Pentapeptide repeat protein QnrB1 requires ATP hydrolysis to rejuvenate poisoned gyrase complexes

Abstract DNA gyrase, a type II topoisomerase found predominantly in bacteria, is the target for variety of ‘poisons’, namely natural product toxins (e.g. albicidin, microcin B17) and clinically important synthetic molecules fluoroquinolones). Resistance to both groups can be mediated by pentapeptide repeat proteins (PRPs). Despite long-term studies, mechanism action these protective PRPs not known. We show that PRP, QnrB1 provides specific protection against fluoroquinolones, which strictly requires ATP hydrolysis gyrase. binds GyrB protein stimulates ATPase activity isolated N-terminal domain (GyrB43). probed binding site using site-specific incorporation photoreactive amino acid mapped crosslinks GyrB43 protein. propose model allosterically promotes dissociation fluoroquinolone molecule from cleavage complex.