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 pH8 in bilayers and is virtually inactive at external pH7.4 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 pH7.4 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.