Elliptical <scp> <i>β</i> </scp> ‐barrel deformation underlies gating in <scp>VDAC1</scp>

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 β1-β5 and β19, as well as in cytosolic loops connecting β6-β7 and β8-β9. 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.