# Structural Transition from Closed to Open for the Influenza A M2 Proton Channel as Observed by Proton-Detected Solid-State NMR

## TL;DR

This study uses advanced NMR techniques to observe how the influenza A M2 proton channel transitions from a closed to an open state under different pH conditions.

## Contribution

The study provides new insights into the structural dynamics of the M2 proton channel during acid activation using proton-detected solid-state NMR.

## Key findings

- At pH 7.8, the M2 channel is closed with rigid H37 side chains and conformational heterogeneity.
- At pH 6.0, H37 side chains become dynamic, enabling proton transport.
- At pH 4.5, the channel adopts a well-defined open state with dynamic H37 and amphipathic helix regions.

## Abstract

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.