title: Ciprofloxacin affects conformational equilibria of DNA gyrase A in the presence of magnesium ions.
abstract: The conformational equilibria of the A subunit of DNA gyrase (GyrA), of its 59 kDa N-terminal fragment (GyrA59) and of the quinolone-resistant Ser-Trp83 mutant (GyrATrp83), were investigated in the presence of mono- and divalent metal ions and ciprofloxacin, a clinically useful antibacterial quinolone. The stability of the proteins was estimated from temperature denaturation, monitoring unfolding with circular dichroism spectroscopy. Two transitions were observed in GyrA and GyrATrp83, which likely reflect unfolding of the N and C-terminal protein domains. Accordingly, one thermal transition is observed for GyrA59. The melting profile of the GyrA subunit is dramatically affected by monovalent and divalent metal ions, both transitions being shifted to lower temperature upon increasing salt concentration. This effect is much more pronounced with divalent ions (Mg(2+)) and cannot be accounted for by changes in ionic strength only. The presence of ciprofloxacin shifts the melting transitions of the wild-type subunit to higher temperatures when physiological concentrations of Mg(2+) are present. In contrast, both the mutant protein and the 59 kDa fragment do not show evidence for quinolone-driven changes. These data suggest that ciprofloxacin binds to the wild-type subunit in an interaction that involves Ser83 of GyrA and that both C and N-terminal domains may be required for effective drug-protein interactions. The bell-shaped dependence of the binding process upon Mg(2+) concentration, with a maximum centred at 3-4 mM [Mg(2+)], is consistent with a metal-ion mediated GyrA-quinolone-interaction. Affinity chromatography data fully support these findings and additionally confirm the requirement for a free carboxylate to elicit binding of the quinolone to GyrA. We infer that the Mg(2+)-GyrA interaction at physiological metal ion concentration could bear biological relevance, conferring more conformational flexibility to the active enzyme. The results obtained in the presence of ciprofloxacin additionally suggest that the Mg(2+)-mediated quinolone binding to the enzyme might be involved in the mechanism of action of this family of drugs.
authors.affiliation: Department of Pharmaceutical Sciences, University of Padova, via Marzolo 5, Padova, 35100, Italy.
authors.forename: C
authors.initials: C
authors.lastname: Sissi
authors.forename: E
authors.initials: E
authors.lastname: Perdonà
authors.forename: E
authors.initials: E
authors.lastname: Domenici
authors.forename: A
authors.initials: A
authors.lastname: Feriani
authors.forename: A J
authors.initials: AJ
authors.lastname: Howells
authors.forename: A
authors.initials: A
authors.lastname: Maxwell
authors.forename: M
authors.initials: M
authors.lastname: Palumbo
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D000890:Anti-Infective Agents / Q000494:pharmacology*; D002118:Calcium / Q000494:pharmacology; D002413:Cations, Divalent / Q000494:pharmacology*; D002846:Chromatography, Affinity; D002939:Ciprofloxacin / Q000494:pharmacology*; D002942:Circular Dichroism; D004250:DNA Topoisomerases, Type II / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004352:Drug Resistance, Microbial; D004795:Enzyme Stability / Q000187:drug effects; D008274:Magnesium / Q000494:pharmacology*; D008970:Molecular Weight; D009154:Mutation / Q000235:genetics; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D021122:Protein Subunits; D012492:Salts / Q000494:pharmacology; D013696:Temperature; D013816:Thermodynamics
chemical_list: D000890:Anti-Infective Agents; D002413:Cations, Divalent; D010446:Peptide Fragments; D021122:Protein Subunits; D012492:Salts; D002939:Ciprofloxacin; D004250:DNA Topoisomerases, Type II; D008274:Magnesium; D002118:Calcium