title: The interaction of DNA gyrase with the bacterial toxin CcdB: evidence for the existence of two gyrase-CcdB complexes.
abstract: CcdB is a bacterial toxin that targets DNA gyrase. Analysis of the interaction of CcdB with gyrase reveals two distinct complexes. An initial complex (alpha) is formed by direct interaction between GyrA and CcdB; this complex can be detected by affinity column and gel-shift analysis, and has a proteolytic signature which is characterised by a 49 kDa fragment of GyrA. Surface plasmon resonance shows that CcdB binds to the N-terminal domain of GyrA with high affinity. In this mode of binding, CcdB does not affect the ability of gyrase to hydrolyse ATP or promote supercoiling. Incubation of this initial complex with ATP in the presence of GyrB and DNA slowly converts it to a second complex (beta), which has a lower rate of ATP hydrolysis and is unable to catalyse supercoiling. The efficiency of formation of this inactive complex is dependent on the concentrations of ATP and CcdB. We suggest that the conversion between the two complexes proceeds via an intermediate, whose formation is dependent on the rate of ATP hydrolysis.
authors.affiliation: Department of Biochemistry, University of Leicester, Leicester, LE1 7RH, UK.
authors.forename: S C
authors.initials: SC
authors.lastname: Kampranis
authors.forename: A J
authors.initials: AJ
authors.lastname: Howells
authors.forename: A
authors.initials: A
authors.lastname: Maxwell
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D001427:Bacterial Toxins / Q000378:metabolism*; D001665:Binding Sites; D002846:Chromatography, Affinity; D004250:DNA Topoisomerases, Type II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004278:DNA, Superhelical / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D008970:Molecular Weight; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D020349:Surface Plasmon Resonance; D059005:Topoisomerase II Inhibitors; D014357:Trypsin / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; C068356:CcdB protein, Plasmid F; D004278:DNA, Superhelical; D010446:Peptide Fragments; D059005:Topoisomerase II Inhibitors; D000255:Adenosine Triphosphate; D014357:Trypsin; D000251:Adenosine Triphosphatases; D004250:DNA Topoisomerases, Type II