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.