A Dimeric Dual-Topology Microbial Fluoride Channel

Abstract

Fluoride ion has pervaded the environment (10-100 micromolar in sea, ground water, and soil) over evolutionary time, and inhibits several essential phosphoryl group transfer enzymes involved in energy metabolism and nucleic acid synthesis with Ki values around 100 micromolar. Widespread microorganisms have evolved defenses against F- ion, including a newly discovered family of membrane transport proteins composed of four transmembrane helices, called Flucs. Using single channel electrical recordings of purified, reconstituted proteins in planar lipid bilayers, we show that Flucs are constitutively open electrodiffusive F- channels, with main state conductance of ∼10 pS, and possess unprecedentedly high and biologically required 10,000-fold selectivity for F- over Cl-. Using SEC-UV/RI/LS, single molecule TIRF photobleaching, and a Poisson dump assay for calculating the molecular weight of membrane transporters in liposomes, we show that Flucs function as homodimers, an unusually small number of subunits for a channel. For deeper mechanistic studies, we have selected a panel of monobody binders to Fluc proteins from a phage-display library of 1010 fibronection-domain scaffolds with randomly varied loops. These monobodies block single-Fluc F- current with nanomolar affinity and simple bimolecular kinetics, and most likely inhibit F- current by blocking the pore. In single channel recordings, we show that the monobodies block the same single Fluc channel from both sides of the bilayer, which can only occur if the subunits of the dimer are arranged in an antiparallel orientation relative to each other, and thus present the same epitope on both sides of the membrane. This result emphatically supports earlier indications of antiparallel topology in Flucs, including lysine cross-linking experiments and genetically fused constructs. Dual-topology architecture is a surprising departure from the familiar barrel-stave plan that many other channels are built on, but is reminiscent of inverted structural repeats in modern-day transporters.