Structural basis for proton conduction and inhibition by the influenza M2 protein

Abstract

The influenza M2 protein forms an acid-activated and drug-sensitive proton channel in the virus envelope that is important for the virus lifecycle. The functional properties and high-resolution structures of this proton channel have been extensively studied to understand the mechanisms of proton conduction and drug inhibition. We review biochemical and electrophysiological studies of M2 and discuss how high-resolution structures have transformed our understanding of this proton channel. Comparison of structures obtained in different membrane-mimetic solvents and under different pH using X-ray crystallography, solution NMR, and solid-state NMR spectroscopy revealed how the M2 structure depends on the environment and showed that the pharmacologically relevant drug-binding site lies in the transmembrane (TM) pore. Competing models of proton conduction have been evaluated using biochemical experiments, high-resolution structural methods, and computational modeling. These results are converging to a model in which a histidine residue in the TM domain mediates proton relay with water, aided by microsecond conformational dynamics of the imidazole ring. These mechanistic insights are guiding the design of new inhibitors that target drug-resistant M2 variants and may be relevant for other proton channels.

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

“…The M2 channel is known to have at least two conformational states that are populated to differing extents at low versus high pH (1,10,12). One, seen primarily at high pH, has been characterized extensively by solution NMR (21,22), solid-state NMR (SSNMR) (10,12), and X-ray crystallography (9).…”

Acid activation mechanism of the influenza A M2 proton channel

“…Structures determined using X-ray crystallography, solution NMR, and solid-state NMR (SSNMR) support this acid-activated transporter-like mechanism (9,10,(18)(19)(20)(21)(22)(23)(24)(25)(26). Near neutral pH the channel is closed in the C closed conformation ( Fig.…”

“…The differences in the Q2 water density profiles near the N-terminal helices between the 3LBW and 4QKL equilibrated structures (black and purple curves in Fig. S2B, respectively) are likely due to the fact that the 4QKL structure has three more residues (22)(23)(24) in the N-terminal region than the 3LBW. This addition causes the N-terminal helices in the equilibrated 4QKL structure to be more closely packed than in the equilibrated 3LBW structure in the Q2 state, resulting in lower water density in the N terminal for the former.…”

2D IR spectroscopy reveals the role of water in the binding of channel-blocking drugs to the influenza M2 channel

“…Peptides were synthesized by introducing 13 The peptides were synthesized by manual fluorenylmethyloxycarbonyl (Fmoc) solid phase synthesis on a 0.1 mmol scale using a Rink Amide-MBHA resin (GL Biochem) with a substitution level of 0.61 mmol/g using a procedure optimized for hydrophobic sequences. 40 Activation of the free amino acids (5-fold excess, 1.5-fold excess in case of N-Fmoc-1-13 C= 18 O labeled amino acids) was achieved with 0.95 equivalents (relative to the amino acid) of 1H-benzotriazolium 1-[bis(dimethylamino)methylene]-5-chlorohexafluorophosphate (1-)-3-oxide (HCTU) in the presence of 10 equivalents of diisopropylethylamine (DIEA); a 5% (v/v) piperazine solution was used for deprotection.…”

“…[6][7][8][9][10][11][12][13][14][15][16] The tetrad formed by the His37 residues is believed to play a key role in controlling the transition between the closed and open states, via a protonation/deprotonation mechanism. 10,12,13,[17][18][19][20] At neutral pH the equilibrium distribution of conformers is dominated by the doubly protonated (or +2) state of the His37 tetrad, while at acidic pH the +3 state is also formed. Protons transit through the M2 channel via a mechanism that involves: (1) protonation of the +2 state to generate the +3 state, (2) redistribution of the conformational equilibrium to favor opening of the Trp41 "gate," 21 and (3) release of a proton to the viral interior.…”