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In Bacillus subtilis, a ParB-like nucleoid occlusion protein (Noc) binds specifically to Noc-binding sites (NBSs) on the chromosome to help coordinate chromosome segregation and cell division. Noc does so by binding to CTP to form large membrane-associated nucleoprotein complexes to physically inhibit the assembly of the cell division machinery. The site-specific binding of Noc to NBS DNA is a prerequisite for CTP-binding and the subsequent formation of a membrane-active DNA-entrapped protein complex. Here, we solve the structure of a C-terminally truncated B. subtilis Noc bound to NBS DNA to reveal the conformation of Noc at this crucial step. Our structure reveals the disengagement between the N-terminal CTP-binding domain and the NBS-binding domain of each DNA-bound Noc subunit; this is driven, in part, by the swapping of helices 4 and 5 at the interface of the two domains. Site-specific crosslinking data suggest that this conformation of Noc-NBS exists in solution. Overall, our results lend support to the recent proposal that parS/NBS binding catalyzes CTP binding and DNA entrapment by preventing the reengagement of the CTP-binding domain and the DNA-binding domain from the same ParB/Noc subunit., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of the article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Proper chromosome segregation is essential in all living organisms. The ParA-ParB- parS system is widely employed for chromosome segregation in bacteria. Previously, we showed that Caulobacter crescentus ParB requires cytidine triphosphate to escape the nucleation site parS and spread by sliding to the neighboring DNA (Jalal et al., 2020). Here, we provide the structural basis for this transition from nucleation to spreading by solving co-crystal structures of a C-terminal domain truncated C. crescentus ParB with parS and with a CTP analog. Nucleating ParB is an open clamp, in which parS is captured at the DNA-binding domain (the DNA-gate). Upon binding CTP, the N-terminal domain (NTD) self-dimerizes to close the NTD-gate of the clamp. The DNA-gate also closes, thus driving parS into a compartment between the DNA-gate and the C-terminal domain. CTP hydrolysis and/or the release of hydrolytic products are likely associated with reopening of the gates to release DNA and recycle ParB. Overall, we suggest a CTP-operated gating mechanism that regulates ParB nucleation, spreading, and recycling., Competing Interests: AJ, CS, AC, AB, DL, TL None, NT none, (© 2021, Jalal et al.)

DNA gyrase, a type II topoisomerase, introduces negative supercoils into DNA using ATP hydrolysis. The highly effective gyrase-targeted drugs, fluoroquinolones (FQs), interrupt gyrase by stabilizing a DNA-cleavage complex, a transient intermediate in the supercoiling cycle, leading to double-stranded DNA breaks. MfpA, a pentapeptide-repeat protein in mycobacteria, protects gyrase from FQs, but its molecular mechanism remains unknown. Here, we show that Mycobacterium smegmatis MfpA (MsMfpA) inhibits negative supercoiling by M. smegmatis gyrase (Msgyrase) in the absence of FQs, while in their presence, MsMfpA decreases FQ-induced DNA cleavage, protecting the enzyme from these drugs. MsMfpA stimulates the ATPase activity of Msgyrase by directly interacting with the ATPase domain (MsGyrB47), which was confirmed through X-ray crystallography of the MsMfpA-MsGyrB47 complex, and mutational analysis, demonstrating that MsMfpA mimics a T (transported) DNA segment. These data reveal the molecular mechanism whereby MfpA modulates the activity of gyrase and may provide a general molecular basis for the action of other pentapeptide-repeat proteins., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Specific interactions between proteins and DNA are essential to many biological processes. Yet, it remains unclear how the diversification in DNA-binding specificity was brought about, and the mutational paths that led to changes in specificity are unknown. Using a pair of evolutionarily related DNA-binding proteins, each with a different DNA preference (ParB [Partitioning Protein B] and Noc [Nucleoid Occlusion Factor], which both play roles in bacterial chromosome maintenance), we show that specificity is encoded by a set of four residues at the protein-DNA interface. Combining X-ray crystallography and deep mutational scanning of the interface, we suggest that permissive mutations must be introduced before specificity-switching mutations to reprogram specificity and that mutational paths to new specificity do not necessarily involve dual-specificity intermediates. Overall, our results provide insight into the possible evolutionary history of ParB and Noc and, in a broader context, might be useful for understanding the evolution of other classes of DNA-binding proteins., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Mycobacterium tuberculosis produces glycogen (also known as α-glucan) to help evade human immunity. This pathogen uses the GlgE pathway to generate glycogen rather than the more well known glycogen synthase GlgA pathway, which is absent in this bacterium. Thus, the building block for this glucose polymer is α-maltose-1-phosphate rather than an NDP-glucose donor. One of the routes to α-maltose-1-phosphate is now known to involve the GlgA homologue GlgM, which uses ADP-glucose as a donor and α-glucose-1-phosphate as an acceptor. To help compare GlgA (a GT5 family member) with GlgM enzymes (GT4 family members), the X-ray crystal structure of GlgM from Mycobacterium smegmatis was solved to 1.9 Å resolution. While the enzymes shared a GT-B fold and several residues responsible for binding the donor substrate, they differed in some secondary-structural details, particularly in the N-terminal domain, which would be expected to be largely responsible for their different acceptor-substrate specificities., (open access.)

