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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.

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 A. 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.

The Fluc family of F− ion channels protects prokaryotes and lower eukaryotes from the toxicity of environmental F−. In bacteria, these channels are built as dual-topology dimers whereby the two subunits assemble in antiparallel transmembrane orientation. Recent crystal structures suggested that Fluc channels contain two separate ion-conduction pathways, each with two F− binding sites, but no functional correlates of this unusual architecture have been reported. Experiments here fill this gap by examining the consequences of mutating two conserved F−-coordinating phenylalanine residues. Substitution of each phenylalanine specifically extinguishes its associated F− binding site in crystal structures and concomitantly inhibits F− permeation. Functional analysis of concatemeric channels, which permit mutagenic manipulation of individual pores, show that each pore can be separately inactivated without blocking F− conduction through its symmetry-related twin. The results strongly support dual-pathway architecture of Fluc channels. DOI: http://dx.doi.org/10.7554/eLife.18767.001

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 “CLC F ” 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 19 F NMR, osmotic response assays, and planar lipid bilayer recordings reveal four mechanistic traits that set CLC F proteins apart from all other known CLCs. First, CLC F s lack conserved residues that form the anion binding site in canonical CLCs. Second, CLC F s exhibit high anion selectivity for F - over Cl - . Third, at a residue thought to distinguish CLC channels and transporters, CLC F s 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.

Aggrecanases are now believed to be the principal proteinases responsible for aggrecan degradation in osteoarthritis. Given their potential as a drug target, we solved crystal structures of the two most active human aggrecanase isoforms, ADAMTS4 and ADAMTS5, each in complex with bound inhibitor and one wherein the enzyme is in apo form. These structures show that the unliganded and inhibitor-bound enzymes exhibit two essentially different catalytic-site configurations: an autoinhibited, nonbinding, closed form and an open, binding form. On this basis, we propose that mature aggrecanases exist as an ensemble of at least two isomers, only one of which is proteolytically active.

A search of prokaryotic genomes uncovered a gene from Mesorhizobium loti homologous to eukaryotic K+ channels of the S4 superfamily that also carry a cyclic nucleotide binding domain at the COOH terminus. The gene was cloned from genomic DNA, and the protein, denoted MloK1, was overexpressed in Escherichia coli and purified. Gel filtration analysis revealed a heterogeneous distribution of protein sizes which, upon inclusion of cyclic nucleotide, coalesces into a homogeneous population, eluting at the size expected for a homotetramer. As followed by a radioactive 86Rb+ flux assay, the putative channel protein catalyzes ionic flux with a selectivity expected for a K+ channel. Ion transport is stimulated by cAMP and cGMP at submicromolar concentrations. Since this bacterial homologue does not have the “C-linker” sequence found in all eukaryotic S4-type cyclic nucleotide-modulated ion channels, these results show that this four-helix structure is not a general requirement for transducing the cyclic nucleotide-binding signal to channel opening.

MAP KAP kinase 2 (MK2), a Ser/Thr kinase, plays a crucial role in the inflammatory process. We have determined the crystal structures of a catalytically active C-terminal deletion form of human MK2, residues 41–364, in complex with staurosporine at 2.7 Å and with ADP at 3.2 Å, revealing overall structural similarity with other Ser/Thr kinases. Kinetic analysis reveals that the Km for ATP is very similar for MK2 41–364 and p38-activated MK2 41–400. Conversely, the catalytic rate and binding for peptide substrate are dramatically reduced in MK2 41–364. However, phosphorylation of MK2 41–364 by p38 restores the Vmax and Km for peptide substrate to values comparable to those seen in p38-activated MK2 41–400, suggesting a mechanism for regulation of enzyme activity.

