Purification, crystallization and preliminary X-ray diffraction experiments on the breakage-reunion domain of the DNA gyrase from Mycobacterium tuberculosis

Abstract

Mycobacterium tuberculosis DNA gyrase, a nanomachine that is involved in the regulation of DNA topology, is the only type II topoisomerase present in this organism and hence is the sole target for fluoroquinolone action. The breakage-reunion domain of the A subunit plays an essential role in DNA binding during the catalytic cycle. Two constructs of 53 and 57 kDa (termed GA53BK and GA57BK) corresponding to this domain have been overproduced, purified and crystallized. Diffraction data were collected from four crystal forms. The resolution limits ranged from 4.6 to 2.7 angstrom depending on the crystal form. The best diffracting crystals belonged to space group C2, with a biological dimer in the asymmetric unit. This is the first report of the crystallization and preliminary X-ray diffraction analysis of the breakage-reunion domain of DNA gyrase from a species containing one unique type II topoisomerase.

Figure 1

(a) Crystal form I of GA53BK obtained in 100 mM sodium cacodylate pH 6.5, 10 mM magnesium acetate and 1.3 M lithium sulfate. After one week, the crystal size was 60 × 60 × 60 µm. (b) Crystal form II of GA53BK obtained in 100 mM bicine pH 9, 150 mM sodium chloride, 9% PEG 550 MME and 9% ethylene glycol. After 2 d, the crystal size was 300 × 30 × 30 µm. (c) Crystal form III of GA57BK obtained in 100 mM Na HEPES pH 7.5, 200 mM calcium chloride and 22% PEG 400. After two weeks, the crystal size was 150 × 100 × 100 µm. (d) Crystal form IV of GA57BK obtained in 100 mM HEPES pH 7.5, 4% PEG 4000 and 30% MPD. After 2 d, the crystal size was 200 × 40 × 40 µm. One single crystal was isolated to collect the complete data set described in Table 2 ▶.

Figure 2

Typical X-ray diffraction pattern of crystal form IV. The oscillation angle was 0.5°.

Figure 3

Probability distributions (Kantardjieff & Rupp, 2003 ▶) of the different possible numbers of molecules in the asymmetric unit for the four crystal forms.