Observation of the Imidazole-Imidazolium Hydrogen Bonds Responsible for Selective Proton Conductance in the Influenza A M2 Channel

Abstract

The integral membrane M2 protein is a 97-residue membrane protein that assembles as a tetramer to conduct protons at a slow rate (10²-10³/s) when activated by low pH. The proton conductance mechanism has been extensively debated in the literature, but it is accepted that the proton conductance is facilitated by hydrogen bonds involving the His37 residues. However, the hydrogen bonding partnership remains unresolved. Here, we report on the measurement of ¹⁵N-¹⁵N <i>J</i>-couplings of ¹⁵N His37-labeled full length M2 (M2FL) protein from <i>Influenza A</i> virus embedded in synthetic liquid crystalline lipid bilayers using two-dimensional <i>J</i>-resolved NMR spectroscopy. We experimentally observed the hydrogen-bond mediated <i>J</i>-couplings between N_δ1 and N_ε2 of adjacent His37 imidazole rings, providing direct evidence for the existence of various imidazolium-imidazole hydrogen-bonding geometries in the histidine tetrad at low pH, thus validating the proton conduction mechanism in the M2FL protein by which the proton is transferred through the breaking and reforming of the hydrogen bonds between pairs of His37 residues.