Two CI Ions and a Glu Compete for a Helix Cage in the CLC Proton/Cl- Antiporter

The ubiquitously expressed CLC chloride transporters are involved in a great variety of physiological functions. The CLC protein fold is shared by Cl-channels and 2Cl-:1H(+) antiporters. The antiporters pump three charges per cycle across the membrane with two CI- ions moving in the opposite direction of one proton. Multiconformational continuum electrostatics was used to calculate the coupled thermodynamics of the protonation of the extracellular-facing gating Glu (E-x) and Cl-binding to the external (Sr) and central (S-c) sites in CLC-ec1, the Escherichia coli exchanger. S-x, S-c, and E-x are buried within the protein where the intersection of two helix N-termini creates a region with a strong, localized positive potential for anion binding. Our chemical potential titrations describe the thermodynamic linkage for binding the Cl-to each site and protons to E-x. We find that the 2Cl(-):1 H+ binding stoichiometry is a result of Cl(-)binding to S-x requiring H+ binding to E-x, whereas Cl-binding to S-c does not lead to proton uptake. When Sx binds a Cl-, the protonated E-x moves upward, out of the positive helix cage. The increasing E-x proton affinity on binding the first Cl reduces the cost of binding the second Cl at either S-x or S-c. Despite the repulsion among the anions, the lowest energy states have two anions bound in the helix cage. The state with no Cl-is not favored electrostatically, but relies on E-x blocking S-x and on the central residues Y445 and S107 blocking S-c.