The influenza A M2 protein is an acid-activated proton channel and an established pharmaceutical target for antiflu drugs. Here, we studied the conductance domain of the tetrameric M2 channel (construct 18–60) using proton-detected solid-state NMR under native-like conditions in lipid bilayers. We obtained results at different pH values relevant to the virus life cycle: pH 7.8 (nonconducting, closed), pH 6.0 (opening), and pH 4.5 (conducting, fully open). In the closed state at pH 7.8, we detected two sets of resonances of the functionally important side chain of H37. Employing quantum mechanics/molecular mechanics (QM/MM) simulations, we assigned them to hydrogen-bonded and free H37 side chains occurring in varying ratios in the tetrameric arrangement. Additionally, some backbone signals also appear twice, suggesting conformational heterogeneity. The arrangement appears rather rigid, explaining the nonconducting nature of the channel. Lowering the pH to 6.0 leads to increased dynamics of the side chains, as manifested by their disappearance in CP based solid-state NMR spectra. This dynamic arrangement, which results from additional protonation of the four H37 side chains, allows for the efficient transport of protons through the channel. Finally, at pH 4.5, the conformational heterogeneity observed at higher pH values disappears completely, and a unique set of highly resolved resonances becomes visible. This suggests a well-defined acid-activated state of the M2 channel. Notably, in this state, the signals of the His37 side chains are absent due to dynamics, as well as the signals of the amphipathic helix (residues 45–52). This study provides strong evidence to a model of proton conduction through M2 which relies on dynamic vs rigid H37 side chains and furthermore lays the basis for an atomic structure of the acid-activated state of M2.
