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-Trp 83 mutant (GyrATrp83), were investigated in the presence of mono- and dival ent metal ions and ciprofloxacin, a clinically useful antibacterial quinolo ne. The stability of the proteins was estimated from temperature denaturati on, monitoring unfolding with circular dichroism spectroscopy. Two transiti ons were observed in GyrA and GyrATrp83, which likely reflect unfolding of the N and C-terminal protein domains. Accordingly, one thermal transition i s observed for GyrA59. The melting profile of the GyrA subunit is dramatically affected by monoval ent and divalent metal ions, both transitions being shifted to lower temper ature upon increasing salt concentration. This effect is much more pronounc ed with divalent ions (Mg2+) and cannot be accounted for by changes in ioni c strength only. The presence of ciprofloxacin shifts the melting transitio ns of the wild-type subunit to higher temperatures when physiological conce ntrations of Mg2+ are present. In contrast, both the mutant protein and the 59 kDa fragment do not show evidence for quinolone-driven changes. These d ata suggest that ciprofloxacin binds to the wild-type subunit in an interac tion that involves Ser83 of GyrA and that both C and N-terminal domains may be required for effective drug-protein interactions. The bell-shaped depen dence of the binding process upon Mg2+ concentration, with a maximum centre d at 3-4 mM [Mg2+], 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 bindi ng of the quinolone to GyrA. We infer that the Mg2+-GyrA interaction at physiological metal ion concentr ation could bear biological relevance, conferring more conformational flexi bility to the active enzyme. The results obtained in the presence of ciprof loxacin additionally suggest that the Mg2+-mediated quinolone binding to th e enzyme might be involved in the mechanism of action of this family of dru gs. (C) 2001 Academic Press.