Ciprofloxacin affects conformational equilibria of DNA gyrase A in the presence of magnesium ions¹

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 (Mg²⁺) 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 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 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 Mg²⁺ concentration, with a maximum centred 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 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 flexibility to the active enzyme. The results obtained in the presence of ciprofloxacin additionally suggest that the Mg²⁺-mediated quinolone binding to the enzyme might be involved in the mechanism of action of this family of drugs.

Introduction

DNA gyrase is a prokaryotic type II topoisomerase that catalyses both relaxation and supercoiling of DNA.1 The process involves the formation of a reversible protein-DNA complex in which gyrase is covalently linked to the polynucleotide chain. As a result, a DNA double-strand break forms, through which another DNA segment can be transported. Resealing of the break leads to a nucleic acid structure with different topology. In its physiologically active form, gyrase is a tetramer formed by the combination of subunits A and B (A₂B₂).2, 3 Each subunit carries different functional protein domains. In particular, the A subunit (GyrA) is functionally divided into a 64 kDa N-terminal and a 33 kDa C-terminal domain, principally involved in DNA breakage-reunion and DNA wrapping, respectively. The B subunit (GyrB) comprises an N-terminal domain containing the ATPase site and a C-terminal domain involved in interactions with the A protein and DNA.4, 5, 6

Together with topoisomerase IV, DNA gyrase represents a primary target for quinolone antibacterials, as shown by analysis of DNA gyrase deriving from drug-resistant bacterial strains.7, 8, 9 Quinolones are believed to interfere with the catalytic cycle of gyrase by stabilizing the gyrase-DNA cleavable intermediate.10 The molecular details of the mechanism of enzyme poisoning are still the subject of debate. A number of models have been proposed in the literature, which mostly focus on drug-DNA interactions, and deduce the drug sites interacting with the enzyme only indirectly.11, 12 In this connection, the possible role of Mg²⁺ in mediating drug-DNA interaction has emerged.13, 14 This fact is intriguing, as gyrase needs the same metal ion as a cofactor to perform its catalytic activity.15, 16 Indeed, little is known about pharmacologically relevant drug-protein contacts. These are likely to occur mainly within the A subunit, since mutations causing resistance to quinolones map in this subunit most frequently,17, 18 although some mutations are also known to map to GyrB.19 Although the crystal structure of a 59 kDa N-terminal GyrA fragment is available,20 structural information about possible interactions of GyrA with quinolones is lacking, as yet. The DSC thermal denaturation profile exhibited by GyrA has been interpreted in terms of two transitions21 arising from the two protein domains mentioned above. Addition of ciprofloxacin to the 64 kDa domain induced some subtle changes in the shape of the curve, and led to a slightly sharper transition, apparently detecting an interaction between the quinolone and the GyrA fragment. However, no clear-cut conclusion could be drawn from DSC experiments.

To further address the issue of quinolone-gyrase interactions, in the present work we have examined the conformational properties and equilibria of wt GyrA, of the quinolone-resistant Ser-Trp83 mutant (GyrATrp83) and of the 59 kDa N-terminal fragment (GyrA59), containing the catalytic site, by spectroscopic and affinity chromatography techniques. In particular, we have investigated the effects of mono- and divalent metal ions upon the conformational transitions of the enzyme subunit in the presence and absence of the clinically useful quinolone ciprofloxacin. Our results suggest that the conformational stability of DNA gyrase A is largely governed by ionic contacts, and that the quinolone interacts with the wt, but not with the mutant, protein.