Structure and mechanism of proton transport through the transmembrane tetrameric M2 protein bundle of the influenza A virus

Abstract

The M2 proton channel from influenza A virus is an essential protein that mediates transport of protons across the viral envelope. This protein has a single transmembrane helix, which tetramerizes into the active channel. At the heart of the conduction mechanism is the exchange of protons between the His37 imidazole moieties of M2 and waters confined to the M2 bundle interior. Protons are conducted as the total charge of the four His37 side chains passes through 2(+) and 3(+) with a pK(a) near 6. A 1.65 A resolution X-ray structure of the transmembrane protein (residues 25-46), crystallized at pH 6.5, reveals a pore that is lined by alternating layers of sidechains and well-ordered water clusters, which offer a pathway for proton conduction. The His37 residues form a box-like structure, bounded on either side by water clusters with well-ordered oxygen atoms at close distance. The conformation of the protein, which is intermediate between structures previously solved at higher and lower pH, suggests a mechanism by which conformational changes might facilitate asymmetric diffusion through the channel in the presence of a proton gradient. Moreover, protons diffusing through the channel need not be localized to a single His37 imidazole, but instead may be delocalized over the entire His-box and associated water clusters. Thus, the new crystal structure provides a possible unification of the discrete site versus continuum conduction models.

Fig. 1.

Structure of M2TM′. (A) The backbone of three monomers is drawn in light gray, and the pore-lining side chain groups are shown as sticks. From the N-terminus (viral exterior) to the C-terminus (viral interior), these are: the Val27 valve (blue), Ser31 (dark gray), the His-box (orange), the Trp-basket (purple), and the Asp/Arg-box (light blue). Water molecules belonging to the entry, bridging, and exit clusters are represented by red spheres. Black lines indicate the observed water–protein H-bonds. The uppermost dimer of waters in the entry cluster exists in two nearly equally occupied configurations in the crystal lattice, related by a 90° rotation down the central axis of the channel. Only one of the two orientations is shown in the figure. In the exit cluster the fifth water molecule (showing a high Debye–Waller factor) is drawn transparent and with a larger radius. The wireframe represents the peak region of the diffuse electron density detected right under the Val27 valve. (B) A closer view of the His-box surrounded by the entry and bridging clusters. (C) A closer view of the exit cluster.

Fig. 2.

M2TM helix bundle in different experimental structures, with Val27, His37, Trp41, Asp44, and Arg45 color coded as in Fig. 1. (A) TM portion of the previously reported NMR structure at pH 7.5–8 (17) (Left). The X-ray structure at pH 6.5 presented here (Center). The previously reported X-ray structure at pH 5.3 (16) (Right). The blue and red cylinders in the top row highlight, respectively, the N- and C-terminal portions of the helices and their angle with respect to the bundle axis. In the X-ray structure presented here the C-terminal portion shows the same angle as the NMR structure (17), whereas the N-terminal one closely resembles that of the previous X-ray structure (16). There is a 12° bend between these two helical sections through several residues around Ala34. The Val27 valve constricts with decreasing pH, whereas the Trp-basket opens up. Importantly, the Trp sidechain rotamer is different in the high pH NMR structure (Lower Left) than in the intermediate pH structure reported here (Lower Center). (B) M2TM′ crystal structure with its backbone B-factors represented by color and helix thickness. B-factors for the crystal structure were normalized, (B - 〈B〉)/σ(B), percent ranked, and averaged over the four helices.

Fig. 3.

Spatial distribution of pore waters and structural stability of the bundle. (A) Average number of water molecules localized within the pocket between the His and the Trp tetrads. (B) The water oxygen density from the simulation of the state (light blue shading) is plotted together with the experimental one (red surfaces). The overall structure of the bundle is also presented (gray shading) and the sidechains of some of functionally relevant residues are highlighted as sticks: Val27 (blue), His37 (orange), and Trp41 (green). (C) The rmsd of the heavy atoms from the initial (X-ray) structure is plotted as a function of MD simulation time for the protonation states (red), (gold), (purple), and 4⁺ (blue), respectively.

Fig. 4.

Effect of mutations of pore-lining residues. (A) The density of water oxygen as a function of the location in the channel pore (z) for the wild type (WT) and 2 mutants (G34A and G34V) is reported in black, red, and blue, respectively. Density profiles were obtained from MD simulations of the 2_ε state of each variant. Shaded areas mark peaks conserved in all mutants. The shape of the channel pore is depicted in blue shading along with the sidechains of pore-lining residues. The density in the region adjacent to residue 34 decreases with the increased size of the sidechain and drops to zero for the G34V mutant. (B) Specific activity measurements for M2 wild type, G34A and G34V in Xenopus laevis oocytes. The amplitude of the channel activity (I) was plotted as a function of the immunosignal intensity for each tested oocyte and a straight line was fitted to the data. The slope of each plot represents the relative specific activity for the protein: 6.6 ± 1.5 for M2 and 2.7 ± 0.4 for M2-G34A. M2-G34V showed membrane expression comparable to the wild type, but no detectable channel activity. (C) Comparison of channel activity and proton selectivity between full-length M2, and the G34A and G34V mutants. The channel activity was evoked by rapid exchange of nonactivating solution (pH 8.5) with activating solution (pH 5.5). The reversal voltages were measured in Na⁺, K⁺ and NMDG⁺—based activating solutions and showed no significant differences in the ion selectivity and channel properties between the M2 wild type and the G34A mutant. G34V mutant (marked with *) did not show any pH induced channel activity. Each bar is the mean (± SD) of 5 independent experiments.

Fig. 5.

M2TM conformational ensemble. (A) Cartoon illustration of the change in the energy landscape as a function of the pH at the N-terminus. Three conformational states of M2TM have been identified by X-ray crystallography and NMR at different pH (16, 17). They are: the O_Out, intermediate (INT), and O_In states, depicted in panels B, C, and D, respectively. Each conformation can exist in a variety of protonation states: in this paper we have performed a study of the INT state. As the pH decreases the favored state changes from the high pH O_Out state to INT and then to O_In. The O_Out and INT states feature different tilt angles of the N-terminal portions of the helices (see also Fig. 2) and rotamers of the Trp41 side chains. The INT and O_In states instead show different conformations of the C-terminal portions.