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Instruments / Technical Area	Publication Details	Published
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	Structural and mechanistic analysis of ATPase inhibitors targeting mycobacterial DNA gyrase Sara R. Henderson , Clare E. M. Stevenson , Brandon Malone , Yelyzaveta Zholnerovych , Lesley A Mitchenall , Mark Pichowicz , David H Mcgarry , Ian R Cooper , Cedric Charrier , Anne-marie Salisbury , David M. Lawson , Anthony Maxwell Journal Of Antimicrobial Chemotherapy 22 DOI: 10.1093/jac/dkaa286 Diamond Proposal Number(s): [18565] Open Access Show Abstract ▼ Abstract: Objectives: To evaluate the efficacy of two novel compounds against mycobacteria and determine the molecular basis of their action on DNA gyrase using structural and mechanistic approaches. Methods: Redx03863 and Redx04739 were tested in antibacterial assays, and also against their target, DNA gyrase, using DNA supercoiling and ATPase assays. X-ray crystallography was used to determine the structure of the gyrase B protein ATPase sub-domain from Mycobacterium smegmatis complexed with the aminocoumarin drug novobiocin, and structures of the same domain from Mycobacterium thermoresistibile complexed with novobiocin, and also with Redx03863. Results: Both compounds, Redx03863 and Redx04739, were active against selected Gram-positive and Gram-negative species, with Redx03863 being the more potent, and Redx04739 showing selectivity against M. smegmatis. Both compounds were potent inhibitors of the supercoiling and ATPase reactions of DNA gyrase, but did not appreciably affect the ATP-independent relaxation reaction. The structure of Redx03863 bound to the gyrase B protein ATPase sub-domain from M. thermoresistibile shows that it binds at a site adjacent to the ATP- and novobiocin-binding sites. We found that most of the mutations that we made in the Redx03863-binding pocket, based on the structure, rendered gyrase inactive. Conclusions: Redx03863 and Redx04739 inhibit gyrase by preventing the binding of ATP. The fact that the Redx03863-binding pocket is distinct from that of novobiocin, coupled with the lack of activity of resistant mutants, suggests that such compounds could have potential to be further exploited as antibiotics.	Jul 2020
I02-Macromolecular Crystallography I03-Macromolecular Crystallography I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Architecture of microcin B17 synthetase: an octameric protein complex converting a ribosomally synthesized peptide into a DNA gyrase poison Dmitry Ghilarov , Clare E. M. Stevenson , Dmitrii Y. Travin , Julia Piskunova , Marina Serebryakova , Anthony Maxwell , David M. Lawson , Konstantin Severinov Molecular Cell DOI: 10.1016/j.molcel.2018.11.032 Diamond Proposal Number(s): [9475, 13467] Open Access Show Abstract ▼ Abstract: 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 B4C2D2 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.	Jan 2019
I03-Macromolecular Crystallography	A new class of antibacterials, the imidazopyrazinones, reveal structural transitions involved in DNA gyrase poisoning and mechanisms of resistance Thomas Germe , Judit Voros , Frederic Jeannot , Thomas Taillier , Robert A. Stavenger , Eric Bacqué , Anthony Maxwell , Benjamin D. Bax Nucleic Acids Research 16 DOI: 10.1093/nar/gky181 Diamond Proposal Number(s): [5799] Open Access Show Abstract ▼ Abstract: Imidazopyrazinones (IPYs) are a new class of compounds that target bacterial topoisomerases as a basis for their antibacterial activity. We have characterized the mechanism of these compounds through structural/mechanistic studies showing they bind and stabilize a cleavage complex between DNA gyrase and DNA (‘poisoning’) in an analogous fashion to fluoroquinolones, but without the requirement for the water–metal–ion bridge. Biochemical experiments and structural studies of cleavage complexes of IPYs compared with an uncleaved gyrase–DNA complex, reveal conformational transitions coupled to DNA cleavage at the DNA gate. These involve movement at the GyrA interface and tilting of the TOPRIM domains toward the scissile phosphate coupled to capture of the catalytic metal ion. Our experiments show that these structural transitions are involved generally in poisoning of gyrase by therapeutic compounds and resemble those undergone by the enzyme during its adenosine triphosphate-coupled strand-passage cycle. In addition to resistance mutations affecting residues that directly interact with the compounds, we characterized a mutant (D82N) that inhibits formation of the cleavage complex by the unpoisoned enzyme. The D82N mutant appears to act by stabilizing the binary conformation of DNA gyrase with uncleaved DNA without direct interaction with the compounds. This provides general insight into the resistance mechanisms to antibiotics targeting bacterial type II topoisomerases.	Mar 2018
