Elliptical β ‐barrel deformation underlies gating in VDAC1

Abstract

Gating by voltage‐dependent anion channels (VDAC) regulates mitochondrial metabolite flux, yet the structural mechanism underlying the open‐to‐closed transition remains unresolved. Here, we combine atomistic molecular dynamics (MD) simulations with double electron–electron resonance (DEER), using hydrostatic pressure as a reversible thermodynamic perturbation to shift conformational equilibria and stabilize low‐population states. MD simulations reveal localized intrinsic flexibility within β ‐strands β 1– β 5 and β 19, as well as in cytosolic loops connecting β 6– β 7 and β 8– β 9. High‐pressure DEER measurements in lipid nanodiscs corroborate these predictions, identifying reversible, pressure‐dependent distance changes within the pore lumen consistent with asymmetric deformation of the β ‐barrel. DEER‐informed analysis of unbiased MD trajectories reveals an elliptical β ‐barrel conformation aligned parallel to the N‐terminal helix that corresponds to the pressure‐stabilized experimental state. ATP permeation simulations identify a free‐energy barrier to metabolite translocation in this elliptical geometry, whereas diffusion through the circular open state is energetically favorable. These findings indicate that the elliptical conformation represents a transient gating‐competent state rather than a fully closed channel. Together, our results support a gating mechanism driven by reversible β ‐barrel deformation and establish pressure‐perturbed DEER integrated with MD as a general strategy for capturing transient, functionally relevant conformations of membrane channels.