The small multidrug resistance (SMR) family of membrane proteins is prominent because of its rare dual topology architecture, simplicity, and small size. Its best studied member, EmrE, is an important model system in several fields related to membrane protein biology, from evolution to mechanism. But despite decades of work on these multidrug transporters, the native function of the SMR family has remained a mystery, and many highly similar SMR homologs do not transport drugs at all. Here we establish that representative SMR proteins, selected from each of the major clades in the phylogeny, function as guanidinium ion exporters. Drug-exporting SMRs are all clustered in a single minority clade. Using membrane transport experiments, we show that these guanidinium exporters, which we term Gdx, are very selective for guanidinium and strictly and stoichiometrically couple its export with the import of two protons. These findings draw important mechanistic distinctions with the notably promiscuous and weakly coupled drug exporters like EmrE.

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.

No abstract

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.

No abstract