pH-induced conformational change of the influenza M2 protein C-terminal domain

Abstract

The M2 protein from influenza A is a pH-activated proton channel that plays an essential role in the viral life cycle and serves as a drug target. Using spin labeling EPR spectroscopy, we studied a 38-residue M2 peptide spanning the transmembrane region and its C-terminal extension. We obtained residue-specific environmental parameters under both high- and low-pH conditions for nine consecutive C-terminal sites. The region forms a membrane surface helix at both high and low pH, although the arrangement of the monomers within the tetramer changes with pH. Both electrophysiology and EPR data point to a critical role for residue Lys 49.

FIGURE 1

Effect of cysteine substitutions on ion channel function of M2. Channel activity and reversal potential (V_rev) were measured at pH 5.5 in Xenopus laevis oocytes expressing full-length wild-type M2, cysteine-less M2, and M2 constructs derived from cysteine-scanning mutagenesis. * indicates that the V_rev of K49C construct could not be determined accurately due to its very low specific activity.

FIGURE 2

pH-dependent conformational change. (A and B) CW-EPR spectra of spin labels at positions 48–56 on the M2TMC peptide reconstituted into POPC/POPG (4:1) lipid bilayers at pH 7.8 (A) and pH 5.6 (B). Overlay of spectra of fully labeled tetrameric channels (black) and spectra of dilute-labeled channels (grey). (C and D) Superposition of lipid (C) and water (D) accessibility profiles at pH 7.8 and pH 5.6. Details of best-fit sin waves to accessibility data are in the Supporting Information. (E) Superposition of per-residue intersubunit proximity patterns based on the interaction parameter measured at pH 7.8 and pH 5.6. Data for position 49 are boxed in grey to highlight that the K49C mutation causes loss of channel function.

FIGURE 3

Ensemble of models selected using EPR data. (A) Superposition of six most favorable scoring models using EPR data collected at pH 7.8 where the channel is in the resting, non-conductive state. (B) Superposition of six most favorable scoring models using EPR data collected at pH 5.6 where the channel is activated for proton conduction.