Abstract
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 because of 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 cross-linked cleaved complexes with mutant GyrB-Cys(466) gyrase as evidenced by resistance to reversal by both EDTA and thermal treatments. Surprisingly, cross-linking was also readily seen with complexes formed by mutant GyrA-G81C gyrase, thereby revealing a novel drug-gyrase interaction not observed in crystal structures. The cross-link between fluoroquinolone and GyrA-G81C gyrase correlated with exceptional bacteriostatic activity for Cip-AcCl with a quinolone-resistant GyrA-G81C variant of Escherichia coli and its Mycobacterium smegmatis equivalent (GyrA-G89C). Cip-AcCl-mediated, irreversible inhibition of DNA replication provided further evidence for a GyrA-drug cross-link. Collectively these data establish the existence of interactions between the fluoroquinolone C-7 ring and both GyrA and GyrB. Because the GyrA-Gly(81) and GyrB-Glu(466) residues are far apart (17 Å) 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.
Highlights
X-ray crystal structures of fluoroquinolone-gyrase-DNA complexes reveal a single drug-binding mode
Cleaved Complexes Formed with Cip-AcCl and Purified GyrB-E466C Gyrase Resist EDTA-mediated Reversal—To study the immediate surroundings of fluoroquinolones within cleaved complexes, we prepared cysteine substitutions in the B subunit of gyrase near the drug binding site defined by crystallography [14] (Fig. 1B)
The chloroacetyl group of Cip-AcCl is expected to be in close proximity to GyrB amino acid Glu466 such that a GyrBE466C variant gyrase would cross-link with Cip-AcCl
Summary
X-ray crystal structures of fluoroquinolone-gyrase-DNA complexes reveal a single drug-binding mode. DNA gyrase and topoisomerase IV control bacterial DNA topology by breaking DNA, passing duplex DNA through the break, and 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. As expected from x-ray crystallography, a thiol-reactive, C-7-modified chloroacetyl derivative of ciprofloxacin (CipAcCl) formed cross-linked cleaved complexes with mutant GyrB-Cys466 gyrase as evidenced by resistance to reversal by both EDTA and thermal treatments. Cip-AcClmediated, irreversible inhibition of DNA replication provided further evidence for a GyrA-drug cross-link. These data establish the existence of interactions between the fluoroquinolone C-7 ring and both GyrA and GyrB. 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
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