Aydin Deniz, Chien Chih-Ta, Kreiter Jürgen, Nava Amy, Portasikova Jasmina M, Fojtik Lukas, Salcedo Catalina Mosquera, Man Petr, Dror Ron O, Chiu Wah, Maduke Merritt
Stanford University, Department of Molecular and Cellular Physiology.
Department of Computer Science, Stanford University, Stanford, CA 94305.
bioRxiv. 2025 May 9:2025.05.08.652968. doi: 10.1101/2025.05.08.652968.
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.
普遍存在的CLC膜转运蛋白在阴离子与阳离子交换能力方面独具特色。尽管已进行了广泛研究，但尚无一个机制模型能完全解释其2:1的Cl⁻/H⁺化学计量交换机制。在此，我们提供了这样一个模型。通过使用差分氢-氘交换质谱、冷冻电镜结构测定和分子动力学模拟，我们在CLC-ec1中发现了新的构象动力学，CLC-ec1是一种细菌CLC同源物，一直是该转运蛋白家族的范例。基于pH 3时的冷冻电镜结构进行的模拟揭示了转运机制中的关键步骤，包括Cl⁻离子向细胞外侧的释放、内门的打开以及促进H⁺转运的新型水线。令人惊讶的是，这些水线的出现独立于Cl⁻结合，这促使我们重新评估Cl⁻结合与Cl⁻/H⁺偶联之间的关系。通过对等温滴定量热法和对Cl⁻结合亲和力降低的突变体进行定量通量测定，我们得出结论，虽然Cl⁻结合对于偶联是必要的，但即使是弱结合也能支持Cl⁻/H⁺偶联。通过将我们的研究结果与现有文献相结合，我们建立了一个完整且高效的CLC 2:1 Cl⁻/H⁺交换机制。
