PH Titration and Acid Activation of the Full-Length Influenza a M2 Proton Channel in Lipid Bilayers

Abstract

The Influenza A M2 protein forms an acid-activated proton-selective tetrameric channel, in which His37 acts as both the pH sensor and selectivity filter. Here we report on the exquisite pH-dependent changes in conformation and dynamics of the His37 tetrad, for the first time, in the full-length (FL) protein, as probed by solid-state NMR spectroscopy (2D 13C-13C, 15N-13C, and 1D 15N) in lipid bilayers. From pH 8.8 to 7.3, the NMR signals show three different tau His37 sidechain conformations and one pai tautomer, with one of the tau Ne2 peaks significantly down-field shifted implicating a strong interaction (e.g., hydrogen bonding) at this site. At pH 6.6 the His37 tetrad starts to be protonated with the arising of new signals from the charged His37 and at below pH 5.8 the tetrad starts to enter the triply protonated state by quantification calculation. The narrow pH range over which the first two protons are added suggests cooperativity. Linewidths are significantly broadened at pH 5.8; this and additional data indicate considerable dynamics and conformational exchange. In addition, a cross peak between a tau Ce1 and a charged Cd2 appears at pH 5.8, indicating a short distance between them. The more complex spectra of M2FL than that of the M2 truncated versions reveal a functional mechanism in which the channel is blocked by His37-His37 hydrogen bonding at neutral pHs, whereas at low pHs sidechain dynamics allows the permeating proton to transfer from the extracellular side of the His37 tetrad to the intracellular side.