Fluoroquinolone-Gyrase-DNA Complexes: Two Modes of Drug Binding [Microbiology]

February 4th, 2014 by Mustaev, A., Malik, M., Zhao, X., Kurepina, N., Luan, G., Oppegard, L. M., Hiasa, H., Marks, K. R., Kerns, R. J., Berger, J. M., Drlica, K.

DNA gyrase and topoisomerase IV control bacterial DNA topology by breaking DNA, passing duplex DNA through the break, and then resealing the break. This process is subject to reversible corruption by fluoroquinolones, antibacterials that form drug-enzyme-DNA complexes in which the DNA is broken. The complexes, called cleaved complexes due to the presence of DNA breaks, have been crystallized and found to have the fluoroquinolone C-7 ring system facing the GyrB/ParE subunits. As expected from X-ray crystallography, a thiol-reactive, C-7-modified chloroacetyl derivative of ciprofloxacin (Cip-AcCl) formed crosslinked cleaved complexes with mutant GyrB-466Cys gyrase, as evidenced by resistance to reversal by both EDTA and thermal treatments. Surprisingly, crosslinking was also readily seen with complexes formed by mutant GyrA-81Cys gyrase, thereby revealing a novel drug-gyrase interaction not observed in crystal structures. The crosslink between fluoroquinolone and GyrA-81Cys gyrase correlated with exceptional bacteriostatic activity for Cip-AcCl with a quinolone-resistant GyrA-81Cys variant of Escherichia coli and its Mycobacterium smegmatis equivalent (GyrA-89Cys). Cip-AcCl-mediated, irreversible inhibition of DNA replication provided further evidence for a GyrA-drug crosslink. Collectively these data establish the existence of interactions between the fluoroquinolone C-7 ring and both GyrA and GyrB. Since the GyrA-81 and GyrB-466 residues are far apart (17 A) in the crystal structure of cleaved complexes, two modes of quinolone binding must exist. The presence of two binding modes raises the possibility that multiple quinolone-enzyme-DNA complexes can form, a discovery that opens new avenues for exploring and exploiting relationships between drug structure and activity with type II DNA topoisomerases.