Resistance-Mutation (N31) Effects on Drug Orientation and Channel Hydration in Amantadine-Bound Influenza A M2.

The mechanism of amantadine binding to the S31 variant of the M2 protein of Influenza A is well understood, but the reasons behind N31 M2 amantadine insensitivity remain under investigation. Many molecular dynamics studies have evaluated the influence of amantadine position within the channel pore on its ability to inhibit proton conductance in M2, but little is known about the influence of amantadine rotational orientation. Replica-exchange umbrella sampling, steered, and classic molecular dynamics simulations were performed on amantadine in the solid-state NMR structure of S31 M2 and an N31 M2 homologue, both in the homotetramer configuration, to explore the effects of the position and tilt angle of amantadine on inhibition of the M2 channel. Steered simulations show that amantadine rotates with the amine toward the bulk water as it passes into the hydrophobic entryway lined by Val27 side chains. Results from all simulation types performed indicate that amantadine has a strong, specific orientation with the amine turned inward toward the central cavity in the S31 M2 pore but has variable orientation and a strong propensity to remain outward pointing in N31 M2. Free energy profiles from umbrella sampling, measured relative to bulk water, show amantadine binds more strongly to the S31 M2 pore by 8 kcal/mol in comparison to amantadine in the N31 pore, suggesting that it can escape more readily from the N31 channel through the Val27 secondary gate, whereas it is captured by the S31 channel in the same region. Lower water density and distribution near amantadine in S31 M2 reveal that the drug inhibits proton conductance in S31 M2 because of its inward-pointing configuration, whereas in N31 M2, amantadine forms hydrogen bonds with an N31 side chain and does not widely occlude water occupancy in any configuration. Both amantadine's weaker binding to and weaker water occlusion in N31 M2 might contribute to its inefficacy as an inhibitor of the mutant protein.

Why bound amantadine fails to inhibit proton conductance according to simulations of the drug-resistant influenza A M2 (S31N).

The mechanisms responsible for drug resistance in the Asn31 variant of the M2 protein of influenza A are not well understood. Molecular dynamics simulations were performed on wild-type (Ser31) and S31N influenza A M2 in the homotetramer configuration. After evaluation of 13 published M2 structures, a solid-state NMR structure with amantadine bound was selected for simulations, an S31N mutant structure was developed and equilibrated, and the native and mutant structures were used to determine the binding behavior of amantadine and the dynamics of water in the two channels. Amantadine is stable in the plugging region of wild-type M2, with the adamantane in contact with the Val27 side chains, while amantadine in S31N M2 has more variable movement and orientation, and spontaneously moves lower into the central cavity of the channel. Free energy profiles from umbrella sampling support this observation. In this configuration, water surrounds the drug and can easily transport protons past it, so the drug binds without blocking proton transport in the S31N M2 channel.