The CLC superfamily of anion transporters and channels has been studied for decades, but large swaths of the superfamily remain unexamined. One of those unstudied branches was recently found to be a family of fluoride/proton antiporters (CLCF), evolved to protect bacteria against intracellular fluoride accumulation. In addition to the clearly altered selectivity, these transporters also have a different transport stoichiometry from the chloride transporters (1 F- / 1 proton, vs 2 Cl- / 1 proton), suggesting a change in the transport mechanism. We have crystallized and solved the structure of CLCF-Eca, the fluoride CLC from Enterococcus casseliflavus, in complex with monobody crystallization chaperones at 3.28 A. To our knowledge this is the first structure of a fluoride CLC, and also the first structure of a non-chloride CLC-transporter. While some critical functional elements from the chloride CLCs remain, such as the external glutamate gate, other regions involved in both selectivity and ion coupling have been significantly altered.

The coordination geometry of zeolite-encapsulated copper(II)-histidine (CuHis) complexes, prepared by ion exchange of the complexes from aqueous solutions into zeolite NaY, was determined by a combination of UV-vis-NIR diffuse reflectance spectroscopy (DRS), X-band EPR, electron-spin-echo envelope modulation (ESEEM), and high field (W-band) pulsed ENDOR techniques. X-band EPR spectroscopy detected two distinct complexes, A and B, which are different from the prevailing Cu(II) bis-His complex in the exchange solution (pH 7.3 with a His:Cu(II) ratio of 5:1). Moreover, the relative amount of complex B was found to increase with increasing Cu(II) concentration. The EPR parameters of complex A are g┴ ) 2.054, g| ) 2.31, and A| ) 15.8 mT, whereas those of complex B are g┴ ) 2.068, g| ) 2.25, A| ) 18.3 mT, and A┴( 14 `) 1.3 mT. The presence of the 14 N superhyperfine splitting shows that in complex B three nitrogens are coordinated to the Cu(II). Furthermore, DRS exhibits a shift of the d-d absorption band of Cu(II) from 15 200 cm -1 in complex A to 15 900 cm -1 in complex B, indicating an increasing ligand field strength in the latter. The coordination of the imino nitrogen of the imidazole group was detected in the two complexes via the ESEEM frequencies of the remote nitrogen. In contrast, only complex A exhibited 27 Al modulation, which indicates that the Cu(II) binds to zeolite framework oxygens. 2 H and 1 H W-band ENDOR measurements on samples where the exchangeable protons were replaced with 2 H, and using specifically labeled histidine (His- R-d-‚-d2), lead to the unambiguous determination of the coordination configuration of the two complexes. Complex A is a mono- His complex where both the amino and imino nitrogens are coordinated and the other equatorial ligands are provided by a zeolite oxygen and a water molecule. Complex B is a bis-His complex, which is situated in the center of the supercage, and all equatorial coordination sites are provided by the His molecules. These are amino and imino nitrogens of one His molecule and the imino nitrogen and carboxylate oxygen of the second His molecule. Complex A can be converted into complex B by stirring the zeolite in a high pH solution, whereas complex B is converted into complex A by using a low pH solution, thus indicating that complex A is stabilized by the presence of intrazeolitic protons. On the basis of the structure of the complexes, the dependence of their relative amounts on the pH and Cu(II) concentration in the exchange solution, the His: Cu(II) ratio in the zeolite, the amount of exchanged Na(I) ions, and the steric constraints imposed by the zeolite framework, a model for the ion exchange processes and the intrazeolite reactions leading to the formation of the two complexes is presented.

W-band (95 GHz) pulsed EPR and electron−nuclear double resonance spectroscopic techniques were used to investigate the manganese S1 binding site of the protein concanavalin A in a frozen solution and in a single crystal. 1H ENDOR spectra were collected by using the Davies ENDOR sequence, whereas Mims ENDOR was applied to record 2H ENDOR spectra. Different EPR transitions were selected by performing the ENDOR measurements at different magnetic fields within the EPR spectrum. This selection, combined with the large thermal polarization achieved at magnetic fields of ∼3.4 T at low temperatures, allowed the assignment of ENDOR signals to their respective MS manifolds, thus providing the sign of the hyperfine couplings. The exchangeable and nonexchangeable protons were differentiated by comparing 1H and 2H ENDOR spectra of a solution of the protein prepared in D2O and of crystals soaked in D2O. The two imidazole protons, located on the carbons flanking the Mn-bound nitrogen, are magnetically equivalent, situat...

