Mechanistic insights from functional characterization of an unnatural His37 mutant of the influenza A/M2 protein

•Proton transport through M2 involves protonation/deprotonation of His37 sidechains.•It is unclear whether the intermediate step is His37 ring flip, tautomerization, or both.•His37 imidazole to 4-thiazole mutant: functional, stable and drug-sensitive•Lower pKa of 4-thiazole mutant results in transport activation at lower pH.•4-Thiazole group cannot tautomerize: transport proceeds without tautomerization.

The influenza A/M2 protein is a homotetrameric single-pass integral membrane protein encoded by the influenza A viral genome. Its transmembrane domain represents both a crucial drug target and a minimalistic model system for transmembrane proton transport and charge stabilization. Recent structural and functional studies of M2 have suggested that the proton transport mechanism involves sequential extraviral protonation and intraviral deprotonation of a highly conserved His37 side chain by the transported proton, consistent with a pH-activated proton shuttle mechanism. Multiple tautomeric forms of His can be formed, and it is not known whether they contribute to the mechanism of proton shuttling. Here we present the thermodynamic and functional characterization of an unnatural amino acid mutant at His37, where the imidazole side chain is substituted with a 4-thiazolyl group that is unable to undergo tautomerization and has a significantly lower solution pKa. The mutant construct has a similar stability to the wild-type protein at pH 8 in bilayers and is virtually inactive at external pH 7.4 in a semiquantitative liposome flux assay as expected from its lower sidechain pKa. However when the external buffer pH is lowered to 4.9 and 2.4, the mutant shows increasing amantadine sensitive flux of a similar magnitude to that of the wild type construct at pH 7.4 and 4.9 respectively. These findings are in line with mechanistic hypotheses suggesting that proton flux through M2 is mediated by proton exchange from adjacent water molecules with the His37 sidechain, and that tautomerization is not required for proton translocation. This article is part of a Special Issue entitled: Viral Membrane Proteins — Channels for Cellular Networking.

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