High-resolution structures of the M2 channel from influenza A virus reveal dynamic pathways for proton stabilization and transduction

Abstract

The matrix 2 (M2) protein from influenza A virus is a proton channel that uses His37 as a selectivity filter. Here we report high-resolution (1.10 Å) cryogenic crystallographic structures of the transmembrane domain of M2 at low and high pH. These structures reveal that waters within the pore form hydrogen-bonded networks or "water wires" spanning 17 Å from the channel entrance to His37. Pore-lining carbonyl groups are well situated to stabilize hydronium via second-shell interactions involving bridging water molecules. In addition, room temperature crystallographic structures indicate that water becomes increasingly fluid with increasing temperature and decreasing pH, despite the higher electrostatic field. Complementary molecular dynamics simulations reveal a collective switch of hydrogen bond orientations that can contribute to the directionality of proton flux as His37 is dynamically protonated and deprotonated in the conduction cycle.

Keywords: influenza M2 channel; membrane proteins; proton channels.

Fig. 1.

Crystal structure of M2TM at 1.10 Å at pH 8.0 (4QK7) under cryogenic conditions. (A) Superposition of this structure (green) with previously solved structures of M2 2LOJ (12) (orange), 3LBW (9) (yellow), and 3BKD (8) (red) shows that the conformation of the N-terminal half of the channel is relatively conserved whereas the conformation of the C terminus varies. Waters from the high-pH cryogenic structure (red spheres) are overlaid with waters from the low-pH cryogenic structure (pink spheres). (B) Density of backbone and side chains of a monomer of M2TM at a contour of 3.0 σ shows features typical of high-resolution structures. (C) Ser31 side chain (conformer A and B) at a contour of 1.0 σ. (D) His37 side chain at a contour of 3.0 σ. (E) Trp41 side chain (conformer A and B) at a contour of 1.5 σ.

Fig. 2.

Waters in the cryogenic crystal structures form ordered water wires whereas room temperature waters are less ordered. The front helix of each tetramer has been removed. (Top) Water wires inferred from crystallographic water positions from the 1.10-Å resolution cryo crystal structures at pH 8.0 (Left, PDB code 4QK7) and pH 5.5 (Right, PDB code 4QKC) form a continuous network of water wires within the N-terminal half of the pore leading to the gating His37 residue, with some positional changes in the waters between the two pH conditions. Alternate occupancy water-wire networks A (light blue) and B (dark blue) intersect at full occupancy waters (red). Continuous water wires span 16.8 Å of the channel pore. (Bottom) Water networks observed under room temperature conditions at pH 8.0 at a 1.71-Å resolution (Left, PDB code 4QKL) and pH 5.5 at a 1.44-Å resolution (Right, PDB code 4QKM) have fewer ordered waters and no longer form a continuous path leading to His37; in the low-pH structure, the waters do not form ordered networks at all and instead appear fluid-like.

Fig. 3.

Electron density comparisons show differences and similarities between the solvent density at high and low pH. The front helices have been removed. Electron density at a contour of 1.5 sigma under (A) cryogenic conditions, (B) room temperature conditions, and (C) calculated electron density from molecular dynamics simulations is shown for both high (green) and low (magenta) pH experiments/simulations (high pH corresponds to a neutral His37 cluster, and low pH to a 4⁺ charge state). Areas where low- and high-pH densities overlap are shown in gray. Electron density from water binding to carbonyl groups and histidines is relatively conserved under all conditions whereas variations in density are seen elsewhere.

Fig. 4.

Hydrogen bond orientation calculated from MD simulations. (A) Schematic representation of the water wires and the hydrogen bond orientation. The oxygens of water molecules observed in the room temperature X-ray structures are shown as van der Waals spheres (orange color) with diameter proportional to the crystallographic B-factors and transparency reflecting the partial occupancy. Hydrogen bonds are displayed as sticks with a color scale denoting their orientation and the thickness proportional to their population. (B) Water wires for a neutral channel. (C) Water wires at the 4⁺ charge state.