Interaction between DNA gyrase and quinolones: effects of alanine mutations at GyrA subunit residues Ser(83) and Asp(87)

Abstract

DNA gyrase is a target of quinolone antibacterial agents, but the molecular details of the quinolone-gyrase interaction are not clear. Quinolone resistance mutations frequently occur at residues Ser(83) and Asp(87) of the gyrase A subunit, suggesting that these residues are involved in drug binding. Single and double alanine substitutions were created at these positions (Ala(83), Ala(87), and Ala(83) Ala(87)), and the mutant proteins were assessed for DNA supercoiling, DNA cleavage, and resistance to a number of quinolone drugs. The Ala(83) mutant was fully active in supercoiling, whereas the Ala(87) and the double mutant were 2.5- and 4- to 5-fold less active, respectively; this loss in activity may be partly due to an increased affinity of these mutant proteins for DNA. Supercoiling inhibition and cleavage assays revealed that the double mutant has a high level of resistance to certain quinolones while the mutants with single alanine substitutions show low-level resistance. Using a drug-binding assay we demonstrated that the double-mutant enzyme-DNA complex has a lower affinity for ciprofloxacin than the wild-type complex. Based on the pattern of resistance to a series of quinolones, an interaction between the C-8 group of the quinolone and the double-mutant gyrase in the region of residues 83 and 87 is proposed.

FIG. 1

QRDR of GyrA. The image at the top right shows a ribbon representation (RasMol) of the 59-kDa N-terminal fragment of GyrA (17). The QRDR is in dark gray. The main image is an expanded version of this region showing the active-site tyrosine (Tyr¹²²) and residues of the QRDR. Ser⁸³ and Asp⁸⁷ are solvent exposed in this structure and have been mutated to Ala in this study.

FIG. 2

DNA supercoiling in the presence of NaCl. Increasing amounts of NaCl were added to standard supercoiling reaction mixtures containing WT or mutant gyrases. Lanes 1, DNA substrate only; 2, no added NaCl; 3, 100 mM NaCl; 4, 150 mM NaCl; 5, 200 mM NaCl; 6, 250 mM NaCl; 7, 300 mM NaCl. WT and mutant GyrA subunits were added at a concentration of 11 nM. Differences in the supercoiling activities of mutants were accounted for by varying incubation times: for WT, 3 min; for Ala⁸³, 2.5 min; for Ala⁸⁷, 7 min; and for Ala⁸³ Ala⁸⁷, 10 min.

FIG. 3

Ca²⁺-induced DNA cleavage by DNA gyrase. CaCl₂ was added to reaction mixtures containing WT or mutant gyrases (final concentrations, 25 nM) and relaxed pBR322 DNA. The amount (shown as a percentage) of cleaved product (linear pBR322) was quantified. Results are averages from three separate experiments and are normalized such that the intensity of the linear band in the WT cleavage reaction is 100% at 2 mM CaCl₂. Symbols: ○, WT; ●, Ala⁸³; □, Ala⁸⁷; ■, Ala⁸³ Ala⁸⁷.

FIG. 4

Cleavage specificity assay. The cleavage of EcoRI-linearized pBR322 by WT and mutant enzymes is shown. Ciprofloxacin was used to stabilize cleavage, and the amount of drug used is indicated (in micromolar concentrations). The enzyme concentration was 50 nM in terms of the GyrA monomer. M, marker; DNA, linear pBR322 DNA.

FIG. 5

Quinolone binding to the gyrase-DNA complex. Spin column chromatography data from a number of experiments show the binding of ciprofloxacin (CFX) to WT and mutant (Ala⁸³ Ala⁸⁷) gyrase-DNA complexes. Reaction mixtures containing [³H]-CFX were passed through Sephadex spin columns, and the amount of drug bound was estimated by scintillation counting. The enzyme concentration was 130 nM in terms of the GyrA monomer. Data were fitted to simple 1:1 ligand binding curves and yielded the following K_d^app values: 7.3 ± 3.1 μM for the WT (○) and 78.0 ± 35.6 μM for the mutant (●).

FIG. 6

Plot demonstrating the possible relationship between the quinolone resistance of the double mutant and the van der Waals radius (relative to ciprofloxacin) of the atom associated with position 8 of the quinolone. Numbers correlate with compound numbers given in Table 1. Enoxacin is not included in this figure.