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.

A Secondary Gate As a Mechanism for Inhibition of the M2 Proton Channel by Amantadine

“…30,31 The X-ray structures confirm the most important features observed in our MD simulations (Supporting Information Figure S6). In particular, the channel pore "is most constricted near Val27", providing direct support of our proposal for a secondary gate.…”

Flu channel drug resistance: a tale of two sites

“…An AM2 construct including residues 18 to 60, AM2(18-60), in dihexanoylphosphocholine (DHPC) detergent micelles met these requirements. This construct, which includes the TM domain as well as 15 residues of the C-terminal extension forms a very stable tetramer and yields high-quality NMR spectra (Schnell and Chou, 2008). It has also been confirmed that when reconstituted into liposomes, AM2(18-60) exhibits specific proton conductance that can be inhibited by as low as 10 μM of rimantadine .…”

“…In 2008, using solution NMR spectroscopy, Schnell and Chou determined the first atomic resolution structure of the closed AM2 channel in the presence of the anti-influenza drug rimantadine (Schnell and Chou, 2008). One of the main challenges of solution NMR study of membrane proteins is finding a combination of protein construct and detergent system that both supports the native function of the protein and results in high quality NMR spectra.…”

“…Therefore, given the available data, we cannot comment on whether the crystal structure in Fig structures, both experimentally determined to high resolution at a similar pH, is the different lengths of constructs used in the two studies. The AM2(22-46) construct used in the crystallographic study does not contain the C-terminal region (residues 47-60) that was shown to be critical for a stable tetramer formation (Schnell and Chou, 2008) and native-like conductance ).…”

“…The NMR pH titration experiment showed that lowering the pH from 7.5 to 6.0 caused severe broadening of most of the TM NMR resonances due to exchange between multiple conformations (Schnell and Chou, 2008). Relaxationcompensated Carr-Purcell-Meiboom-Gill (CPMG) measurement (Loria et al, 1999) of millisecond timescale dynamics of the channel gate also supported a more dynamic view of the open state (Schnell and Chou, 2008). Comparison of the CPMG measurements of the Trp41 indole amine at different pH showed that as the pH was lowered from 7.5 to 6.0, the chemical exchange rate increased by more than four-fold.…”