Glycoside phosphorylases (GPs) with specificity for β-(1 → 3)-gluco-oligosaccharides are potential candidate biocatalysts for oligosaccharide synthesis. GPs with this linkage specificity are found in two families thus far-glycoside hydrolase family 94 (GH94) and the recently discovered glycoside hydrolase family 149 (GH149). Previously, we reported a crystallographic study of a GH94 laminaribiose phosphorylase with specificity for disaccharides, providing insight into the enzyme's ability to recognize its' sugar substrate/product. In contrast to GH94, characterized GH149 enzymes were shown to have more flexible chain length specificity, with preference for substrate/product with higher degree of polymerization. In order to advance understanding of the specificity of GH149 enzymes, we herein solved X-ray crystallographic structures of GH149 enzyme Pro_7066 in the absence of substrate and in complex with laminarihexaose (G6). The overall domain organization of Pro_7066 is very similar to that of GH94 family enzymes. However, two additional domains flanking its catalytic domain were found only in the GH149 enzyme. Unexpectedly, the G6 complex structure revealed an oligosaccharide surface binding site remote from the catalytic site, which, we suggest, may be associated with substrate targeting. As such, this study reports the first structure of a GH149 phosphorylase enzyme acting on β-(1 → 3)-gluco-oligosaccharides and identifies structural elements that may be involved in defining the specificity of the GH149 enzymes., (© 2019 The Authors. Proteins: Structure, Function, and Bioinformatics published by Wiley Periodicals, Inc.)

The introduction of azole heterocycles into a peptide backbone is the principal step in the biosynthesis of numerous compounds with therapeutic potential. One of them is microcin B17, a bacterial topoisomerase inhibitor whose activity depends on the conversion of selected serine and cysteine residues of the precursor peptide to oxazoles and thiazoles by the McbBCD synthetase complex. Crystal structures of McbBCD reveal an octameric B 4 C 2 D 2 complex with two bound substrate peptides. Each McbB dimer clamps the N-terminal recognition sequence, while the C-terminal heterocycle of the modified peptide is trapped in the active site of McbC. The McbD and McbC active sites are distant from each other, which necessitates alternate shuttling of the peptide substrate between them, while remaining tethered to the McbB dimer. An atomic-level view of the azole synthetase is a starting point for deeper understanding and control of biosynthesis of a large group of ribosomally synthesized natural products., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

GlgE is a maltosyltransferase involved in α-glucan biosynthesis in bacteria that has been genetically validated as a target for tuberculosis therapies. Crystals of the Mycobacterium tuberculosis enzyme diffract at low resolution so most structural studies have been with the very similar Streptomyces coelicolor GlgE isoform 1. Although the donor binding site for α-maltose 1-phosphate had been previously structurally defined, the acceptor site had not. Using mutagenesis, kinetics, and protein crystallography of the S. coelicolor enzyme, we have now identified the +1 to +6 subsites of the acceptor/product, which overlap with the known cyclodextrin binding site. The sugar residues in the acceptor subsites +1 to +5 are oriented such that they disfavor the binding of malto-oligosaccharides that bear branches at their 6-positions, consistent with the known acceptor chain specificity of GlgE. A secondary binding site remote from the catalytic center was identified that is distinct from one reported for the M. tuberculosis enzyme. This new site is capable of binding a branched α-glucan and is most likely involved in guiding acceptors toward the donor site because its disruption kinetically compromises the ability of GlgE to extend polymeric substrates. However, disruption of this site, which is conserved in the Streptomyces venezuelae GlgE enzyme, did not affect the growth of S. venezuelae or the structure of the polymeric product. The acceptor subsites +1 to +4 in the S. coelicolor enzyme are well conserved in the M. tuberculosis enzyme so their identification could help inform the design of inhibitors with therapeutic potential., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