Mutagenesis, functional analysis, and crystal structures identify a watery tunnel through which protons enter the interior of a Cl−/H+ antiport protein involved in acid resistance of enteric bacteria., Chloride-transporting membrane proteins of the CLC family appear in two distinct mechanistic flavors: H+-gated Cl− channels and Cl−/H+ antiporters. Transmembrane H+ movement is an essential feature of both types of CLC. X-ray crystal structures of CLC antiporters show the Cl− ion pathway through these proteins, but the H+ pathway is known only inferentially by two conserved glutamate residues that act as way-stations for H+ in its path through the protein. The extracellular-facing H+ transfer glutamate becomes directly exposed to aqueous solution during the transport cycle, but the intracellular glutamate E203, Gluin, is buried within the protein. Two regions, denoted “polar” and “interfacial,” at the intracellular surface of the bacterial antiporter CLC-ec1 are examined here as possible pathways by which intracellular aqueous protons gain access to Gluin. Mutations at multiple residues of the polar region have little effect on antiport rates. In contrast, mutation of E202, a conserved glutamate at the protein–water boundary of the interfacial region, leads to severe slowing of the Cl−/H+ antiport rate. An X-ray crystal structure of E202Y, the most strongly inhibited of these substitutions, shows an aqueous portal leading to Gluin physically blocked by cross-subunit interactions; moreover, this mutation has only minimal effect on a monomeric CLC variant, which necessarily lacks such interactions. The several lines of experiments presented argue that E202 acts as a water-organizer that creates a proton conduit connecting intracellular solvent with Gluin., Author Summary Chloride-proton antiport proteins of the “CLC” superfamily are transmembrane proteins that form homodimers and are used for myriad physiological purposes, all requiring the coordinated movements of Cl− anions and H+ cations in opposite directions across biological membranes. While the pathway for Cl− ions through CLC antiporters is known, we currently have only indirect glimpses of how protons navigate their way through these membrane-embedded proteins. By combining mechanistic and structural approaches, we identify a proton-access pathway in a bacterial Cl−/H+ antiporter that allows intracellular protons to enter the protein interior and engage in the coupled antiport mechanism. We conclude that E202, a highly conserved glutamate residue, serves to organize water molecules and guide protons to the adjacent glutamate E203 (known as “Gluin”), a critical residue for the antiport mechanism.

CLC-ec1 is a bacterial CLC Cl-/H+ exchanger that catalyzes 2:1 exchange of Cl- and H+. E148 is the pH-dependent external gate of both Cl- and H+ transport and E203 mediates proton transport from intracellular solution to the protein interior. Substitution of non-protonatable residues on either glutamate uncouples the exchanger by completely abolishing H+ transport. However, it is not clear how protons in the intracellular solution gain access to E203, which is buried under a cytoplasmic “lid” formed by the protein's N-terminal helix. Truncation of the N-terminal 29 residues removes this lid and preserves 2:1 coupling of Cl- and H+ and absolute transport rate. This result implies that proton transport from intracellular solution to E203 is not rate-limiting in the transport cycle, and that protons are somehow facilitated in their movement to the buried E203. In order to discover proton facilitators, we examined polar residues near E203. Nonpolar mutants of Q207, S446 and R403 only slightly inhibit H+-transport rate. However, mutations on the proximate glutamate, E113, and disruption of a nearby salt-bridge (E117I and R209I) decrease H+-transport rate ∼5-fold. These residues appear to form a water-filled conduit for proton access to E203. Interestingly, nonpolar mutants of E202, located near the dimer interface and near to intracellular solution, show 30-100-fold reduced H+-transport rates compared to wildtype. All mutations here preserve H+-coupled Cl- transport, although those with severely reduced rates display somewhat higher Cl-/H+ stoichiometry, indicative of Cl- slippage. One or two crystallographic water molecules were found between E203 and E202. We propose that these water molecules form an access pathway for H+ from bulk water, which is blocked by bulky, hydrophobic amino acids at E202.

