Revealing an outward-facing open conformational state in a CLC CL^-/H⁺ exchange transporter

CLC secondary active transporters exchange Cl^- for H⁺. Crystal structures have suggested that the conformational change from occluded to outward-facing states is unusually simple, involving only the rotation of a conserved glutamate (Glu_ex) upon its protonation. Using ¹⁹F NMR, we show that as [H⁺] is increased to protonate Gluex and enrich the outward-facing state, a residue ~20 Å away from Glu_ex, near the subunit interface, moves from buried to solvent-exposed. Consistent with functional relevance of this motion, constriction via inter-subunit cross-linking reduces transport. Molecular dynamics simulations indicate that the cross-link dampens extracellular gate-opening motions. In support of this model, mutations that decrease steric contact between Helix N (part of the extracellular gate) and Helix P (at the subunit interface) remove the inhibitory effect of the cross-link. Together, these results demonstrate the formation of a previously uncharacterized ‘outward-facing open’ state, and highlight the relevance of global structural changes in CLC function.