I02-Macromolecular Crystallography I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	The Origins of Specificity in the Microcin-Processing Protease TldD/E Dmitry Ghilarov , Marina Serebryakova , Clare E. M. Stevenson , Stephen J. Hearnshaw , Dmitry Volkov , Anthony Maxwell , David M. Lawson , Konstantin Severinov Structure DOI: 10.1016/j.str.2017.08.006 Diamond Proposal Number(s): [9475] Open Access Show Abstract ▼ Abstract: TldD and TldE proteins are involved in the biosynthesis of microcin B17 (MccB17), an Escherichia coli thiazole/oxazole-modified peptide toxin targeting DNA gyrase. Using a combination of biochemical and crystallographic methods we show that E. coli TldD and TldE interact to form a heterodimeric metalloprotease. TldD/E cleaves the N-terminal leader sequence from the modified MccB17 precursor peptide, to yield mature antibiotic, while it has no effect on the unmodified peptide. Both proteins are essential for the activity; however, only the TldD subunit forms a novel metal-containing active site within the hollow core of the heterodimer. Peptide substrates are bound in a sequence-independent manner through β sheet interactions with TldD and are likely cleaved via a thermolysin-type mechanism. We suggest that TldD/E acts as a “molecular pencil sharpener”: unfolded polypeptides are fed through a narrow channel into the active site and processively truncated through the cleavage of short peptides from the N-terminal end.	Sep 2017
I02-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	Thiophene antibacterials that allosterically stabilize DNA-cleavage complexes with DNA gyrase Pan F. Chan , Thomas Germe , Benjamin Bax , Jianzhong Huang , Reema K. Thalji , Eric Bacqué , Anna Checchia , Dongzhao Chen , Haifeng Cui , Xiao Ding , Karen Ingraham , Lynn Mccloskey , Kaushik Raha , Velupillai Srikannathasan , Anthony Maxwell , Robert A. Stavenger Proceedings Of The National Academy Of Sciences 114, E4492 - E4500 DOI: 10.1073/pnas.1700721114 Diamond Proposal Number(s): [1195] Open Access Show Abstract ▼ Abstract: A paucity of novel acting antibacterials is in development to treat the rising threat of antimicrobial resistance, particularly in Gram-negative hospital pathogens, which has led to renewed efforts in antibiotic drug discovery. Fluoroquinolones are broad-spectrum antibacterials that target DNA gyrase by stabilizing DNA-cleavage complexes, but their clinical utility has been compromised by resistance. We have identified a class of antibacterial thiophenes that target DNA gyrase with a unique mechanism of action and have activity against a range of bacterial pathogens, including strains resistant to fluoroquinolones. Although fluoroquinolones stabilize double-stranded DNA breaks, the antibacterial thiophenes stabilize gyrase-mediated DNA-cleavage complexes in either one DNA strand or both DNA strands. X-ray crystallography of DNA gyrase–DNA complexes shows the compounds binding to a protein pocket between the winged helix domain and topoisomerase-primase domain, remote from the DNA. Mutations of conserved residues around this pocket affect activity of the thiophene inhibitors, consistent with allosteric inhibition of DNA gyrase. This druggable pocket provides potentially complementary opportunities for targeting bacterial topoisomerases for antibiotic development.	May 2017
