Crystallization of the HigBA2 toxin-antitoxin complex from Vibrio cholerae

Abstract

The genome of Vibrio cholerae encodes two higBA toxin-antitoxin (TA) modules that are activated by amino-acid starvation. Here, the TA complex of the second module, higBA2, as well as the C-terminal domain of the corresponding HigA2 antitoxin, have been purified and crystallized. The HigBA2 complex crystallized in two crystal forms. Crystals of form I belonged to space group P2(1)2(1)2, with unit-cell parameters a = 129.0, b = 119.8, c = 33.4 Å, and diffracted to 3.0 Å resolution. The asymmetric unit is likely to contain a single complex consisting of two toxin monomers and one antitoxin dimer. The second crystal form crystallized in space group P3(2)21, with unit-cell parameters a = 134.5, c = 55.4 Å. These crystals diffracted to 2.2 Å resolution and probably contain a complex with a different stoichiometry. Crystals of the C-terminal domain of HigA2 belonged to space group C2, with unit-cell parameters a = 115.4, b = 61.2, c = 73.8 Å, β = 106.7°, and diffracted to 1.8 Å resolution.

Keywords: intrinsic disorder; macromolecular complexes; persistence; ribonucleases; toxin–antitoxin modules.

Figure 1

Expression and purification of proteins encoded by the higBA2 module. Lane 1, molecular-weight standards (labelled in kDa). Lane 2, soluble fraction before induction. Lane 3, soluble fraction after 4 h of induction with IPTG. Lane 4, purified HigBA2 complex. Lane 5, purified HigB2 toxin. Lane 6, purified HigA2 antitoxin.

Figure 2

SAXS curves for the HigA2 antitoxin (a), the HigB2 toxin (b) and the HigBA2 complex (c). The experimental data are drawn in black and the error margins in grey. The insets show the corresponding Kratky plots.

Figure 3

Crystals of HigBA2 and HigA2. (a) Crystals of form I of the HigBA2 complex obtained after microseeding. (b) Zinc-stained SDS–PAGE of redissolved crystals (lane 1, form I crystals of HigBA2; lane 2, form II crystals of HigBA2; lane 3, crystals of HigA2; leftmost lane, molecular-mass markers labelled in kDa). The green arrows indicate bands corresponding to HigB2 (upper) and HigA2 (lower). The red arrows correspond to likely degradation products. The putative HigB2 degradation product is also visible in the mass spectrum shown in Fig. 5 ▶(b) as a peak corresponding to a mass of 13 897 Da. (c) Crystals of form II of the HigBA2 complex. (d) Crystals of the C-terminal domain of the antitoxin. The black bar corresponds to 0.1 mm and the scale is identical in (a), (c) and (d).

Figure 4

Typical diffraction patterns of HigBA2 crystal form I (a), HigBA2 crystal form II (b) and crystals of the C-terminal domain of HigA2 (c). The insets show a magnification of the boxed parts of the diffraction images with rings that indicate the resolution limit for each crystal form.

Figure 5

Mass-spectrometric analysis of crystal contents. (a) ESI ion trap mass spectrum of crystal form I of the HigBA2 complex. A first major peak at 11 566 Da corresponds to the mass of the HigA2 antitoxin within 1 Da experimental error. The second major peak corresponds to a mass of 15 039 Da, again within 1 Da experimental error of the theoretical mass of the HigB2 toxin. (b) Identical ESI ion trap mass spectrum for crystal form II. Note the different ratio of peak intensities suggesting a different stoichiometry of the complex and the presence of a peak at 13 897 Da corresponding to a putative HigB2 degradation product.