Crystallization and X-ray analysis of all of the players in the autoregulation of the ataRT toxin-antitoxin system

Abstract

The ataRT operon from enteropathogenic Escherichia coli encodes a toxin-antitoxin (TA) module with a recently discovered novel toxin activity. This new type II TA module targets translation initiation for cell-growth arrest. Virtually nothing is known regarding the molecular mechanisms of neutralization, toxin catalytic action or translation autoregulation. Here, the production, biochemical analysis and crystallization of the intrinsically disordered antitoxin AtaR, the toxin AtaT, the AtaR-AtaT complex and the complex of AtaR-AtaT with a double-stranded DNA fragment of the operator region of the promoter are reported. Because they contain large regions that are intrinsically disordered, TA antitoxins are notoriously difficult to crystallize. AtaR forms a homodimer in solution and crystallizes in space group P6₁22, with unit-cell parameters a = b = 56.3, c = 160.8 Å. The crystals are likely to contain an AtaR monomer in the asymmetric unit and diffracted to 3.8 Å resolution. The Y144F catalytic mutant of AtaT (AtaT_Y144F) bound to the cofactor acetyl coenzyme A (AcCoA) and the C-terminal neutralization domain of AtaR (AtaR_44-86) were also crystallized. The crystals of the AtaT_Y144F-AcCoA complex diffracted to 2.5 Å resolution and the crystals of AtaR_44-86 diffracted to 2.2 Å resolution. Analysis of these structures should reveal the full scope of the neutralization of the toxin AtaT by AtaR. The crystals belonged to space groups P6₅22 and P3₁21, with unit-cell parameters a = b = 58.1, c = 216.7 Å and a = b = 87.6, c = 125.5 Å, respectively. The AtaR-AtaT-DNA complex contains a 22 bp DNA duplex that was optimized to obtain high-resolution data based on the sequence of two inverted repeats detected in the operator region. It crystallizes in space group C222₁, with unit-cell parameters a = 75.6, b = 87.9, c = 190.5 Å. These crystals diffracted to 3.5 Å resolution.

Keywords: AtaR; AtaT; Escherichia coli; acetyltransferase; protein–DNA complexes; toxin–antitoxin.

Figure 1

Purification of AtaR–AtaT and AtaT_Y144F. (a) Coomassie-stained SDS–PAGE gel of protein purification on Ni Sepharose. Lanes 1–6, purification of HisTEV-AtaR–AtaT; lanes 7–13, purification of HisTEV-AtaT_Y144F. Lanes 1 and 7, protein extract; lanes 2 and 8, flowthrough; lane 9, wash; lanes 3–6 and 10–13, elution fractions. (b) SDS gel (16%) and Coomassie staining (left) or Western blotting with anti-His antibody (right) of HisTEV-AtaR–AtaT before and after TEV cleavage (lanes 1 and 2), HisTEV-AtaT_Y144F (lanes 3 and 4). Lane M, molecular-weight marker (labelled in kDa). (c) Analytical SEC on a Superdex 200 Increase SEC column; the measurements were performed in 50 mM Tris–HCl pH 8.5, 100 mM NaCl.

Figure 2

Crystals of AtaR and AtaT. (a) Crystals of HisTEV-AtaT_Y144F. (b) Crystals of tag-free AtaT_Y144F. (c) Crystals of AtaR. (d) SDS–PAGE (16%) analysis of the AtaR crystals; only one band is observed in lane 1 next to the 10 kDa band from the molecular-weight markers (lane 2; labelled in kDa). This suggests that only the antitoxin AtaR is present in the crystal.

Figure 3

(a) Sequence of the AtaR antitoxin; the functional modules of the antitoxin (N-terminal DNA-binding domain and C-terminal neutralization domain) are labelled in the figure. The arrow marks the start of the C-terminal domain of AtaR used in this study. (b) Bacterial growth assay on inducers indicating that the C-terminal domain of AtaR is sufficient to neutralize AtaT-His expression. Overnight cell cultures were serially diluted and spotted on LB-agar plates without (left) or with inducer (right). (c) AtaT-His expression was confirmed by assaying protein extracts 3 h after induction with 0.5 mM IPTG by SDS–PAGE (16%) and Coomassie staining (left) or Western blotting (right). Lane 1, AtaR–AtaT-His; lane 2, AtaR_Cter–AtaT-His; 3, control (empty expression vector). (d) Typical crystals of AtaT_Y144F in complex with the synthetic AtaR_44–88 peptide. (e) Schematic representation of the ataRT promoter region. The transcription start site is marked +1 and shown in bold in the sequence. The −10 and −35 promoter elements and the start of the open reading frame of the ataR gene are indicated at the top of the sequence and the ATG start codon is shown in bold. The red arrows indicate the inverted repeat regions of the ataRT operator. (f) Sequences of the fragments used for crystallization. The sequence of the optimized DNA fragment that led to high-resolution diffraction is shown in bold. (g) Crystals of AtaR–AtaT in complex with double-stranded DNA. (h) 16% SDS–PAGE (left) and 2% agarose gel (right) of the dissolved AtaT–AtaR–DNA crystals, showing the presence of both proteins and of DNA.

Figure 4

Initial 2mF _o − DF _c map (σ = 1) for AtaR after density modification and automated tracing with SHELXE. The RHH dimer backbone autotraced with SHELXE and generated by crystallographic symmetry is shown as a ribbon.