Ciprofloxacin affects conformational equilibria of DNA gyrase A in the presence of magnesium ions

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 cipro¯oxacin, 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 re¯ect unfolding of the N and C-terminal protein domains. Accordingly, one thermal transition is observed for GyrA59. The melting pro®le 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 (Mg2‡) and cannot be accounted for by changes in ionic strength only. The presence of cipro¯oxacin shifts the melting transitions of the wild-type subunit to higher temperatures when physiological concentrations of Mg2‡ 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 cipro¯oxacin 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 Mg2‡ concentration, with a maximum centred at 3-4 mM [Mg2‡], is consistent with a metal-ion mediated GyrA-quinolone-interaction. Af®- nity chromatography data fully support these ®ndings and additionally con®rm the requirement for a free carboxylate to elicit binding of the quinolone to GyrA. We infer that the Mg2‡-GyrA interaction at physiological metal ion concentration could bear biological relevance, conferring more conformational ¯exibility to the active enzyme. The results obtained in the presence of cipro¯oxacin additionally suggest that the Mg2‡-mediated quinolone binding to the enzyme might be involved in the mechanism of action of this family of drugs.