Two Cl 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 Cl 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 (S_x) 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^-:1H⁺ 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 S_x 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.