BPS2026 – Structural basis of uncoupling in a CLC transporter

CLC Cl − channels and Cl − /H + transporters are vital for human physiology and their mutations can cause genetic disorders. In transporters, the Cl − and H + pathways intersect at a conserved glutamic acid, Egate, which serves as the Cl − gate. Egate sidechain rotates between the two pathways enabling H + movement, and its mutations abolish H + coupling. However, the structural basis of Cl − /H + coupling remains poorly understood. To address this question, we take advantage of the finding that large polyatomic anions, such as NO3 − and SCN − , impair H + coupling in transporters. CryoEM imaging of CLC-ec1, the CLC transporter from E. coli, shows that in Cl − , Egate (E148 in CLC-ec1) and conserved F357 can adopt two rotameric states in a pH-dependent manner: R1, favored at high pH, where E148 lines the Cl- pathway and F357 is “down” state that renders the pathway more hydrophobic, thus preventing H + transport, and R2 at low pH, where E148 lines the H + pathway and F357 in an “up” state which favors H + pathway hydration. In contrast, the uncoupling anions favor the H + -transport incompetent state R1 only, pH independently. MD simulations show that with Cl − at the central binding site, Scen, E148, and F357 can adopt R1 and R2, and the H + pathway is more hydrated in R2, likely facilitating H + transport. This suggests that F357 acts as a gate for the H + pathway and that its movements are energetically coupled to those of E148. In contrast, SCN- binding at Scen decouples the two gating residues, while F357 exclusively adopts a down conformation, independent of the state of E148, and H + pathway remains dehydrated. These findings explain how SCN- specifically inhibits H + movement in CLC-ec1 while leaving anion transport intact, and reveal the precise molecular steps underlying coupled exchange in CLCs.