Randy B. Stockbridge

Widespread Genetic Switches and Toxicity Resistance Proteins for Fluoride

Fluoride Riboswitch Riboswitches are found in prokaryote and eukaryote messenger RNAs (mRNAs), where they regulate expression of the linked mRNA through ligand binding and conformational change. Baker et al. (p. 233, published online 22 December) analyzed the binding properties of the “crcB motif” found in the noncoding RNA at the 5′ end of a diverse collection of prokaryotic genes. A crcB motif from Pseudomonas syringae was capable of selectively sensing the very small and highly charged fluoride ion. Some of the crcB and eriC genes associated with the fluoride riboswitch showed evidence of being fluoride transporters. The bacterium Methylobacterium extorquens DM4, which can use halogenated hydrocarbons as an energy source, was found to encode at least 10 fluoride riboswitches in its genome. A fluoride-sensing riboswitch regulates the expression of putative fluoride channels in prokaryotes. Most riboswitches are metabolite-binding RNA structures located in bacterial messenger RNAs where they control gene expression. We have discovered a riboswitch class in many bacterial and archaeal species whose members are selectively triggered by fluoride but reject other small anions, including chloride. These fluoride riboswitches activate expression of genes that encode putative fluoride transporters, enzymes that are known to be inhibited by fluoride, and additional proteins of unknown function. Our findings indicate that most organisms are naturally exposed to toxic levels of fluoride and that many species use fluoride-sensing RNAs to control the expression of proteins that alleviate the deleterious effects of this anion.

Fluoride resistance and transport by riboswitch-controlled CLC antiporters

A subclass of bacterial CLC anion-transporting proteins, phylogenetically distant from long-studied CLCs, was recently shown to be specifically up-regulated by F-. We establish here that a set of randomly selected representatives from this “CLCF” clade protect Escherichia coli from F- toxicity, and that the purified proteins catalyze transport of F- in liposomes. Sequence alignments and membrane transport experiments using 19F NMR, osmotic response assays, and planar lipid bilayer recordings reveal four mechanistic traits that set CLCF proteins apart from all other known CLCs. First, CLCFs lack conserved residues that form the anion binding site in canonical CLCs. Second, CLCFs exhibit high anion selectivity for F- over Cl-. Third, at a residue thought to distinguish CLC channels and transporters, CLCFs bear a channel-like valine rather than a transporter-like glutamate, and yet are F-/H+ antiporters. Finally, F-/H+ exchange occurs with 1∶1 stoichiometry, in contrast to the usual value of 2∶1.

A family of fluoride-specific ion channels with dual-topology architecture

Fluoride ion, ubiquitous in soil, water, and marine environments, is a chronic threat to microorganisms. Many prokaryotes, archea, unicellular eukaryotes, and plants use a recently discovered family of F− exporter proteins to lower cytoplasmic F− levels to counteract the anion’s toxicity. We show here that these ‘Fluc’ proteins, purified and reconstituted in liposomes and planar phospholipid bilayers, form constitutively open anion channels with extreme selectivity for F− over Cl−. The active channel is a dimer of identical or homologous subunits arranged in antiparallel transmembrane orientation. This dual-topology assembly has not previously been seen in ion channels but is known in multidrug transporters of the SMR family, and is suggestive of an evolutionary antecedent of the inverted repeats found within the subunits of many membrane transport proteins. DOI: http://dx.doi.org/10.7554/eLife.01084.001

Crystal structures of a double-barrelled fluoride ion channel

To contend with hazards posed by environmental fluoride, microorganisms export this anion through F−-specific ion channels of the Fluc family. Since the recent discovery of Fluc channels, numerous idiosyncratic features of these proteins have been unearthed, including strong selectivity for F− over Cl− and dual-topology dimeric assembly. To understand the chemical basis for F− permeation and how the antiparallel subunits convene to form a F−-selective pore, here we solve the crystal structures of two bacterial Fluc homologues in complex with three different monobody inhibitors, with and without F− present, to a maximum resolution of 2.1 Å. The structures reveal a surprising ‘double-barrelled’ channel architecture in which two F− ion pathways span the membrane, and the dual-topology arrangement includes a centrally coordinated cation, most likely Na+. F− selectivity is proposed to arise from the very narrow pores and an unusual anion coordination that exploits the quadrupolar edges of conserved phenylalanine rings.

