Side Chain Structures of the Proton-Selective Histidine and Gating Tryptophan in Influenza BM2 Reveal Both Conservation and Variation of the Proton Conduction Mechanism.

Aromatic residues play important roles in protein structure and function, but are difficult to study at atomic resolution by NMR because of their low spectral sensitivity and resolution. The M2 proton channels of influenza A and B viruses use a histidine for proton selection and a tryptophan for gating. High-resolution structures and dynamics of His37 and Trp41 side chains in AM2 have provided detailed insights into the proton conduction mechanism of AM2. However, the side chain structures of the corresponding His19 and Trp23 in BM2 have not been established. Here, we directly determine the side chain conformations of His19 and Trp23 using C-N and C-F distance measurements. Interestingly, we find that His19 adopts a distribution of χ torsion angles: the major conformer places the imidazole ring in a tilted and partly transverse orientation from the channel axis, while a minor population orients the imidazole ring parallel to the channel axis, similar to His37 in AM2. Trp23 adopts χ and χ angles similar to those of Trp41 in AM2, but the indole ring orientation inside the pore differs moderately from that of Trp41 due to backbone conformational differences. Finally, a membrane-surface histidine in BM2, His27, is dynamic, consistent with its distinct function from His19. These results provide new insights into the structural basis for the different proton conduction behaviors of influenza AM2 and BM2 and illustrate how interactions among aromatic residues influence the structure and function of membrane proteins.