Constant-pH molecular dynamics simulations of closed and open states of a proton-gated ion channel

Although traditional molecular dynamics simulations successfully capture a variety of different molecular interactions, the protonation states of titratable residues are kept static. A recent constant-pH molecular dynamics implementation in the GROMACS package allows pH effects to be captured dynamically and promises to provide both the accuracy and computational performance required for studying pH-mediated conformational dynamics in large, complex systems containing hundreds of titratable residues. Here, we demonstrate the applicability of this constant-pH implementation by simulating the proton-gated ion channel GLIC at resting and activating pH, starting from closed and open structures. Our simulations identify residues E26 and E35 as especially pH sensitive and reveal state-dependent pK_a shifts at multiple residues, as well as side-chain and domain rearrangements in line with the early stages of gating. Our results are consistent with several previous experimental findings, demonstrating the applicability of constant-pH simulations to elucidate pH-mediated activation mechanisms in multidomain membrane proteins, likely extensible to other complex systems.