Mechanisms Underlying Proton Release in CLC-type F^-/H⁺ Antiporters

The CLC family of anion channels and transporters includes Cl^-/H⁺ exchangers (blocked by F^-) and F^-/H⁺ exchangers (or CLC^Fs). CLC^Fs contain a glutamate (E318) in the central anion-binding site that is absent in CLC Cl^-/H⁺ exchangers. The X-ray structure of the protein from Enterococcus casseliflavus (CLC^F-eca) shows that E318 tightly binds to F^- when the gating glutamate (E118; highly conserved in the CLC family) faces the extracellular medium. Here, we use classical and DFT-based QM/MM metadynamics simulations to investigate proton transfer and release by CLC^F-eca. After up to down movement of protonated E118, both glutamates combine with F^- to form a triad, from which protons and F^- anions are released as HF. Our results illustrate how glutamate insertion into the central anion-binding site of CLC^F-eca permits the release of H⁺ to the cytosol as HF, thus enabling a net 1:1 F^-/H⁺ stoichiometry.