Mapping simocyclinone D8 interaction with DNA gyrase: evidence for a new binding site on GyrB.
Simocyclinone D8, a coumarin derivative isolated from Streptomyces antibioticus Tü 6040, represents an interesting new antiproliferative agent. It was originally suggested that this drug recognizes the GyrA subunit and interferes with the gyrase catalytic cycle by preventing its binding to DNA. To further characterize the mode of action of this antibiotic, we investigated its binding to the reconstituted DNA gyrase (A(2)B(2)) as well as to its GyrA and GyrB subunits and the individual domains of these proteins, by performing protein melting and proteolytic digestion studies as well as inhibition assays. Two binding sites were identified, one (anticipated) in the N-terminal domain of GyrA (GyrA59) and the other (unexpected) at the C-terminal domain of GyrB (GyrB47). Stabilization of the A subunit appears to be considerably more effective than stabilization of the B subunit. Our data suggest that these two distinct sites could cooperate in the reconstituted enzyme.
Department of Pharmaceutical Sciences, University of Padova, Via Marzolo 5, Padova 35131, Italy. claudia.sissi@unipd.it
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Sissi
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Vazquez
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Chemello
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Mitchenall
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Maxwell
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Palumbo
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001665:Binding Sites; D001667:Binding, Competitive / Q000187:drug effects; D002942:Circular Dichroism; D003374:Coumarins / Q000494:pharmacology; D027081:DNA Gyrase / Q000737:chemistry*; D004269:DNA, Bacterial / Q000187:drug effects / Q000378:metabolism; D004278:DNA, Superhelical / Q000187:drug effects / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D006027:Glycosides / Q000494:pharmacology; D013816:Thermodynamics; D059005:Topoisomerase II Inhibitors
D000900:Anti-Bacterial Agents; D003374:Coumarins; D004269:DNA, Bacterial; D004278:DNA, Superhelical; D006027:Glycosides; D059005:Topoisomerase II Inhibitors; C416814:simocyclinone D8; D027081:DNA Gyrase