Fluc family fluoride channels protect microbes against ambient environmental fluoride by undermining the cytoplasmic accumulation of this toxic halide. These proteins are structurally idiosyncratic, and thus the permeation pathway and mechanism have no analogy in other known ion channels. Although fluoride binding sites were identified in previous structural studies, it was not evident how these ions access aqueous solution, and the molecular determinants of anion recognition and selectivity have not been elucidated. Using x-ray crystallography, planar bilayer electrophysiology and liposome-based assays, we identify additional binding sites along the permeation pathway. We use this information to develop an oriented system for planar lipid bilayer electrophysiology and observe anion block at one of these sites, revealing insights into the mechanism of anion recognition. We propose a permeation mechanism involving alternating occupancy of anion binding sites that are fully assembled only as the substrate approaches.

Significance: This provides a specific example of how interactions between lipid headgroups and membrane-bound proteins can influence function.

No abstract

Microorganisms contend with numerous and unusual chemical threats and have evolved a catalog of resistance mechanisms in response. One particularly ancient, pernicious threat is posed by fluoride ion (F−), a common xenobiotic in natural environments that causes broad-spectrum harm to metabolic pathways. This review focuses on advances in the last ten years toward understanding the microbial response to cytoplasmic accumulation of F−, with a special emphasis on the structure and mechanisms of the proteins that microbes use to export fluoride: the CLCF family of F−/H+ antiporters and the Fluc/FEX family of F− channels. Expected final online publication date for the Annual Review of Biochemistry, Volume 90 is June 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Summary Crystallization of dual topology fluoride (Fluc) channels requires small, soluble crystallization chaperones known as monobodies, which act as primary crystal lattice contacts. Previous structures of Flucs have been solved in the presence of monobodies that inhibit fluoride currents in single-channel electrophysiological recordings. These structures have revealed two-fold symmetric, doubly-bound arrangements, with one monobody on each side of the membrane. The combined electrohysiological and structural observations raise the possibility that chaperone binding allosterically closes the channel, altering the structure from its conducting form. To address this, we identify and solve the structure with a different monobody that only partially blocks fluoride currents. The structure of the channel-monobody complex is asymmetric, with monobody bound to one side of the channel only. The channel conformation is nearly identical on the bound- and uncomplexed sides, and to all previously solved structures, providing direct structural evidence that monobody binding does not induce local structural changes.