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Instruments / Technical Area	Publication Details	Published
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
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

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