Multiscale Simulation of an Influenza A M2 Channel Mutant Reveals Key Features of Its Markedly Different Proton Transport Behavior
The influenza A M2 channel, a prototype for the viroporin class of viral channels, is an acid-activated viroporin that conducts protons across the viral membrane, a critical step in the viral life cycle. As the protons enter from the viral exterior, four central His37 residues control the channel activation by binding subsequent protons, which opens the Trp41 gate and allows proton flux to the viral interior. Asp44 is essential for maintaining the Trp41 gate in a closed state at high pH, which results in asymmetric conduction. The prevalent D44N mutant disrupts this gate and opens the C-terminal end of the channel, resulting in overall increased conduction in the physiologically relevant pH range and a loss of this asymmetric conduction. Here, we use extensive Multiscale Reactive Molecular Dynamics (MS-RMD) and Quantum Mechanics/Molecular mechanics (QM/MM) simulations with an explicit, reactive excess proton to calculate the free energy of proton transport in the M2 mutant and to study the dynamic molecular-level behavior of D44N M2. We find that this mutation significantly lowers the barrier of His37 deprotonation in the activated state and shifts the barrier for entry up to the Val27 tetrad. These free energy changes are reflected in structural shifts. Additionally, we show that the increased hydration around the His37 tetrad diminishes the effect of the His37 charge on the channels water structure, facilitating proton transport and enabling activation from the viral interior. Altogether, this work provides key insight into the fundamental characteristics of PT in WT M2 and how the D44N mutation alters this PT mechanism, and it expands our understanding of the role of emergent mutations in viroporins.