A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site.
DNA gyrase is the only topoisomerase able to introduce negative supercoils into DNA. Absent in humans, gyrase is a successful target for antibacterial drugs. However, increasing drug resistance is a serious problem and new agents are urgently needed. The naturally-produced Escherichia coli toxin CcdB has been shown to target gyrase by what is predicted to be a novel mechanism. CcdB has been previously shown to stabilize the gyrase 'cleavage complex', but it has not been shown to inhibit the catalytic reactions of gyrase. We present data showing that CcdB does indeed inhibit the catalytic reactions of gyrase by stabilization of the cleavage complex and that the GyrA C-terminal DNA-wrapping domain and the GyrB N-terminal ATPase domain are dispensable for CcdB's action. We further investigate the role of specific GyrA residues in the action of CcdB by site-directed mutagenesis; these data corroborate a model for CcdB action based on a recent crystal structure of a CcdB-GyrA fragment complex. From this work, we are now able to present a model for CcdB action that explains all previous observations relating to CcdB-gyrase interaction. CcdB action requires a conformation of gyrase that is only revealed when DNA strand passage is taking place.
Smith
Andrew B
AB
Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK.
Maxwell
Anthony
A
D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000494:pharmacology*; D001427:Bacterial Toxins / Q000737:chemistry / Q000494:pharmacology*; D001665:Binding Sites; D002939:Ciprofloxacin / Q000494:pharmacology; D027081:DNA Gyrase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004278:DNA, Superhelical / Q000378:metabolism; D020128:Inhibitory Concentration 50; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D009711:Nucleotides / Q000378:metabolism; D017434:Protein Structure, Tertiary
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D001427:Bacterial Toxins; C068355:CcdA protein, Bacteria; C068356:CcdB protein, Plasmid F; D004278:DNA, Superhelical; D009711:Nucleotides; D002939:Ciprofloxacin; D027081:DNA Gyrase