Constant-pH molecular dynamics simulations of progressive activation in a proton-gated ion channel

Abstract

AbstractAllosteric activation of ligand-gated ion channels is critical to electrochemical signal transduction, yet the details of how structural transitions are induced remain unclear. The proton-gatedGloeobacter violaceusligand-gated ion channel (GLIC) offers an invaluable model system to study this process, and with high-resolution structures available it should in principle be possible to approach computationally. However, molecular dynamics has traditionally struggled to accurately describe numerous dynamic protonation states with scalable efficiency. Here, we employ a new constant-pH method to simulate GLIC at resting and activating pH, starting from closed and open structures. Our simulations identify E26 and E35 as early protonation sites in gating, and reveal state-dependent shifts in pKa at multiple residues, as well as side chain and domain rearrangements supporting a progressive activation mechanism. Our results demonstrate the applicability of constant-pH simulation to substantiate detailed activation mechanisms in a multidomain membrane protein, likely extensible to other complex systems.Significance statementElectrostatic interactions play important roles in protein structure and function. Since changes in pH will (de)protonate residues and thereby modify such interactions, pH itself is a critical environment parameter. However, protonation states of titratable residues are static during classical molecular dynamics simulations. Recently, aconstant-pHalgorithm was implemented in the GROMACS package, allowing pH-effects to be captured dynamically. Here, we used this implementation to perform constant-pH simulations of a proton-gated ion channel, providing insight into its activation mechanism by revealing state-dependent shifts in protonation as well as pH-dependent side chain and domain-level rearrangements. The results show that constant-pH simulations are both accurate and capable of modeling dozens of titratable sites, with important implications e.g. for drug design.