Molecular mechanism of exchange coupling in CLC chloride/proton antiporters

Abstract 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 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 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.