Abstract
Proton transfer through membrane-bound ion channels is mediated by both water and polar residues of proteins, but the detailed molecular mechanism is challenging to determine. The tetrameric influenza A and B virus M2 proteins form canonical proton channels that use an HxxxW motif for proton selectivity and gating. The BM2 channel also contains a second histidine (His), H27, equidistant from the gating tryptophan, which leads to a symmetric H(19)xxxW(23)xxxH(27) motif. The proton-dissociation constants (pK(a)'s) of H19 in BM2 were found to be much lower than the pK(a)'s of H37 in AM2. To determine if the lower pK(a)'s result from H27-facilitated proton dissociation of H19, we have now investigated a H27A mutant of BM2 using solid-state NMR. (15)N NMR spectra indicate that removal of the second histidine converted the protonation and tautomeric equilibria of H19 to be similar to the H37 behavior in AM2, indicating that the peripheral H27 is indeed the origin of the low pK(a)'s of H19 in wild-type BM2. Measured interhelical distances between W23 sidechains indicate that the pore constriction at W23 increases with the H19 tetrad charge but is independent of the H27A mutation. These results indicate that H27 both accelerates proton dissociation from H19 to increase the inward proton conductance and causes the small reverse conductance of BM2. The proton relay between H19 and H27 is likely mediated by the intervening gating tryptophan through cation-π interactions. This relayed proton transfer may exist in other ion channels and has implications for the design of imidazole-based synthetic proton channels.