Mapping simocyclinone D8 interaction with DNA gyrase: evidence for a new binding site on GyrB

Abstract

Simocyclinone D8, a coumarin derivative isolated from Streptomyces antibioticus Tü 6040, represents an interesting new antiproliferative agent. It was originally suggested that this drug recognizes the GyrA subunit and interferes with the gyrase catalytic cycle by preventing its binding to DNA. To further characterize the mode of action of this antibiotic, we investigated its binding to the reconstituted DNA gyrase (A(2)B(2)) as well as to its GyrA and GyrB subunits and the individual domains of these proteins, by performing protein melting and proteolytic digestion studies as well as inhibition assays. Two binding sites were identified, one (anticipated) in the N-terminal domain of GyrA (GyrA59) and the other (unexpected) at the C-terminal domain of GyrB (GyrB47). Stabilization of the A subunit appears to be considerably more effective than stabilization of the B subunit. Our data suggest that these two distinct sites could cooperate in the reconstituted enzyme.

FIG. 1.

Chemical structures of the DNA gyrase ligands tested.

FIG. 2.

Melting profiles of DNA gyrase acquired by reading the protein CD signal at 220 nm in 10 mM Tris-HCl (pH 7.5)-20 mM KCl-4 mM MgCl₂ in the absence (solid line) and in the presence (dashed line) of 50 μM SD8. The protein concentration was 0.05 μM. Mol. Ell., molar ellipticity.

FIG. 3.

SD8 modulation of the thermal stability of GyrA59 in 10 mM Tris-HCl (pH 7.5)-20 mM KCl-4 mM MgCl₂. (A) Melting profiles of GyrA59 acquired by reading the protein CD signal at 220 nm in the absence (solid line) and in the presence (dashed line) of SD8. The protein concentration was 0.2 μM. (B) Variation of the T_m for the thermal transition of GyrA59 as a function of SD8 concentration.

FIG. 4.

Modulation of the melting profiles of GyrB and related subdomains acquired by recording the protein CD signal at 220 nm in 10 mM Tris-HCl (pH 7.5)-20 mM KCl-4 mM MgCl₂. (A) Effects of 20 μM ligands on the melting profile of GyrB; (B) effects of increasing SD8 concentrations on the melting profile of GyrB; (C) effects of 20 μM SD8 on the melting profile of GyrB43; (D) melting profile of GyrB47 in the presence or absence of SD8. The protein concentration was 0.2 μM.

FIG. 5.

Incremental variation of the melting temperature (ΔT/ΔT_max, where ΔT is the change in temperature and ΔT_max is the change in the maximum temperature) for the higher-temperature thermal transition of GyrB as a function of the ligand concentrations. The data refer to the melting profiles acquired by reading the CD signal at 220 nm in 10 mM Tris-HCl (pH 7.5)-20 mM KCl in the presence (full symbols) or the absence (empty symbols) of 4 mM MgCl₂. In the presence of ADPNP, it was not possible to calculate this value when Mg²⁺ was not included in the buffer (see Fig. S1 in the supplemental material).

FIG. 6.

Increments of GyrB melting temperature (ΔT) resulting from the protein CD signal at 220 nm in 10 mM Tris-HCl (pH 7.5)-20 mM KCl-4 mM MgCl₂ in the presence or the absence of different ligand (20 μM) combinations, as indicated.

FIG. 7.

Kinetics of GyrB (3 μg) cleavage by trypsin in the absence (A) or the presence (B) of 100 μM SD8. Lanes M, molecular mass markers; lanes B, GyrB in the absence of trypsin. (C) Quantitative determination of the 25 kDa GyrB fragment formation; (D) GyrB (3 μg) cleavage patterns after 30 min of incubation with trypsin in the absence or the presence of the ligand (0.1 or 1 mM) combinations.

FIG. 8.

Effect of SD8 on gyrase-DNA complex formation. DNA was incubated with increasing protein concentrations in the presence or the absence of 100 μM SD8 (A). (B) The percentage of DNA bound by DNA gyrase (400 nM) as a function of the SD8 concentration in the reaction mixture. The buffer was made up of 10 mM Tris-HCl (pH 7.5), 20 mM KCl, and 4 mM MgCl₂.