Structure and function of the influenza A M2 proton channel

Abstract

The M2 protein of influenza A viruses forms a tetrameric pH-activated proton-selective channel that is targeted by the amantadine class of antiviral drugs. Its ion channel function has been extensively studied by electrophysiology and mutagenesis; however, the molecular mechanism of proton transport is still elusive, and the mechanism of inhibition by amantadine is controversial. We review the functional data on proton channel activity, molecular dynamics simulations of the proton conduction mechanism, and high-resolution structural and dynamical information of this membrane protein in lipid bilayers and lipid-mimetic detergents. These studies indicate that elucidation of the structural basis of M2 channel activity and inhibition requires thorough examination of the complex dynamics and conformational plasticity of the protein in different lipid bilayers and lipid-mimetic environments.

Fig. 1

Amino acid sequence of influenza A/Udorn/72 M2 protein. The TM domain containing the crucial His₃₇ and Trp₄₁ residues (red) are underlined.

Fig. 2

(a) Interhelical ¹⁹F-¹⁹F spin diffusion data of 5-¹⁹F-Trp₄₁ WT M2 (red) and 4-¹⁹F-A30F M2 (black) in DMPC bilayers (59, 67). The best-fit distances confirm the tetrameric state of the peptide, and constrain the Trp₄₁ sidechain (χ₁, χ₂) torsion angles (inset). (b) Trp₄₁ t90 and His₃₇ t-160 rotamers proposed from the distance data.

Fig. 3

¹³C MAS spectra of DLPC-bound M2(22–46). (a) Variable temperature ¹³C MAS spectra indicate large-amplitude intermediate timescale motion at ambient temperature (54). (b) Amantadine narrows the linewidths of most residues at 313 K (55).

Fig. 4

Uniaxial diffusion of M2(22–46) helical bundles around the bilayer normal. (a) ¹⁵N CSA of L26 is uniaxially averaged from the rigid-limit pattern (dashed line) (54). (b) L26 N–H dipolar coupling is reduced from the rigid-limit coupling. (c) Amantadine decreases ¹H T_1ρ relaxation rates at high temperature, indicating it speeds up protein motion (71). (d) Schematic of M2 uniaxial diffusion. Amantadine binding creates better packed tetramers, thus speeding up rotational diffusion.

Fig. 5

High-resolution structures of the TM domain of AM2. (a) Amantadine-bound orientational structure of M2(22–46) in DMPC bilayers at pH 8.8 from oriented-membrane SSNMR (PDB: 2H95) (48). The drug binding site was not directly studied but was implicitly assumed to be in the pore at the N-terminus side. (b) Amantadine-bound crystal structure of M2(22–46) in OG at pH 5.3 (PDB: 3C9J) (35). (c) Rimantadine-bound solution NMR structure of M2(18–60) in DHPC micelles at pH 7.5 (PDB: 2RLF) (34). Only the TM part is shown. (d) Amantadine-bound structure of M2(22–46) in DLPC bilayers at pH 7.5 from MAS SSNMR (PDB: 2KAD) (36). The amantadine position is hypothesized based on chemical shift perturbation of S31. The bilayer planes are drawn for reference. In each structure, two of the Trp₄₁ indole rings (green) are shown, and the drug molecules are shown in red. IN (c), only two rimantadine molecules are shown for clarity.

Fig. 6

SSNMR evidence for amantadine binding near S31. (a) 2D ¹⁵N-¹³C correlation spectra of DLPC-bound M2(22–46) in the absence (black) and presence (red) of amantadine (36). (b) Chemical shift perturbation by amantadine in bilayer-bound M2(22–46).