SimC7 is a polyketide ketoreductase involved in biosynthesis of the angucyclinone moiety of the gyrase inhibitor simocyclinone D8 (SD8). SimC7, which belongs to the short-chain dehydrogenase/reductase (SDR) superfamily, catalyzes reduction of the C-7 carbonyl of the angucyclinone, and the resulting hydroxyl is essential for antibiotic activity. SimC7 shares little sequence similarity with characterized ketoreductases, suggesting it might have a distinct mechanism. To investigate this possibility, we determined the structures of SimC7 alone, with NADP(+), and with NADP(+) and the substrate 7-oxo-SD8. These structures show that SimC7 is distinct from previously characterized polyketide ketoreductases, lacking the conserved catalytic triad, including the active-site tyrosine that acts as central acid-base catalyst in canonical SDR proteins. Taken together with functional analyses of active-site mutants, our data suggest that SimC7 catalyzes a substrate-assisted, two-step reaction for reduction of the C-7 carbonyl group involving intramolecular transfer of a substrate-derived proton to generate a phenolate intermediate., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The widespread second messenger molecule cyclic di-GMP (cdG) regulates the transition from motile and virulent lifestyles to sessile, biofilm-forming ones in a wide range of bacteria. Many pathogenic and commensal bacterial-host interactions are known to be controlled by cdG signaling. Although the biochemistry of cyclic dinucleotide metabolism is well understood, much remains to be discovered about the downstream signaling pathways that induce bacterial responses upon cdG binding. As part of our ongoing research into the role of cdG signaling in plant-associated Pseudomonas species, we carried out an affinity capture screen for cdG binding proteins in the model organism Pseudomonas fluorescens SBW25. The flagella export AAA+ ATPase FliI was identified as a result of this screen and subsequently shown to bind specifically to the cdG molecule, with a KD in the low micromolar range. The interaction between FliI and cdG appears to be very widespread. In addition to FliI homologs from diverse bacterial species, high affinity binding was also observed for the type III secretion system homolog HrcN and the type VI ATPase ClpB2. The addition of cdG was shown to inhibit FliI and HrcN ATPase activity in vitro. Finally, a combination of site-specific mutagenesis, mass spectrometry, and in silico analysis was used to predict that cdG binds to FliI in a pocket of highly conserved residues at the interface between two FliI subunits. Our results suggest a novel, fundamental role for cdG in controlling the function of multiple important bacterial export pathways, through direct allosteric control of export ATPase proteins., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

The crystal structure of the GH78 family α-rhamnosidase from Klebsiella oxytoca (KoRha) has been determined at 2.7 Å resolution with rhamnose bound in the active site of the catalytic domain. Curiously, the putative catalytic acid, Asp 222, is preceded by an unusual non-proline cis-peptide bond which helps to project the carboxyl group into the active centre. This KoRha homodimeric structure is significantly smaller than those of the other previously determined GH78 structures. Nevertheless, the enzyme displays α-rhamnosidase activity when assayed in vitro, suggesting that the additional structural domains found in the related enzymes are dispensible for function., (© 2015 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.)

Consistent with their complex lifestyles and rich secondary metabolite profiles, the genomes of streptomycetes encode a plethora of transcription factors, the vast majority of which are uncharacterized. Herein, we use Surface Plasmon Resonance (SPR) to identify and delineate putative operator sites for SCO3205, a MarR family transcriptional regulator from Streptomyces coelicolor that is well represented in sequenced actinomycete genomes. In particular, we use a novel SPR footprinting approach that exploits indirect ligand capture to vastly extend the lifetime of a standard streptavidin SPR chip. We define two operator sites upstream of sco3205 and a pseudopalindromic consensus sequence derived from these enables further potential operator sites to be identified in the S. coelicolor genome. We evaluate each of these through SPR and test the importance of the conserved bases within the consensus sequence. Informed by these results, we determine the crystal structure of a SCO3205-DNA complex at 2.8 Å resolution, enabling molecular level rationalization of the SPR data. Taken together, our observations support a DNA recognition mechanism involving both direct and indirect sequence readout.