I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	The role of monovalent cations in the ATPase reaction of DNA gyrase Stephen Hearnshaw , Terence Chung , Clare Stevenson , Anthony Maxwell , David Lawson Acta Crystallographica Section D 71, 996-1005 DOI: 10.1107/S1399004715002916 PMID: 25849408 Diamond Proposal Number(s): [1219, 7641] Open Access Show Abstract ▼ Abstract: Abstract We have determined four new crystal structures of the ATPase domain of the GyrB subunit of Escherichia coli DNA gyrase. One of these, solved in the presence of K+, is the highest resolution structure reported so far for this domain, and, in conjunction with the three other structures, reveals new insights into the function of this domain. We show evidence for the existence of two monovalent cation-binding sites: site 1, which preferentially binds a K+ ion that interacts directly with the Ñ-phosphate of ATP, and site 2, which preferentially binds a Na+ ion, whose functional significance is not clear. The crystallographic data are corroborated by ATPase data, and we compare our structures with those of homologues to investigate the broader conservation of these sites.	Feb 2015
I04-1-Macromolecular Crystallography (fixed wavelength) I24-Microfocus Macromolecular Crystallography	A new crystal structure of the bifunctional antibiotic simocyclinone D8 bound to DNA gyrase gives fresh insight into the mechanism of inhibition Stephen Hearnshaw , Marcus Edwards , Clare Stevenson , David Lawson , Anthony Maxwell Journal Of Molecular Biology DOI: 10.1016/j.jmb.2014.02.017 PMID: 24594357 Diamond Proposal Number(s): [1219, 7641] Open Access Show Abstract ▼ Abstract: Simocyclinone D8 (SD8) is an antibiotic produced by Streptomyces antibioticus that targets DNA gyrase. A previous structure of SD8 complexed with the N-terminal domain of the DNA gyrase A protein (GyrA) suggested that four SD8 molecules stabilized a tetramer of the protein; subsequent mass spectrometry experiments suggested that a protein dimer with two symmetry-related SD8s was more likely. This work describes the structures of a further truncated form of the GyrA N-terminal domain fragment with and without SD8 bound. The structure with SD8 has the two SD8 molecules bound within the same GyrA dimer. This new structure is entirely consistent with the mutations in GyrA that confer SD8 resistance and, by comparison with a new apo structure of the GyrA N-terminal domain, reveals the likely conformation changes that occur upon SD8 binding and the detailed mechanism of SD8 inhibition of gyrase. Isothermal titration calorimetry experiments are consistent with the crystallography results and further suggest that a previously observed complex between SD8 and GyrB is ~ 1000-fold weaker than the interaction with GyrA.	Mar 2014
I02-Macromolecular Crystallography I03-Macromolecular Crystallography I04-Macromolecular Crystallography	Structures of the TetR-like simocyclinone efflux pump repressor, SimR, and the mechanism of ligand-mediated derepression Tung Le , Clare Stevenson , Hans-peter Fiedler , Anthony Maxwell , David M. Lawson , Mark J. Buttner Journal Of Molecular Biology DOI: 10.1016/j.jmb.2011.02.035 PMID: 21354180 Show Abstract ▼ Abstract: Simocyclinone D8, a potent DNA gyrase inhibitor produced by Streptomyces antibioticus, is exported from the producing organism by the SimX efflux pump. The expression of simX is under the control of SimR, a member of the TetR family of transcriptional regulators (TFRs). SimR represses simX transcription by binding to operators in the intergenic region between simR and simX. Previously we have shown that the mature antibiotic, simocyclinone D8, or its biosynthetic intermediate, simocyclinone C4, can dissociate SimR from its operators, leading to derepression of simX and export of simocyclinone D8 from the cell. This provides a mechanism that couples the biosynthesis of the antibiotic to its export. Here we report crystal structures of SimR alone and in complex with either simocyclinone D8 or simocyclinone C4. The ligand-binding pocket is unusual compared to those of other characterised TFRs: the structures show an extensive ligand-binding pocket spanning both monomers in the functional dimeric unit, with the aminocoumarin moiety of simocyclinone D8 buried in the protein core, while the angucyclic polyketide moiety is partially exposed to bulk solvent. Through comparisons of the structures, we postulate a derepression mechanism for SimR that invokes rigid body motions of the subunits relative to one another, coupled with a putative locking mechanism to restrict further conformational change.	Feb 2011

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