipathic helices on this transition and shedding light on the proton-transport mechanism. This work demonstrates the importance of including the amphipathic helices in future experimental and theoretical studies of ion channels. Finally, our work shows that explicit solvent CpHMD^MSλD provides a realistic pH-dependent model for membrane proteins.

Keywords: M2 transmembrane protein; amphipathic helix; explicit solvent CpHMD.

Fig. 1.

Structure of M2. (A) M2CD construct (residues 22 to 62) contains TM and AH domains. (B) M2TM construct (residues 22 to 46) only contains TM domain. Phosphorous atoms of the lipid bilayer are presented in green van der Waals (VDW) spheres. His37, Asp44, and Arg45 are shown in licorice, and Gly34 is shown in VDW spheres.

Fig. 2.

Characterization of the His37 tetrad in the M2CD and M2TM constructs. Unprotonated fraction (S_unprot) (A) and the interhelical distance for histidine tetrad as a function of pH in M2TM and M2CD constructs (B). The dashed blue and red lines show the average values within the corresponded pH range.

Fig. 3.

The hydrogen-bond analysis. (A) Hydrogen bond between two pairs of histidine dimers in M2CD and M2TM constructs. (B) Hydrogen bond between H_δ and H_ε of histidine residues and water molecules in M2CD constructs.

Fig. 4.

Structural comparison of the M2TM and M2CD constructs. The χ₂ angle for the histidine tetrad (A) and distance between Asp44 and Arg45 in M2TM and M2CD at different pH conditions (B) are shown.

Fig. 5.

The effect of the amphipathic domain on conformational transition of M2CD construct. (A) Tilt angle of the C-terminal portion of the TM domain in M2CD and M2TM constructs. The dashed blue and red lines show the averaged value in the corresponding pH range. (B) Tilt angle of amphipathic helices. The values are averages over the two opposite helices. (C and D) The principal components of motion calculated using PCA in the open state (C) and the closed state (D) show the magnitude and direction of the TM and amphipathic helices’ mobility.