The interaction of DNA gyrase with the bacterial toxin CcdB: Evidence for the existence of two gyrase-CcdB complexes

CcdB is a bacterial toxin that targets DNA gyrase. Analysis of the interact ion 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 prote olytic signature which is characterised by a 49 kDa fragment of GyrA. Surfa ce 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 abil ity of gyrase to hydrolyse Am or promote supercoiling. Incubation of this i nitial complex with Am 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 inact ive 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 Am hydrolysis. (C) 1999 Acade mic Press.