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

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 J-couplings of 15N His37-labeled full length M2 (M2FL) protein from Influenza A virus embedded in synthetic liquid crystalline lipid bilayers using two-dimensional J-resolved NMR spectroscopy. We experimentally observed the hydrogen-bond mediated J-couplings between Nδ1 and Ne2 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 transferr...