Abstract
Fluoride/proton antiporters of the CLCF family combat F- toxicity in bacteria by exporting this halide from the cytoplasm. These transporters belong to the widespread CLC superfamily but display transport properties different from those of the well-studied Cl-/H+ antiporters. Here, we report a structural and functional investigation of these F--transport proteins. Crystal structures of a CLCF homolog from Enterococcus casseliflavus are captured in two conformations with simultaneous accessibility of F- and H+ ions via separate pathways on opposite sides of the membrane. Manipulation of a key glutamate residue critical for H+ and F- transport reverses the anion selectivity of transport; replacement of the glutamate with glutamine or alanine completely inhibits F- and H+ transport while allowing for rapid uncoupled flux of Cl-. The structural and functional results lead to a 'windmill' model of CLC antiport wherein F- and H+ simultaneously move through separate ion-specific pathways that switch sidedness during the transport cycle.