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.

Department of Biochemistry Faculty of Science Charles University Hlavova 2030 8 128 43 Prague 2 Czech Republic

Department of Bioengineering and Department of Microbiology and Immunology Stanford University Stanford 94305

Department of Computer Science Stanford University Stanford CA 94305

Department of Structural Biology Stanford University Stanford CA 94305

Division of CryoEM and Bioimaging SSRL SLAC National Accelerator Laboratory Stanford University Menlo Park 94025

Institute for Computational and Mathematical Engineering Stanford University Stanford CA 94305

Institute of Microbiology of the Czech Academy of Sciences Division BioCeV Prumyslova 595 252 50 Vestec Czech Republic

National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health Bethesda MD 20892

Stanford University Department of Molecular and Cellular Physiology

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