Molecular mechanism of exchange coupling in CLC chloride/proton antiporters

The ubiquitous CLC membrane transporters are unique in their ability to exchange anions for cations. Despite extensive study, there is no mechanistic model that fully explains their 2:1 Cl^‒/H⁺ stoichiometric exchange mechanism. Here, we provide such a model. Using differential hydrogen-deuterium exchange mass spectrometry, cryo-EM structure determination, and molecular dynamics simulations, we uncovered new conformational dynamics in CLC-ec1, a bacterial CLC homolog that has served as a paradigm for this family of transporters. Simulations based on a cryo-EM structure at pH 3 revealed critical steps in the transport mechanism, including release of Cl^‒ ions to the extracellular side, opening of the inner gate, and novel water wires that facilitate H⁺ transport. Surprisingly, these water wires occurred independently of Cl^‒ binding, prompting us to reassess the relationship between Cl^‒ binding and Cl^‒/H⁺ coupling. Using isothermal titration calorimetry and quantitative flux assays on mutants with reduced Cl^‒ binding affinity, we conclude that, while Cl^‒ binding is necessary for coupling, even weak binding can support Cl^‒/H⁺ coupling. By integrating our findings with existing literature, we establish a complete and efficient CLC 2:1 Cl^‒/H⁺ exchange mechanism.