The crystal structure of Sco5413 from Streptomyces coelicolor A3(2) has been determined at 1.25 Å resolution, the highest resolution reported for a MarR family transcriptional regulator. Putative orthologs are encoded by the majority of sequenced actinomycete genomes, and may play roles in regulating growth and antibiotic production, but they have yet to be assigned a precise function. Sco5413 forms a homodimer and, through comparisons with other MarR family protein structures, we postulate that it adopts a conformation compatible with DNA binding, and that a channel at the dimer interface, lined by well-conserved residues, is the binding site of an unidentified effector ligand., (Copyright © 2012 Wiley Periodicals, Inc.)

Pac17 is an uncharacterized protein from the pacidamycin gene cluster of the soil bacterium Streptomyces coeruleorubidus. It is implicated in the biosynthesis of the core diaminobutyric acid residue of the antibiotic, although its precise role is uncertain at present. Given that pacidamycins inhibit translocase I of Pseudomonas aeruginosa, a clinically unexploited antibiotic target, they offer new hope in the search for antibacterial agents directed against this important pathogen. Crystals of Pac17 were grown by vapour diffusion and X-ray data were collected at a synchrotron to a resolution of 1.9 Å from a single crystal. The crystal belonged to space group C2, with unit-cell parameters a = 214.12, b = 70.88, c = 142.22 Å, β = 92.96°. Preliminary analysis of these data suggests that the asymmetric unit consists of one Pac17 homotetramer, with an estimated solvent content of 49.0%.

SimR, a TetR-family transcriptional regulator (TFR), controls the export of simocyclinone, a potent DNA gyrase inhibitor made by Streptomyces antibioticus. Simocyclinone is exported by a specific efflux pump, SimX and the transcription of simX is repressed by SimR, which binds to two operators in the simR-simX intergenic region. The DNA-binding domain of SimR has a classical helix-turn-helix motif, but it also carries an arginine-rich N-terminal extension. Previous structural studies showed that the N-terminal extension is disordered in the absence of DNA. Here, we show that the N-terminal extension is sensitive to protease cleavage, but becomes protease resistant upon binding DNA. We demonstrate by deletion analysis that the extension contributes to DNA binding, and describe the crystal structure of SimR bound to its operator sequence, revealing that the N-terminal extension binds in the minor groove. In addition, SimR makes a number of sequence-specific contacts to the major groove via its helix-turn-helix motif. Bioinformatic analysis shows that an N-terminal extension rich in positively charged residues is a feature of the majority of TFRs. Comparison of the SimR-DNA and SimR-simocyclinone complexes reveals that the conformational changes associated with ligand-mediated derepression result primarily from rigid-body rotation of the subunits about the dimer interface.

Crystals of SimR were grown by vapour diffusion. The protein crystallized with trigonal symmetry and X-ray data were recorded to a resolution of 2.3 Å from a single crystal at the synchrotron. SimR belongs to the TetR family of bacterial transcriptional regulators. In the absence of the antibiotic simocyclinone, SimR represses the transcription of a divergently transcribed gene encoding the simocyclinone efflux pump SimX in Streptomyces antibioticus by binding to operators in the simR-simX intergenic region. Simocyclinone binding causes SimR to dissociate from its operators, leading to expression of the SimX efflux pump. Thus, SimR represents an intimate link between the biosynthesis of simocyclinone and its export, which may also provide the mechanism of self-resistance to the antibiotic in the producer strain.

Crystals of recombinant NovN, an O-carbamoyltransferase from Streptomyces spheroides, were grown by vapour diffusion. The protein crystallized in two different crystal forms. Crystal form I belonged to space group C2 and native data were collected to 2.9 A resolution in-house. Crystal form II had I-centred orthorhombic symmetry and native data were recorded to a resolution of 2.3 A at a synchrotron. NovN catalyses the final step in the biosynthesis of the aminocoumarin antibiotic novobiocin that targets the essential bacterial enzyme DNA gyrase.

