Fluoroquinolone-gyrase-DNA complexes: two modes of drug binding.
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.
From the Public Health Research Institute and Department of Microbiology and Molecular Genetics, New Jersey Medical School, Rutgers Biomedical and Health Sciences, Newark, New Jersey 07103.
Arkady
A
Mustaev
Muhammad
M
Malik
Xilin
X
Zhao
Natalia
N
Kurepina
Gan
G
Luan
Lisa M
LM
Oppegard
Hiroshi
H
Hiasa
Kevin R
KR
Marks
Robert J
RJ
Kerns
James M
JM
Berger
Karl
K
Drlica
D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D002939:Ciprofloxacin / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D018360:Crystallography, X-Ray; D027081:DNA Gyrase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004250:DNA Topoisomerases, Type II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D024841:Fluoroquinolones / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D046911:Macromolecular Substances / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation; D020102:Mycobacterium smegmatis / Q000187:drug effects; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D059005:Topoisomerase II Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D024841:Fluoroquinolones; D046911:Macromolecular Substances; D059005:Topoisomerase II Inhibitors; D002939:Ciprofloxacin; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II
Antibiotic Action; Bacteria; Bacterium; Cleaved Complexes; Cross-linking; DNA Topoisomerase; Enzyme Inhibitors; Escherichia coli; Mycobacterium smegmatis; Resistance Mutations