Structure and mechanism of the M2 proton channel of influenza A virus

Abstract

The integral membrane protein M2 of influenza virus forms pH-gated proton channels in the viral lipid envelope. The low pH of an endosome activates the M2 channel before haemagglutinin-mediated fusion. Conductance of protons acidifies the viral interior and thereby facilitates dissociation of the matrix protein from the viral nucleoproteins--a required process for unpacking of the viral genome. In addition to its role in release of viral nucleoproteins, M2 in the trans-Golgi network (TGN) membrane prevents premature conformational rearrangement of newly synthesized haemagglutinin during transport to the cell surface by equilibrating the pH of the TGN with that of the host cell cytoplasm. Inhibiting the proton conductance of M2 using the anti-viral drug amantadine or rimantadine inhibits viral replication. Here we present the structure of the tetrameric M2 channel in complex with rimantadine, determined by NMR. In the closed state, four tightly packed transmembrane helices define a narrow channel, in which a 'tryptophan gate' is locked by intermolecular interactions with aspartic acid. A carboxy-terminal, amphipathic helix oriented nearly perpendicular to the transmembrane helix forms an inward-facing base. Lowering the pH destabilizes the transmembrane helical packing and unlocks the gate, admitting water to conduct protons, whereas the C-terminal base remains intact, preventing dissociation of the tetramer. Rimantadine binds at four equivalent sites near the gate on the lipid-facing side of the channel and stabilizes the closed conformation of the pore. Drug-resistance mutations are predicted to counter the effect of drug binding by either increasing the hydrophilicity of the pore or weakening helix-helix packing, thus facilitating channel opening.

Figure 1

Structure of the M2 channel. a, An ensemble of 15 low energy structures derived from NMR restraints. Since residues 47–50 are unstructured, the TM helices (residues 25–46) and the AP helices (residues 51–59) are superimposed separately. The backbone rmsd for the TM and AP helices are 0.30 Å and 0.56 Å, respectively. b, A ribbon representation of a typical structure from the ensemble in a, showing the left-handed packing of the TM helices, right-handed packing of the AP helices, the sidechains of His37 and Trp41, as well as the drug rimantadine (colored in red). c, A close-up view from the C-terminal side of the channel showing the Trp41 gate and how it is stabilized by the inter-monomer hydrogen bond between Trp41 Hε1 of one TM helix and Asp44 carboxyl of the adjacent TM helix. d, The surface representation of the rimantadine binding pocket, showing the Asp44, the indole amine of Trp41, and Arg45 that form the polar patch, as well as the hydrophobic wall composed of Leu40, Ile42, and Leu43.

Figure 2

Water accessibility of the M2 channel. a, Distribution of water NOEs relative to the structure. Amide protons colored in blue have a NOE crosspeak to water. Those that do not are colored red. b, The pore surface calculated using the program HOLE. The region of the channel colored in green is only wide enough to allow passing of a water molecule, where as the blue portion can accommodate two or more water molecules.

Figure 3

Low-pH induced destabilization of the channel and opening of the Trp41 gate. a, ¹H-¹⁵N TROSY spectra of reconstituted M2(18–60) tetramer at pH 6.0, 6.5, 7.0, and 7.5, in the absence of rimantadine, recorded at 500 MHz ¹H frequency and 30 °C. The coloring of the peaks is: green – TM helix; pink – AP helix; black – N-terminal loop. b, The ¹⁵N R₂ (pure R₂ + R_ex) of the Trp41 Nε1 as a function of the frequency of refocusing (1/τ_cp) of chemical shift evolution obtained at pH 7.5, 7.0, and 6.0, showing faster timescale motion of the Trp41 gate as the channel is activated. c, Comparsion between R₂(τ_cp) at pH 7.0 in the absence (blue) and presence (black) of rimantadine, demonstrating that the drug slows down the gate flipping at this pH.

Figure 4

Schematic illustration of M2 channel activation. At high pH, the TM helices are packed tightly and the tryptophan gate is locked through intermolecular interactions with Asp44. At low pH, protonation of the His37 imidazoles destabilizes the TM helix packing, allowing hydration of the channel pore, and proton conductance. The C-terminal base of the tetramer and N-terminal disulfide bonds keep the channel from completely disassembling. For clarity, only two of the four monomers are shown.