Inside and Out of the Pore: Comparing Interactions and Molecular Dynamics of Influenza A M2 Viroporin Complexes in...

Ion channels located at viral envelopes (viroporins) have a critical function for the replication of infectious viruses and are important drug targets. Over the last decade, the number and duration of molecular dynamics (MD) simulations of the influenza A M2 ion channel owing to the increased computational efficiency. Here, we aimed to define the system setup and simulation conditions for the correct description of the protein-pore and the protein-lipid interactions for influenza A M2 in comparison with experimental data. We performed numerous MD simulations of the influenza A M2 protein in complex with adamantane blockers in standard lipid bilayers using OPLS2005 and CHARMM36 (C36) force fields. We explored the effect of varying the M2 construct (M2(22-46) and M2(22-62)), the lipid buffer size and type (stiffer DMPC or softer POPC with or without 20% cholesterol), the simulation time, the H37 protonation site (N_δ or Ν_ε), the conformational state of the W41 channel gate, and M2's cholesterol binding sites (BSs). We report that the 200 ns MD with M2(22-62) (having N_ε Η37) in the 20 Å lipid buffer with the C36 force field accurately describe: (a) the M2 pore structure and interactions inside the pore, that is, adamantane channel blocker location, water clathrate structure, and water or chloride anion blockage/passage from the M2 pore in the presence of a channel blocker and (b) interactions between M2 and the membrane environment as reflected by the calculation of the M2 bundle tilt, folding of amphipathic helices, and cholesterol BSs. Strikingly, we also observed that the C36 1 μs MD simulations using M2(22-62) embedded in a 20 Å POPC:cholesterol (5:1) scrambled membrane produced frequent interactions with cholesterol, which when combined with computational kinetic analysis, revealed the experimentally observed BSs of cholesterol and suggested three similarly long-interacting positions in the top leaflet that have previously not been observed experimentally. These findings promise to be useful for other viroporin systems.