BPS2026 – Evaluating three models of the hHv1 dimer interface

The voltage-gated proton channel, Hv1, is responsible for maintaining pH homeostasis and many other crucial physiological processes. Its activity has been reported to hinder recovery from ischemic stroke, traumatic brain injury, spinal cord injury, and to increase the metastatic potential of different types of cancer, thereby motivating the development of Hv1 antagonists as potential neuroprotective agents and anti-cancer drugs. Hv1 contains a voltage-sensing domain composed of four transmembrane helices (S1 through S4). Conserved arginines on S4 respond to changes in membrane potential by moving within the membrane electric field via the water-catalyzed exchange of salt-bridges with acidic residues in S1-S3. Hv1 is expressed as a dimer made of two VSDs, each containing a gated proton permeation pathway. The S4 segments are connected to a C-terminal coiled-coil domain. While monomeric models have been proposed and validated, the architecture of the hHv1 dimer remains debated. Cross-linking experiments and mutational scans have suggested three different dimer interfaces along S1 helices (S1S1 model), along the S4 helices (S4S4 model), or involving both the S1 and S4 helices (S1S4 model). Here, guided by experimental data and AlphaFold predictions, we constructed structural models of each proposed interface and performed microsecond-timescale all-atom molecular dynamics simulations of the dimer embedded in lipid bilayers under an applied membrane potential. The simulations revealed differences in protein stability and dynamics among the models, suggesting that one interface may better explain available experimental data. Ongoing analyses aim to refine this picture and establish a framework for probing small-molecule modulation of Hv1.