David M. Lawson

Identification of the ferroxidase centre in ferritin

Ferroxidase activity in human H‐chain ferritin has been studied with the aid of site‐directed mutagenesis. A site discovered by X‐ray crystallography has now been identified as the ferroxidase centre. This centre is present only in H‐chains and is located within the four‐helix bundle of the chain fold.

The ATP-Binding Site of Type II Topoisomerases as a Target for Antibacterial Drugs

DNA topoisomerases are essential enzymes in all cell types and have been found to be valuable drug targets both for antibacterial and anti-cancer chemotherapy. Type II topoisomerases possess a binding site for ATP, which can be exploited as a target for chemo-therapeutic agents. High-resolution structures of protein fragments containing this site complexed with antibiotics or an ATP analogue have provided vital information for the understanding of the action of existing drugs and for the potential development of novel anti-bacterial agents. In this article we have reviewed the structure and function of the ATPase domain of DNA gyrase (bacterial topoisomerase II), particularly highlighting novel information that has been revealed by structural studies. We discuss the efficacy and mode of action of existing drugs and consider the prospects for the development of novel agents.

Unprecedented Proximal Binding of Nitric Oxide to Heme: Implications for Guanylate Cyclase

Microbial cytochromes c′ contain a 5‐coordinate His‐ligated heme that forms stable adducts with nitric oxide (NO) and carbon monoxide (CO), but not with dioxygen. We report the 1.95 and 1.35 Å resolution crystal structures of the CO‐ and NO‐bound forms of the reduced protein from Alcaligenes xylosoxidans. NO disrupts the His–Fe bond and binds in a novel mode to the proximal face of the heme, giving a 5‐coordinate species. In contrast, CO binds 6‐coordinate on the distal side. A second CO molecule, not bound to the heme, is located in the proximal pocket. Since the unusual spectroscopic properties of cytochromes c′ are shared by soluble guanylate cyclase (sGC), our findings have potential implications for the activation of sGC induced by the binding of NO or CO to the heme domain.

A crystal structure of the bifunctional antibiotic simocyclinone D8, bound to DNA gyrase.

Targeting DNA Gyrase DNA gyrase, an enzyme that unwinds double-stranded DNA, is essential in bacteria, but missing in humans, and is thus an important antibiotic target. DNA gyrase is inhibited by the well-known fluoroquinolines and aminocoumarins antibiotics, as well as by symocyclinones—bifunctional antibiotics comprising an aminocoumarin and a polyketide group. Surprisingly, symocyclinones, unlike aminocoumarin inhibitors, do not inhibit DNA gyrase GTPase activity, but instead inhibit binding to DNA. Now Edwards et al. (p. 1415) use biochemical and structural studies to show that the two functional groups of the antibiotic bind in separate pockets on the gyrase. Each group is a relatively weak inhibitor that together potently inhibit DNA binding. The molecular mechanism is revealed by which an antibiotic prevents DNA binding by a bacterial DNA gyrase. Simocyclinones are bifunctional antibiotics that inhibit bacterial DNA gyrase by preventing DNA binding to the enzyme. We report the crystal structure of the complex formed between the N-terminal domain of the Escherichia coli gyrase A subunit and simocyclinone D8, revealing two binding pockets that separately accommodate the aminocoumarin and polyketide moieties of the antibiotic. These are close to, but distinct from, the quinolone-binding site, consistent with our observations that several mutations in this region confer resistance to both agents. Biochemical studies show that the individual moieties of simocyclinone D8 are comparatively weak inhibitors of gyrase relative to the parent compound, but their combination generates a more potent inhibitor. Our results should facilitate the design of drug molecules that target these unexploited binding pockets.

Identification and structure of the anti-sigma factor-binding domain of the disulphide-stress regulated sigma factor σR from Streptomyces coelicolor

The extracytoplasmic function (ECF) sigma factor sigma(R) is a global regulator of redox homeostasis in the antibiotic-producing bacterium Streptomyces coelicolor, with a similar role in other actinomycetes such as Mycobacterium tuberculosis. Normally maintained in an inactive state by its bound anti-sigma factor RsrA, sigma(R) dissociates in response to intracellular disulphide-stress to direct core RNA polymerase to transcribe genes, such as trxBA and trxC that encode the enzymes of the thioredoxin disulphide reductase pathway, that re-establish redox homeostasis. Little is known about where RsrA binds on sigma(R) or how it suppresses sigma(R)-dependent transcriptional activity. Using a combination of proteolysis, surface-enhanced laser desorption ionisation mass spectrometry and pull-down assays we identify an N-terminal, approximately 10kDa domain (sigma(RN)) that encompasses region 2 of sigma(R) that represents the major RsrA binding site. We show that sigma(RN) inhibits transcription by an unrelated sigma factor and that this inhibition is relieved by RsrA binding, reaffirming that region 2 is involved in binding to core RNA polymerase but also demonstrating that the likely mechanism by which RsrA inhibits sigma(R) activity is by blocking this association. We also report the 2.4A resolution crystal structure of sigma(RN) that reveals extensive structural conservation with the equivalent region of sigma(70) from Escherichia coli as well as with the cyclin-box, a domain-fold found in the eukaryotic proteins TFIIB and cyclin A. sigma(RN) has a propensity to aggregate, due to steric complementarity of oppositely charged surfaces on the domain, but this is inhibited by RsrA, an observation that suggests a possible mode of action for RsrA which we compare to other well-studied sigma factor-anti-sigma factor systems.