Crystals of recombinant AbsC (subunit MW = 18 313 Da; 158 amino acids), a novel regulator of antibiotic production from Streptomyces coelicolor, were grown by vapour diffusion. The protein crystallizes in space group P2(1)2(1)2(1), with unit-cell parameters a = 43.53, b = 121.30, c = 143.75 A. Native data to a resolution of 2.25 A were recorded at station PX 14.1 (Daresbury) from a single crystal. Preliminary analysis of these data suggests that the asymmetric unit contains four copies of the AbsC monomer, giving an estimated solvent content of 47.0%. AbsC belongs to the MarR family of proteins that mediate ligand-responsive transcriptional control.

The X-ray structures of the cytoplasmic molybdate-binding protein ModG from Azotobacter vinelandii in two different crystal forms have been determined. For such a small protein it is remarkably complex. Each 14.3 kDa subunit contains two small beta-barrel domains, which display an OB-fold motif, also seen in the related structure of ModE, a molybdenum-dependent transcriptional regulator, and very recently in the Mop protein that, like ModG, has been implicated in molybdenum homeostasis within the cell. In contrast to earlier speculation, the functional unit of ModG is actually not a dimer (as in ModE), but a trimer capable of binding a total of eight molybdate molecules that are distributed between two disparate types of site. All the binding sites are located at subunit interfaces, with one type lying on a crystallographic 3-fold axis, whilst the other lies between pairs of subunits. The two types of site are linked by short hydrogen bond networks that may suggest a cooperative binding mechanism. A superposition of two subunits of the ModG trimer on the apo-ModE dimer allows the probable locations of the molybdate-binding sites of the latter to be assigned. Through structural comparisons with other oxyanion-binding proteins, including Mop and ModE, it is possible to speculate about ligand-binding affinities, selectivity and evolution., (Copyright 12001 Academic Press.)

Resonance Raman (RR) studies have been conducted on Alcaligenes xylosoxidans cytochrome c', a mono-His ligated hemoprotein which reversibly binds NO and CO but not O(2). Recent crystallographic characterization of this protein has revealed the first example of a hemoprotein which can utilize both sides of its heme (distal and proximal) for binding exogenous ligands to its Fe center. The present RR investigation of the Fe coordination and heme pocket environments of ferrous, carbonyl, and nitrosyl forms of cytochrome c' in solution fully supports the structures determined by X-ray crystallography and offers insights into mechanisms of ligand discrimination in heme-based sensors. Ferrous cytochrome c' reacts with CO to form a six-coordinate heme-CO complex, whereas reaction with NO results in cleavage of the proximal linkage to give a five-coordinate heme-NO adduct, despite the relatively high stretching frequency (231 cm(-1)) of the ferrous Fe-N(His) bond. RR spectra of the six-coordinate CO adduct indicate that CO binds to the Fe in a nonpolar environment in line with its location in the hydrophobic distal heme pocket. On the other hand, RR data for the five-coordinate NO adduct suggest a positively polarized environment for the NO ligand, consistent with its binding close to Arg 124 on the opposite (proximal) side of the heme. Parallels between certain physicochemical properties of cytochrome c' and those of heme-based sensor proteins raise the possibility that the latter may also utilize both sides of their hemes to discriminate between NO and CO binding.

Background: All mononuclear molybdoenzymes bind molybdenum in a complex with an organic cofactor termed molybdopterin (MPT). In many bacteria, including Escherichia coli, molybdopterin can be further modified by attachment of a GMP group to the terminal phosphate of molybdopterin to form molybdopterin guanine dinucleotide (MGD). This modification reaction is required for the functioning of many bacterial molybdoenzymes, including the nitrate reductases, dimethylsulfoxide (DMSO) and trimethylamine-N-oxide (TMAO) reductases, and formate dehydrogenases. The GMP attachment step is catalyzed by the cellular enzyme MobA., Results: The crystal structure of the 21.6 kDa E. coli MobA has been determined by MAD phasing with selenomethionine-substituted protein and subsequently refined at 1. 35 A resolution against native data. The structure consists of a central, predominantly parallel beta sheet sandwiched between two layers of alpha helices and resembles the dinucleotide binding Rossmann fold. One face of the molecule bears a wide depression that is lined by a number of strictly conserved residues, and this feature suggests that this is where substrate binding and catalysis take place., Conclusions: Through comparisons with a number of structural homologs, we have assigned plausible functions to several of the residues that line the substrate binding pocket. The enzymatic mechanism probably proceeds via a nucleophilic attack by MPT on the GMP donor, most likely GTP, to produce MGD and pyrophosphate. By analogy with related enzymes, this process is likely to require magnesium ions.

