Crystallization and preliminary X-ray analysis of two variants of the Escherichia coli O157 ParE2-PaaA2 toxin-antitoxin complex

Abstract

The paaR2-paaA2-parE2 operon is a three-component toxin-antitoxin module encoded in the genome of the human pathogen Escherichia coli O157. The toxin (ParE2) and antitoxin (PaaA2) interact to form a nontoxic toxin-antitoxin complex. In this paper, the crystallization and preliminary characterization of two variants of the ParE2-PaaA2 toxin-antitoxin complex are described. Selenomethionine-derivative crystals of the full-length ParE2-PaaA2 toxin-antitoxin complex diffracted to 2.8 Å resolution and belonged to space group P41212 (or P43212), with unit-cell parameters a = b = 90.5, c = 412.3 Å. It was previously reported that the full-length ParE2-PaaA2 toxin-antitoxin complex forms a higher-order oligomer. In contrast, ParE2 and PaaA213-63, a truncated form of PaaA2 in which the first 12 N-terminal residues of the antitoxin have been deleted, form a heterodimer as shown by analytical gel filtration, dynamic light scattering and small-angle X-ray scattering. Crystals of the PaaA213-63-ParE2 complex diffracted to 2.7 Å resolution and belonged to space group P6122 (or P6522), with unit-cell parameters a = b = 91.6, c = 185.6 Å.

Keywords: Escherichia coli O157; ParE2–PaaA2; toxin–antitoxin.

Figure 1

Coding regions of the used expression vectors. The ORFs of paaA2 and parE2 are highlighted by blue and red boxes, respectively. Grey boxes indicate purification tags and protease cleavage sites. The single nucleotide separating the paaA2 and parE2 ORFs is indicated in bold. The resulting amino-acid sequences are also shown in bold and the molecular masses of the produced proteins are indicated.

Figure 2

Morphology of native and of SeMet-derivatized crystals of the full-length ParE2–PaaA2 toxin–antitoxin complex. (a) Microcrystals of the native full-length ParE2–PaaA2 toxin–antitoxin complex obtained in 100 mM MgCl₂, 100 mM MES pH 6.0, 8%(w/v) PEG 6000. (b) Optimization of the initial hit shown in (a) led to a final crystallization condition in 100 mM MgCl₂, 100 mM MES pH 6.1, 8%(w/v) PEG 6000. Typical crystals appear after incubation for around 7 d at 293 K. (c) SeMet-derivatized crystals of the full-length ParE2–PaaA2 toxin–antitoxin complex prepared by micro-seeding with a 10⁻¹ dilution of crushed fragments of native crystals similar to those in (b).

Figure 3

An X-ray fluorescence scan (relative units) of a crystal of an SeMet derivative of the full-length ParE2–PaaA2 complex displays an absorption peak close to 12.66 keV, which is typical for SeMet.

Figure 4

(a) Diffraction pattern of an SeMet-derivatized crystal similar in size to that shown in Fig. 1 ▶(c). It shows diffraction spots to 2.8 Å resolution (the 2.8 Å resolution limit is indicated by an orange circle). (b) Diffraction pattern of the only crystal of the ParE2–PaaA2_13–63 complex. The crystal shows diffraction spots to 2.7 Å resolution (the 2.7 Å resolution limit is indicated by an orange circle).

Figure 5

The ParE2–PaaA2_13–63 complex exists as a heterodimer in solution. (a) Analytical gel filtration. The experiment was performed on a Superdex 75 HR 10/30 column. The black and grey traces indicate the elution profiles for the ParE2–PaaA2_13–63 complex and the Bio-Rad standard (A, bovine thyroglobulin, molecular mass 670 kDa, R _h = 8.6 nm; B, bovine γ-globulin, molecular mass 158 kDa, R _h = 5.1 nm; C, chicken ovalbumin, molecular mass 44 kDa, R _h = 2.8 nm; D, horse myoglobin, molecular mass 17 kDa, R _h = 1.9 nm; E, vitamin B₁₂, molecular mass 1.35 kDa), respectively. The inset shows an SDS–PAGE analysis of the elution peak. PaaA2_13–63 and ParE2 are indicated by blue and red arrows, respectively. Lane M, Prestained Protein Molecular Weight Marker (Fermentas); lanes 1–3, fractions of the ParE2–PaaA2_13–63 complex elution peak. (b) DLS. The black dots and grey line represent the experimental data and the fit, respectively. The residuals are shown below. The percentage of mass as a function of the R _h distribution is given in the inset. (c) The final scattering curve of the ParE2–PaaA2_13–63 complex. The experimental data are shown in black, while the error margins are shown in grey. The inset shows the Guinier region of the scattering curve (experimental data in black and the Guinier fit in grey). (d) The normalized Kratky plot of the recorded data set indicates the presence of flexibility in the scattering particle. (e) An analysis of the p(r) function demonstrates that the scattering particle has a maximum intra-particle distance of around 75 Å.