Conformational changes in DNA gyrase revealed by limited proteolysis.
We have used limited proteolysis to identify conformational changes in DNA gyrase. Gyrase exhibits a proteolytic fingerprint dominated by two fragments, one of approximately 62 kDa, deriving from the A protein, and another of approximately 25 kDa from the B protein. Quinolone binding to the enzyme-DNA complex induces a conformational change which is reflected in the protection of the C-terminal 47-kDa domain of the B protein. An active site mutant (Tyr122 to Ser in the A protein) that binds quinolones but cannot cleave DNA still gives the quinolone proteolytic pattern, while stabilization of a cleaved-DNA intermediate by calcium ions does not reveal any protection, suggesting that the quinolone-induced conformational change is different from an "open-gate" state of the enzyme. A quinolone-resistant mutant of gyrase fails to give the characteristic quinolone-associated proteolytic signature. The ATP-induced dimerization of the B subunits is a key step of the gyrase mechanism. The proteolytic fingerprint of this conformation (stabilized by the non-hydrolyzable ATP analog 5'-adenylyl-beta, gamma-imidodiphosphate (ADPNP) shows a protection of the 43-kDa N-terminal domain of the B subunit. The presence of quinolones does not prevent dimerization since incubation of the enzyme-DNA complex with both ADPNP and quinolones gives rise to a complex whose proteolytic pattern retains the characteristic signature of dimerization but has lost the quinolone-induced protection. As a result, the quinolone-gyrase complex can still hydrolyze ATP, albeit with different kinetic characteristics. We interpret the proteolytic signatures observed in terms of four complexes of gyrase, each representing a particular conformational state.
Kampranis
S C
SC
Department of Biochemistry, University of Leicester, University Road, Leicester, LE1 7RH, United Kingdom.
Maxwell
A
A
D000255:Adenosine Triphosphate / Q000378:metabolism; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D004250:DNA Topoisomerases, Type II / Q000737:chemistry* / Q000378:metabolism; D019281:Dimerization; D006868:Hydrolysis; D011485:Protein Binding; D011487:Protein Conformation; D015363:Quinolones / Q000378:metabolism
D015363:Quinolones; D000266:Adenylyl Imidodiphosphate; D000255:Adenosine Triphosphate; D004250:DNA Topoisomerases, Type II