Structure and Dynamics of the Proton-Selective Histidine and the Gating Tryptophan in an Inward Rectifying Hybrid Influenza B and A Virus M2 Proton Channel

Abstract

The M2 proteins of influenza A and B viruses form acid-activated proton channels that are essential for the virus lifecycle. Proton selectivity of M2 channels is achieved by a transmembrane (TM) histidine (H) whereas gating is fulfilled by a tryptophan (W) residue. Despite this conserved functional apparatus, AM2 conducts protons exclusively inward whereas BM2 conducts protons in both directions under suitable pH gradients. Previous data showed that residue D44 of AM2 stabilizes the W41 gate: mutation of D44 abolished inward rectification. To elucidate the role of residues C-terminal to the gating tryptophan in proton conduction, here we investigate the structure and dynamics of H19 and W23 in a BM2 mutant, GDR-BM2, in which three BM2 residues are mutated to the corresponding AM2 residues, S16G, G26D and H27R. GDR-BM2 conducts protons with strong inward rectification that is indistinguishable from wild-type AM2 but distinct from wild-type BM2. 15N and 13C solid-state NMR spectra reveal that GDR-BM2 channel contains higher populations of cationic and neutral τ tautomeric H19 compared to wild-type BM2 at pH 5.5. Remarkably, 19F NMR spectra of 5-19F-labeled W23 reveal three well-ordered conformations at acidic pH. Comparison of these 19F chemical shifts with previously measured spectra of AM2 and BM2 peptides suggests that the distinct indole conformations arise from tryptophan interactions with the C-terminal charged residues and the N-terminal cationic histidine. Taken together, these solid-state NMR data show that inward rectification in M2 proton channels is accomplished by tryptophan interactions with charged residues on both its C-terminal side and its N-terminal side. Thus, gating of the influenza M2 proton channels is achieved by a multi-residue complex with finely tuned electrostatic and aromatic interactions.