TY - JOUR

T1 - The interaction of DNA gyrase with the bacterial toxin CcdB

T2 - evidence for the existence of two gyrase-CcdB complexes

AU - Kampranis, S C

AU - Howells, A J

AU - Maxwell, A

N1 - Copyright 1999 Academic Press.

PY - 1999/10/29

Y1 - 1999/10/29

N2 - 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.

AB - 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.

KW - Adenosine Triphosphatases

KW - Adenosine Triphosphate

KW - Amino Acid Sequence

KW - Bacterial Proteins

KW - Bacterial Toxins

KW - Binding Sites

KW - Chromatography, Affinity

KW - DNA Topoisomerases, Type II

KW - DNA, Superhelical

KW - Dimerization

KW - Escherichia coli

KW - Hydrolysis

KW - Kinetics

KW - Models, Molecular

KW - Molecular Weight

KW - Mutation

KW - Peptide Fragments

KW - Protein Binding

KW - Protein Conformation

KW - Structure-Activity Relationship

KW - Surface Plasmon Resonance

KW - Topoisomerase II Inhibitors

KW - Trypsin

U2 - 10.1006/jmbi.1999.3182

DO - 10.1006/jmbi.1999.3182

M3 - Journal article

C2 - 10543963

VL - 293

SP - 733

EP - 744

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

SN - 0022-2836

IS - 3

ER -