Mechanisms Underlying Proton Release in CLC-type F<sup>–</sup>/H<sup>+</sup> Antiporters
Maria Gabriella Chiariello, Mercedes Alfonso‐Prieto, Emiliano Ippoliti, Christoph Fahlke, Paolo Carloni
Abstract
The CLC family of anion channels and transporters includes Cl–/H+ exchangers (blocked by F–) and F–/H+ exchangers (or CLCFs). CLCFs 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 (CLCF-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 CLCF-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 CLCF-eca permits the release of H+ to the cytosol as HF, thus enabling a net 1:1 F–/H+ stoichiometry.