Conformational heterogeneity of the M2 proton channel and a structural model for channel activation

Abstract

The M2 protein of influenza virus A is a proton-selective ion channel activated by pH. Structure determination by solid-state and solution NMR and X-ray crystallography has contributed significantly to our understanding, but channel activation may involve conformations not captured by these studies. Indeed, solid-state NMR data demonstrate that the M2 protein possesses significant conformational heterogeneity. Here, we report molecular dynamics (MD) simulations of the M2 transmembrane domain (TMD) in the absence and presence of the antiviral drug amantadine. The ensembles of MD conformations for both apo and bound forms reproduced the NMR data well. The TMD helix was found to kink around Gly-34, where water molecules penetrated deeply into the backbone. The amantadine-bound form exhibited a single peak approximately 10 degrees in the distribution of helix-kink angle, but the apo form exhibited 2 peaks, approximately 0 degrees and 40 degrees . Conformations of the apo form with small and large kink angles had narrow and wide pores, respectively, around the primary gate formed by His-37 and Trp-41. We propose a structural model for channel activation, in which the small-kink conformations dominate before proton uptake by His-37 from the exterior, and proton uptake makes the large-kink conformations more favorable, thereby priming His-37 for proton release to the interior.

Fig. 1.

Comparison between experimental and MD-derived PISEMA resonances of the M2 TMD. For the apo form, the experimental data are taken from ref. . Values for Ser-31, Gly-34, and His-37 were not available. For the amantadine-bound form, the experimental data are taken from ref. . Values for Ser-31 and His-37 were not available. Typical differences between experimental and MD resonances amount to <5° errors in peptide-plane orientations. The helix tilt angle obtained from the MD results as a whole agrees with the experimental counterpart to within ≈2°; the helix rotation angle agrees to within ≈10°.

Fig. 2.

Normalized distributions of the cosine of helix-kink angle for apo and amantadine-bound M2 TMD. For the apo form, dot and hash patterns under the curve indicate the small- and large-kink populations, respectively (separated at cosθ = 0.91).

Fig. 3.

PISEMA resonances of 5 Leu residues (at positions 26, 36, 38, 40, and 43) calculated from the MD trajectories. There are 8 data points for each residue: the M2 TMD contains helices that are identical in sequence, and 2 independent MD trajectories were generated.

Fig. 4.

Water-backbone hydrogen bonding around Gly-34 in the absence (Left) and presence (Right) of amantadine. For clarity, only 2 of the 4 helices are shown, with Gly-34 presented as ball and stick. Typically each water molecule participated in a single water–Gly-34 hydrogen bond; sometimes a water molecule was found to bridge between helices. One water molecule, present in the apo form and shown as yellow spheres, simultaneously served as an acceptor to a backbone amide of Gly-34 and as a donor to a carbonyl of a neighboring residue in the same helix. Such water molecules are referred to as SDA.

Fig. 5.

Structural model for channel activation by pH. Vertical arrows indicate equilibration between small- and large-kink conformations; the lengths of these arrows indicate the magnitudes of tendencies. Horizontal arrows indicate proton release or uptake. The curly brackets signify that the small- and large-kink conformations are in fast exchange on the time scales of proton release and uptake. Note that in the protonated state it is the minority population of large-kink conformations that is most likely to release the proton.