Metabolism of Free Guanidine in Bacteria Is Regulated by a Widespread Riboswitch Class

The guanidyl moiety is a component of fundamental metabolites, including the amino acid arginine, the energy carrier creatine, and the nucleobase guanine. Curiously, reports regarding the importance of free guanidine in biology are sparse, and no biological receptors that specifically recognize this compound have been previously identified. We report that many members of the ykkC motif RNA, the longest unresolved riboswitch candidate, naturally sense and respond to guanidine. This RNA is found throughout much of the bacterial domain of life, where it commonly controls the expression of proteins annotated as urea carboxylases and multidrug efflux pumps. Our analyses reveal that these proteins likely function as guanidine carboxylases and guanidine transporters, respectively. Furthermore, we demonstrate that bacteria are capable of endogenously producing guanidine. These and related findings demonstrate that free guanidine is a biologically relevant compound, and several gene families that can alleviate guanidine toxicity exist.

Proof of dual-topology architecture of Fluc F − channels with monobody blockers

Fluc-type F- channels — used by microorganisms for resisting fluoride toxicity — are unusual in their quaternary architecture: They are thought to associate as dimers with the two subunits in antiparallel transmembrane orientation. Here we subject this unusual structural feature to a direct test. Single purified Fluc channels recorded in planar lipid bilayers are constitutively open, with rare, short-lived closings. Using combinatorial libraries, we generated synthetic binding proteins, “monobodies,” that specifically bind to Fluc homologues with nanomolar affinity. Reversible binding of monobodies to two different Fluc channel homologues is seen in single-channel recordings as long-lived nonconducting events that follow bimolecular kinetics. By applying monobodies sequentially to the two sides of the bilayer in a double-sided perfusion maneuver, we show that Fluc channels present monobody-binding epitopes to both sides of the membrane. The result establishes that Fluc subunits are arranged in dimeric antiparallel orientation.

Fluoride-dependent interruption of the transport cycle of a CLC Cl(-)/H(+) antiporter.

Cl−/H+ antiporters of the CLC superfamily transport anions across biological membranes in varied physiological contexts. These proteins are weakly selective among anions commonly studied, including Cl−, Br−, I−,NO3−, and SCN−, but appear to be very selective against F−. The recent discovery of a new CLC clade of F−/H+ antiporters, which are highly selective for F− over Cl−, led us to investigate the mechanism of Cl−-over-F− selectivity by a CLC Cl−/H+ antiporter, CLC-ec1. By subjecting purified CLC-ec1 to anion transport measurements, electrophysiological recording, equilibrium ligand-binding studies, and x-ray crystallography, we show that F− binds in the Cl− transport pathway with affinity similar to Cl−, but stalls the transport cycle. Examination of various mutant antiporters implies a “lock-down” mechanism of F− inhibition, in which F−, by virtue of its unique H-bonding chemistry, greatly retards a proton-linked conformational change essential for the transport cycle of CLC-ec1.

Bacterial fluoride resistance, Fluc channels, and the weak acid accumulation effect

Fluc channels protect bacteria from accumulating F− in acidic environments.

F−/Cl− selectivity in CLCF-type F−/H+ antiporters

Two subclades of the CLCF-type F−/H+ antiporters show differences in F−/Cl− selectivity.

Two-sided block of a dual-topology F− channel

Significance The studys significance is in its novelty along several lines. First, the ion channel studied, a Fluc F− channel, is recently discovered and has been only sparsely studied yet; this work adds a piece of the puzzle to the channels mechanistic landscape. Second, this is the first channel, to our knowledge, for which two-sided blocking has been observed; this type of blocking is a consequence of the unusual dual-topology assembly of Fluc channels. Third, the mathematical analysis of this kind of block is novel (necessarily so). Fourth, the main result—that the two ends of the pore can be simultaneously occupied by blockers but with negative cooperativity in binding—provokes future structural and mechanistic experiments. The Fluc family is a set of small membrane proteins forming F−-specific electrodiffusive ion channels that rescue microorganisms from F− toxicity during exposure to weakly acidic environments. The functional channel is built as a dual-topology homodimer with twofold symmetry parallel to the membrane plane. Fluc channels are blocked by nanomolar-affinity fibronectin-domain monobodies originally selected from phage-display libraries. The unusual symmetrical antiparallel dimeric architecture of Flucs demands that the two chemically equivalent monobody-binding epitopes reside on opposite ends of the channel, a double-sided blocking situation that has never before presented itself in ion channel biophysics. However, it is not known if both sites can be simultaneously occupied, and if so, whether monobodies bind independently or cooperatively to their transmembrane epitopes. Here, we use direct monobody-binding assays and single-channel recordings of a Fluc channel homolog to reveal a novel trimolecular blocking behavior that reveals a doubly occupied blocked state. Kinetic analysis of single-channel recordings made with monobody on both sides of the membrane shows substantial negative cooperativity between the two blocking sites.