Year: 2026
Type: preprint
Abstract: Many protein functions are coupled to proton transfer, yet the underlying mechanisms remain extremely difficult to resolve by experiment or conventional molecular dynamics (MD) simulations. The human voltage-gated proton channel H V 1 (hH V 1) plays key roles in human physiology, but how voltage and pH together drive its activation remains unresolved. Experimental structures of hH V 1 are limited to the X-ray structure of a mouse chimera and NMR models of a truncated hH V 1, both representing an intermediate resting state. Here we determine the activation pathway of hH V 1, together with structural models of its resting and activated states, using an approach that integrates all-atom continuous constant-pH MD (CpHMD), weighted-ensemble (WE) path sampling, and AlphaFold modeling. Starting from an AlphaFold model selected for consistency with resting-state experimental data, over 100 µ s of simulations reveal a mechanism in which transmembrane voltage, assisted by proton titration, drives an outward displacement of the transmembrane segment S4 accompanied by a one-click reorganization of countercharge pairings. These events are enabled by a delicately balanced electrostatic landscape and pH-modulated breathing motions in the resting state that prime the channel for protonation-state switches upon voltage sensing, a principle that may underlie other proton-coupled voltage-gating processes. The simulations also uncover lipid-binding sites at which POPG selectively stabilizes the S4-up conformation, whereas POPE can stabilize either state. These predictions are validated against extensive electrophysiology, mutagenesis data, and new smFRET experiments, which support the one-click S4 displacement and the lipid effects. WE-CpHMD, implemented in the AMBER package, is applicable to other proton-coupled processes that remain elusive.
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W7220861493