Dynamic Short Hydrogen Bonds in Histidine Tetrad of Full-Length M2 Proton Channel Reveal Tetrameric Structural Heterogeneity and Functional Mechanism

The tetrameric M2 protein from influenza A conducts protons into the virus upon acid activation of its His37 tetrad and is a proven drug target. Here, in studies of full-length M2 protein solubilized in native-like liquid-crystalline lipid bilayers, a pH titration monitored by solid-state nuclear magnetic resonance revealed a clustering of the first three His37 pKas (6.3, 6.3, and 5.5). When the +2 state of the tetrad accepts a third proton from the externally exposed portion of the channel pore and releases a proton to the internally exposed pore, successful proton conductance is achieved, but more frequently the tetrad accepts and returns the proton to the externally exposed pore, resulting in a futile cycle. Both dynamics and conformational heterogeneity of the His37 tetrad featuring short hydrogen bonds between imidazolium-imidazole pairs are characterized, and the heterogeneity appears to reflect oligomeric helix packing and the extent of transmembrane helical bending around Gly34. [Display omitted] •Unique His37 tetrad chemistry in full-length M2 proton channel defines function•His37 imidazolium-imidazole short hydrogen bonds exchange with water•Multiple His37 tetrad conformations reflect heterogeneous tetrameric helix packing•Hydrogen-bond exchange leads to both futile and proton conducting cycles Miao et al. have characterized the full-length M2 proton channel from influenza A by solid-state NMR in liquid-crystalline lipid bilayers. Hydronium ions attack short hydrogen-bonded His37 pairs, followed by proton release to yield either a futile or proton conducting cycle through the charged histidine tetrad.