Molecular basis for differential anion binding and proton coupling in the Cl^-/H⁺ exchanger ClC-ec1

Cl^-/H⁺ transporters of the CLC superfamily form a ubiquitous class of membrane proteins that catalyze stoichiometrically coupled exchange of Cl^- and H⁺ across biological membranes. CLC transporters exchange H⁺ for halides and certain polyatomic anions, but exclude cations, F^-, and larger physiological anions, such as PO₄^3- and SO₄^2-. Despite comparable transport rates of different anions, the H⁺ coupling in CLC transporters varies significantly depending on the chemical nature of the transported anion. Although the molecular mechanism of exchange remains unknown, studies on bacterial ClC-ec1 transporter revealed that Cl^- binding to the central anion-binding site (S_cen) is crucial for the anion-coupled H⁺ transport. Here, we show that Cl^-, F^-, NO3-, and SCN^- display distinct binding coordinations at the S_cen site and are hydrated in different manners. Consistent with the observation of differential bindings, ClC-ec1 exhibits markedly variable ability to support the formation of the transient water wires, which are necessary to support the connection of the two H⁺ transfer sites (Glu_in and Glu_ex), in the presence of different anions. While continuous water wires are frequently observed in the presence of physiologically transported Cl^-, binding of F^- or NO₃- leads to the formation of pseudo-water-wires that are substantially different from the wires formed with Cl^-. Binding of SCN^-, however, eliminates the water wires altogether. These findings provide structural details of anion binding in ClC-ec1 and reveal a putative atomic-level mechanism for the decoupling of H⁺ transport to the transport of anions other than Cl^-.