Solid-state 19F NMR spectroscopy reveals that Trp41 participates in the gating mechanism of the M2 proton channel of influenza A virus.

The integral membrane protein M2 of influenza A virus assembles as a tetrameric bundle to form a proton-conducting channel that is activated by low pH. The side chain of His37 in the transmembrane alpha-helix is known to play an important role in the pH activation of the proton channel. It has also been suggested that Trp41, which is located in an adjacent turn of the helix, forms part of the gating mechanism. Here, a synthetic 25-residue peptide containing the M2 transmembrane domain was labeled with 6F-Trp41 and studied in lipid membranes by solid-state 19F NMR. We monitored the pH-dependent differences in the 19F dipolar couplings and motionally narrowed chemical shift anisotropies of this 6F-Trp41 residue, and we discuss the pH activation mechanism of the H+ channel. At pH 8.0, the structural parameters implicate an inactivated state, while at pH 5.3 the tryptophan conformation represents the activated state. With the aid of COSMOS force field simulations, we have obtained new side-chain torsion angles for Trp41 in the inactivated state (chi1 = -100 degrees +/- 10 degrees , chi2 = +110 degrees +/- 10 degrees ), and we predict a most probable activated state with chi1 = -50 degrees +/- 10 degrees and chi2 = +115 degrees +/- 10 degrees . We have also validated the torsion angles of His37 in the inactivated state as chi1 = -175 degrees +/- 10 degrees and chi2 = -170 degrees +/- 10 degrees .