Crystallization and initial crystallographic analysis of covalent DNA-cleavage complexes of Staphyloccocus aureus DNA gyrase with QPT-1, moxifloxacin and etoposide

Abstract

Fluoroquinolone drugs such as moxifloxacin kill bacteria by stabilizing the normally transient double-stranded DNA breaks created by bacterial type IIA topoisomerases. Previous crystal structures of Staphylococcus aureus DNA gyrase with asymmetric DNAs have had static disorder (with the DNA duplex observed in two orientations related by the pseudo-twofold axis of the complex). Here, 20-base-pair DNA homoduplexes were used to obtain crystals of covalent DNA-cleavage complexes of S. aureus DNA gyrase. Crystals with QPT-1, moxifloxacin or etoposide diffracted to between 2.45 and 3.15 Å resolution. A G/T mismatch introduced at the ends of the DNA duplexes facilitated the crystallization of slightly asymmetric complexes of the inherently flexible DNAcleavage complexes.

Structural basis of DNA gyrase inhibition by antibacterial QPT-1, anticancer drug etoposide and moxifloxacin

“…As part of a structure-based approach to develop novel bacterial topo2A pharmacophores, we co-crystallized and determined structures 22 of DNA-cleavage complexes of the S. aureus B27-A56(GKdel) fusion truncate (gyrase CORE ) with QPT-1, etoposide and moxifloxacin ( Figs 1 , 2 , 3 , Table 1 , Supplementary Fig. 2 and Supplementary Table 1 ).…”

“…4 ). These complexes (ba_ba' 2–QPT , BA_BA' 2–QPT and BA_BA' 2 − QPT ; see Table 1 ) come from the 2.5- and 3.15-Å QPT-1 co-crystal structures, which were obtained under the same crystallization conditions 22 . When the QPT-1-binding sites are compared ( Fig.…”

“…The optimization of DNA sequences, crystallization and initial structure determinations of the six complexes are described in an accompanying paper 22 .To refine structures, cycles of rebuilding and refinement were carried out with coot 53 , Refmac 54 , Phenix 55 and Buster 56 , to give models with good geometry ( Supplementary Table 1 ). The Ramachandran plots of the structures determined had the following percentages of residues in Ramachandran favoured and Ramachandran outlier regions: (i) 97.7 and 0.15% 2.5 Å QPT-1 structure (pdb code 5CDM), (ii) 96.3 and 0.04% 3.15 Å QPT-1 structure (pdb code 5CDO), (iii) 97.5% and 0.26% 2.8 Å etoposide structure (pdb code 5CDN), (iv) 98.1 and 0.08% 2.45 Å etoposide structure (pdb code 5CDP), (v) 96.7 and 0.64% 2.95 Å moxifloxacin structure (pdb code 5CDQ), and (vi) 97.6 and 0.0% 2.65 Å binary complex (pdb code: 5CDR).…”

Thiophene antibacterials that allosterically stabilize DNA-cleavage complexes with DNA gyrase

“…In parallel with the biochemical and genetic studies, we explored cocrystallization of the inhibitor with DNA gyrase. We have found that the S. aureus DNA gyrase crystallography platform can be used to gain structural information on compounds that target both Grampositive and Gram-negative organisms (15,19,24). Using this crystallographic platform, we obtained a 1.98-Å structure of compound 1 complexed to an S. aureus DNA gyrase core fusion [lacking the gyrase A (GyrA) C-terminal domain, the gyrase B (GyrB) Greek-key domain, and the GyrB N-terminal domain] and DNA ( Fig.…”

“…Proteins were purified and complexes were prepared and crystallized and structures were determined using methods as previously described (19,24). The X-ray data collection (35) and refinement statistics for the two thiophene structures described in this paper are shown in SI Appendix, Table S1.…”