Pore-Bound Water at the Key Residue Histidine 37 in Influenza A M2

Atomic details of structured water molecules are indispensable to understand the thermodynamics of important biological processes including the proton conduction mechanism of the M2 protein. Despite the expectation of structured water molecules based on crystal structures of Influenza A M2, only two water populations have been observed by NMR in reconstituted lipid bilayer samples. These are the bulk- and lipid-associated water populations typically seen in membrane samples. Here, we detect a bound water molecule at a chemical shift of 11 ppm, located near the functional histidine 37 residue in the M2 conductance domain, which comprises residues 18 to 60. Combining 100 kHz magic-angle spinning NMR, dynamic nuclear polarization and density functional theory calculations, we show that the bound water forms a hydrogen bond to the δ1 nitrogen of histidine 37.