# Molecular mechanism of exchange coupling in CLC chloride/proton antiporters

## TL;DR

This study reveals how CLC transporters exchange chloride and protons using a detailed model based on structural and dynamic analyses.

## Contribution

A complete mechanistic model of CLC transporters' 2:1 Cl-/H+ exchange is established through integrated experimental and computational approaches.

## Key findings

- Conformational dynamics in CLC-ec1 were identified using cryo-EM and simulations, revealing Cl- release and H+ transport steps.
- Water wires facilitate H+ transport independently of Cl- binding, challenging previous assumptions about coupling.
- Weak Cl- binding can still support Cl-/H+ coupling, as shown through functional assays on mutant proteins.

## 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.

CLC transporters exchange Cl− for H− with 2:1 stoichiometry. Here, authors integrate hydrogen-deuterium exchange, cryo-EM, MD simulations, ion binding analysis, and functional assays to establish a complete ion-exchange model, explaining how subtle dynamics enable reversible transport.