Several chimeric alpha-amylases genes were constructed by an in vivo recombination technique from the Bacillus amyloliquefaciens and Bacillus licheniformis genes. One of the fusion amylases (hereafter BA2), consisting of residues 1-300 from B. amyloliquefaciens and 301-483 from B. licheniformis, has been extensively studied by X-ray crystallography at resolutions between 2.2 and 1.7 A. The 3-dimensional structure of the native enzyme was solved by multiple isomorphous replacement, and refined at a resolution of 1.7 A. It consists of 483 amino acids, organized similarly to the known B. lichiniformis alpha-amylase structure [Machius et al. (1995) J. Mol. Biol. 246, 545-559], but features 4 bound calcium ions. Two of these form part of a linear cluster of three ions, the central ion being attributed to sodium. This cluster lies at the junction of the A and B domains with one calcium of the cluster structurally equivalent to the major Ca(2+) binding site of fungal alpha-amylases. The third calcium ion is found at the interface of the A and C domains. BA2 contains a fourth calcium site, not observed in the B. licheniformis alpha-amylase structure. It is found on the C domain where it bridges the two beta-sheets. Three acid residues (Glu261, Asp328, and Asp231) form an active site similar to that seen in other amylases. In the presence of TRIS buffer, a single molecule of TRIS occupies the -1 subsite of the enzyme where it is coordinated by the three active-center carboxylates. Kinetic data reveal that BA2 displays properties intermediate to those of its parents. Data for crystals soaked in maltooligosaccharides reveal the presence of a maltotriose binding site on the N-terminal face of the (beta/alpha)(8) barrel of the molecule, not previously described for any alpha-amylase structure, the biological function of which is unclear. Data for a complex soaked with the tetrasaccharide inhibitor acarbose, at 1.9 A, reveal a decasaccharide moiety, spanning the -7 to +3 subsites of the enzyme. The unambiguous presence of three unsaturated rings in the (2)H(3) half-chair/(2)E envelope conformation, adjacent to three 6-deoxypyranose units, clearly demonstrates synthesis of this acarbose-derived decasaccharide by a two-step transglycosylation mechanism.

Crystals of the molbindin ModG (subunit Mr = 14359 Da), a cytoplasmic molybdate-binding protein from Azotobacter vinelandii, were grown by vapour diffusion. Both apo and tungstate-bound forms were crystallized and X-ray data were collected at 100 K. Apo-ModG crystallizes in space group P6322, with unit-cell dimensions a = b = 90.62, c = 79.46 A. Native data to a resolution of 2.5 A were collected from a single crystal, which showed a marked improvement in diffraction quality after annealing. Data from a single-site gold derivative were also collected at 2.7 A resolution. Crystals of the ligand-bound form of ModG belong to space group P321, with unit-cell parameters a = b = 50.57, c = 79.29 A. X-ray data to a resolution of 2.0 A were collected.

Background: . Periplasmic receptors constitute a diverse class of binding proteins that differ widely in size, sequence and ligand specificity. Nevertheless, almost all of them display a common beta/alpha folding motif and have similar tertiary structures consisting of two globular domains. The ligand is bound at the bottom of a deep cleft, which lies at the interface between these two domains. The oxyanion-binding proteins are notable in that they can discriminate between very similar ligands., Results: . Azotobacter vinelandii is unusual in that it possesses two periplasmic molybdate-binding proteins. The crystal structure of one of these with bound ligand has been determined at 1.2 A resolution. It superficially resembles the structure of sulphate-binding protein (SBP) from Salmonella typhimurium and uses a similar constellation of hydrogen-bonding interactions to bind its ligand. However, the detailed interactions are distinct from those of SBP and the more closely related molybdate-binding protein of Escherichia coli., Conclusions: . Despite differences in the residues involved in binding, the volumes of the binding pockets in the A. vinelandii and E. coli molybdate-binding proteins are similar and are significantly larger than that of SBP. We conclude that the discrimination between molybdate and sulphate shown by these binding proteins is largely dependent upon small differences in the sizes of these two oxyanions.