Crystallographic analysis of the MoFe protein of nitrogenase from a nifV mutant of Klebsiella pneumoniae identifies citrate as a ligand to the molybdenum of iron molybdenum cofactor (FeMoco).

The x-ray crystal structure of NifV− Klebsiella pneumoniae nitrogenase MoFe protein (NifV− Kp1) has been determined and refined to a resolution of 1.9 Å. This is the first structure for a nitrogenase MoFe protein with an altered cofactor. Moreover, it is the first direct evidence that the organic acid citrate is not just present, but replaces homocitrate as a ligand to the molybdenum atom of the iron molybdenum cofactor (FeMoco). Subsequent refinement of the structure revealed that the citrate was present at reduced occupancy.

Ligand size is a major determinant of specificity in periplasmic oxyanion-binding proteins: the 1.2 Å resolution crystal structure of Azotobacter vinelandii ModA

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.

The crystal structure of the TetR family transcriptional repressor SimR bound to DNA and the role of a flexible N-terminal extension in minor groove binding

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.

Structure of Streptomyces Maltosyltransferase GlgE, a Homologue of a Genetically Validated Anti-tuberculosis Target

Background: GlgE is a maltosyltransferase involved in bacterial α-glucan biosynthesis and is a genetically validated anti-tuberculosis target. Results: We have determined the catalytic properties of Streptomyces coelicolor GlgE and solved its structure. Conclusion: The enzyme has the same catalytic properties as Mycobacterium tuberculosis GlgE and the structure reveals how GlgE functions. Significance: The structure will help guide the development of inhibitors with therapeutic potential. GlgE is a recently identified (1→4)-α-d-glucan:phosphate α-d-maltosyltransferase involved in α-glucan biosynthesis in bacteria and is a genetically validated anti-tuberculosis drug target. It is a member of the GH13_3 CAZy subfamily for which no structures were previously known. We have solved the structure of GlgE isoform I from Streptomyces coelicolor and shown that this enzyme has the same catalytic and very similar kinetic properties to GlgE from Mycobacterium tuberculosis. The S. coelicolor enzyme forms a homodimer with each subunit comprising five domains, including a core catalytic α-amylase-type domain A with a (β/α)8 fold. This domain is elaborated with domain B and two inserts that are specifically configured to define a well conserved donor pocket capable of binding maltose. Domain A, together with domain N from the neighboring subunit, forms a hydrophobic patch that is close to the maltose-binding site and capable of binding cyclodextrins. Cyclodextrins competitively inhibit the binding of maltooligosaccharides to the S. coelicolor enzyme, showing that the hydrophobic patch overlaps with the acceptor binding site. This patch is incompletely conserved in the M. tuberculosis enzyme such that cyclodextrins do not inhibit this enzyme, despite acceptor length specificity being conserved. The crystal structure reveals two further domains, C and S, the latter being a helix bundle not previously reported in GH13 members. The structure provides a framework for understanding how GlgE functions and will help guide the development of inhibitors with therapeutic potential.

Probing a novel potato lipoxygenase with dual positional specificity reveals primary determinants of substrate binding and requirements for a surface hydrophobic loop and has implications for the role of lipoxygenases in tubers.

A new potato tuber lipoxygenase full-length cDNA sequence (lox1:St:2) has been isolated from potato tubers and used to express in Escherichia coli and characterize a novel recombinant lipoxygenase (potato 13/9-lipoxygenase). Like most plant lipoxygenases it produced carbonyl compounds from linoleate (the preferred substrate) and was purified in the Fe(II) (ferrous) state. Typical of other potato tuber lipoxygenases, it produced 5-HPETE [5(S)-hydroperoxy-(6E, 8Z, 11Z, 14Z)-eicosatetraenoic acid] from arachidonate. In contrast to any other potato tuber lipoxygenase, it exhibited dual positional specificity and produced roughly equimolar amounts of 13- and 9-hydroperoxides (or only a slight molar excess of 9-hydroperoxides) from linoleate. We have used a homology model of pea 9/13-lipoxygenase to superimpose and compare the linoleate-binding pockets of different potato lipoxygenases of known positional specificity. We then tested this model by using site-directed mutagenesis to identify some primary determinants of linoleate binding to potato 13/9-lipoxygenase and concluded that the mechanism determining positional specificity described for a cucumber lipoxygenase does not apply to potato 13/9-lipoxygenase. This supports our previous studies on pea seed lipoxygenases for the role of pocket volume rather than inverse orientation as a determinant of dual positional specificity in plant lipoxygenases. We have also used deletion mutagenesis to identify a critical role in catalysis for a surface hydrophobic loop in potato 13/9-lipoxygenase and speculate that this may control substrate access. Although potato 13/9-lipoxygenase represents only a minor isoform in tubers, such evidence for a single lipoxygenase species with dual positional specificity in tubers has implications for the proposed role of potato lipoxygenases in the plant.