BPS2026 – Protonation-dependent uptake and emergent chloride gating underpin fluoride selectivity in CLCF F−/H+antiporter

CLC-type fluoride/proton antiporters (CLCFs) safeguard cells from fluoride toxicity, yet the physical determinants of fluoride uptake and anion selectivity remain debated. Here, we combine atomistic molecular dynamics with free-energy profiling to dissect how the protonation of two conserved glutamates—Gluex (E118) and Gluin (E318)—controls early fluoride uptake events. We find that the state with a deprotonated E118 and protonated E318 enlarges the intracellular pore and lowers the potential-of-mean-force barrier for fluoride migration from the cytosol to the central site (Scen), promoting efficient uptake. Strikingly, in the presence of chloride at Scen the protein undergoes a helix-to-coil transition within residues 74–87. This local disorder stabilizes dehydrated chloride through favorable interactions while simultaneously impeding its forward translocation. The emergent conformational trap for chloride, together with the fluoride-favorable electrostatics and hydration thermodynamics in the deprotonated E118 and protonated E318 state, rationalizes the fluoride-over-chloride selectivity at the uptake stage. Our results reinforce and refine the original “windmill” mechanism by pinpointing a protonation-coupled uptake state that is selectively permissive to fluoride, and by revealing a chloride-induced conformational trap that disfavors chloride transport. These insights unify mechanistic proposals by showing how protonation, pore size, hydration penalties, and secondary-structure plasticity cooperate to bias flux toward fluoride. More broadly, our work illustrates a general biophysical principle for anion selectivity—coupling ion hydration energetics to localized backbone transitions can gate competing ions without imposing large global rearrangements. We anticipate that this framework will guide future experiments—mutagenesis of E118/E318 and spectroscopy targeting the 74–87 segment—to test how protonation and coil formation sculpt the selectivity landscape in CLCFs.