Structure and inhibition of the drug-resistant S31N mutant of the M2 ion channel of influenza A virus

Abstract

The influenza A virus M2 proton channel (A/M2) is the target of the antiviral drugs amantadine and rimantadine, whose use has been discontinued due to widespread drug resistance. Among the handful of drug-resistant mutants, S31N is found in more than 95% of the currently circulating viruses and shows greatly decreased inhibition by amantadine. The discovery of inhibitors of S31N has been hampered by the limited size, polarity, and dynamic nature of its amantadine-binding site. Nevertheless, we have discovered smallmolecule drugs that inhibit S31N with potencies greater than amantadine's potency against WT M2. Drug binding locks the protein into a well-defined conformation, and the NMR structure of the complex shows the drug bound in the homotetrameric channel, threaded between the side chains of Asn31. Unrestrained molecular dynamics simulations predicted the same binding site. This S31N inhibitor, like other potent M2 inhibitors, contains a charged ammonium group. The ammonium binds as a hydrate to one of three sites aligned along the central cavity that appear to be hotspots for inhibition. These sites might stabilize hydronium-like species formed as protons diffuse through the outer channel to the proton-shuttling residue His37 near the cytoplasmic end of the channel.M2-S31N mutant structure | membrane protein structure | M2-S31N inhibitorT he influenza A virus M2 proton channel (A/M2) is the target of the antiviral drugs amantadine and rimantadine (1-3), which bind directly to the pore of the channel (2-4). Although amantadine has been widely used for several decades, drug resistance has curtailed the use of this family of drugs. Many amantadineresistant influenza viruses can be selected in cell culture (5, 6). A subset of these mutations is found in infected patients undergoing treatment with amantadine (7), and reverse-engineered viruses harboring various pore-lining mutations are competent to replicate in the mouse (8). However, many of these mutations give rise to somewhat attenuated viruses that are less transmissible than WT virus, and they tend to revert in the absence of drug pressure (6, 9). Indeed, large-scale sequencing of transmissible viruses isolated as early as 1918 showed that mutations to pore-lining residues are allowed only within the first turn of the transmembrane (TM) helix at positions 26, 27, and 31 (10). S31N has long been the predominant amantadine-resistant mutation in M2 (11)(12)(13)(14). It predominated in 98-100% of the transmissible amantadineresistant H1N1, H5N1, and H3N2 strains isolated from humans, birds, and swine in the past decade. V27A and L26F are less frequent mutations (10,11,15). Extensive studies of point mutations to the pore-lining residues of M2 have been conducted to understand the paucity of natural variants (16,17). Numerous mutants in the N-terminal aqueous pore retained the ability to conduct protons selectively over other ions, although the magnitude and pH dependence of their conduction varied. However, only a few mutations at the most distal sites, V27A,...

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

“…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). A second form is observed in dynamic equilibrium at lower pH (21)(22)(23), as evidenced by a broadening of magnetic resonances that thus far has made it impractical to determine a high-resolution structure of the protein in this state by SSNMR or solution NMR. X-ray crystallographic studies, however, have provided structures of both states (8,9), which differ primarily in the conformation of the C terminus where protons exit the channel.…”

XFEL structures of the influenza M2 proton channel: Room temperature water networks and insights into proton conduction

“…This conformational transition provides a pathway for proton transfer past the Trp41 gate into the viral interior. In NMR studies at low pH, both conformations are observed in equilibrium with one another (40,43,44 (Fig. S1, Bottom).…”

“…The Inward closed state has also been characterized by solution NMR (39,40) and solid-state NMR (41,42) studies under high pH conditions. The Inward closed conformation can transition to the Inward open conformation by straightening a kink in the TM helix near Gly34 (40,43,44). This conformational transition provides a pathway for proton transfer past the Trp41 gate into the viral interior.…”

Multiscale simulation reveals a multifaceted mechanism of proton permeation through the influenza A M2 proton channel

“…However, the water dynamics in the AM2 protein probed using 2D infrared (2D-IR) spectroscopy revealed that the well-ordered "ice-like" pore water dynamics at pH 8.0 change to more mobile and "liquid-like" dynamics (on the timescale of a few picoseconds) at pH 3.2 (23). This result suggests an interesting pH-dependent behavior of the AM2 protein that is highly relevant for understanding its PT mechanism.Although many computational studies have investigated the features of AM2 that may influence its PT in recent years (12,(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40), only a few have explicitly simulated any aspect of the explicit PT process (24,25,29,37,38). This is because it is challenging to accurately model the charge delocalization and Grotthuss shuttling of the hydrated excess proton in a computationally tractable way.…”