To reach the mammalian gut, enteric bacteria must pass through the stomach. Many such organisms survive exposure to the harsh gastric environment (pH 1.5-4) by mounting extreme acid-resistance responses, one of which, the arginine-dependent system of Escherichia coli, has been studied at levels of cellular physiology, molecular genetics and protein biochemistry. This multiprotein system keeps the cytoplasm above pH 5 during acid challenge by continually pumping protons out of the cell using the free energy of arginine decarboxylation. At the heart of the process is a 'virtual proton pump' in the inner membrane, called AdiC, that imports L-arginine from the gastric juice and exports its decarboxylation product agmatine. AdiC belongs to the APC superfamily of membrane proteins, which transports amino acids, polyamines and organic cations in a multitude of biological roles, including delivery of arginine for nitric oxide synthesis, facilitation of insulin release from pancreatic beta-cells, and, when inappropriately overexpressed, provisioning of certain fast-growing neoplastic cells with amino acids. High-resolution structures and detailed transport mechanisms of APC transporters are currently unknown. Here we describe a crystal structure of AdiC at 3.2 A resolution. The protein is captured in an outward-open, substrate-free conformation with transmembrane architecture remarkably similar to that seen in four other families of apparently unrelated transport proteins.

Chloride-transporting membrane proteins of the CLC family appear in two distinct mechanistic flavors: H+-gated Cl− channels and Cl−/H+ antiporters. Transmembrane H+ movement is an essential feature of both types of CLC. X-ray crystal structures of CLC antiporters show the Cl− ion pathway through these proteins, but the H+ pathway is known only inferentially by two conserved glutamate residues that act as way-stations for H+ in its path through the protein. The extracellular-facing H+ transfer glutamate becomes directly exposed to aqueous solution during the transport cycle, but the intracellular glutamate E203, Gluin, is buried within the protein. Two regions, denoted “polar” and “interfacial,” at the intracellular surface of the bacterial antiporter CLC-ec1 are examined here as possible pathways by which intracellular aqueous protons gain access to Gluin. Mutations at multiple residues of the polar region have little effect on antiport rates. In contrast, mutation of E202, a conserved glutamate at the protein-water boundary of the interfacial region, leads to severe slowing of the Cl−/H+ antiport rate. The x-ray crystal structure of E202Y, the most severely affected mutant, suggests that the aromatic ring of Y202 makes cross-subunit hydrophobic interaction with an I201 sidechain from neighboring subunit, which physically blocks the interfacial pathway. This mechanism is functionally supported by the minimal effect of E202Y mutation in a monomeric variant of the transporter, which does not have such an interaction. The several lines of experiments presented argue that E202 acts as a water-organizer that creates a proton conduit connecting intracellular solvent with Gluin.

AdiC transports arginine and agmatine (the decarboxylation product of arginine) across the membrane of certain enteric bacteria including E. coli, as well as pathognic organisms such as Salmonella. It belongs to the APC (amino acids, polyamines and organic cations) superfamily. Its major role is to maintain the internal pH of the cell in the acidic environment (such as stomach) by functioning as a virtual proton pump - transporting arginine (+1 charge) into and agmatine (decarboxylated arginine, +2 charge) out of the cell, resulting in export of 1 proton out during each turnover.We recently solved the crystal structure of AdiC with a Fab fragment at 3.2 A. The protein is captured in an outward-open, substrate-free conformation. Both structure and functional data have shown that some aromatic residues (Y93, W293 and Y365) are important for the substrate binding and transport, yet we don't have the direct picture of the structure in the presence of either arginine or agmatine. Therefore, our current work is focused on obtaining the conformation with substrate-bound. One approach is to design pair wise cysteines that can crosslink and mimic the conformation with substrate. Comparing the structure of AdiC and several other proteins with similar fold, we are targeting TM2 and TM8 for the cysteine crosslinking now. A second approach, based on our experience with Fab production, is to find another Fab that can stabilize the substrate-bound conformation. Currently we have twelve more monoclonal antibodies that bind AdiC and need to be tested.