Crystallization and X-ray analysis of all of the players in the autoregulation of theataRTtoxin–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 P622, 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) bound to the cofactor acetyl coenzyme A (AcCoA) and the C-terminal neutralization domain of AtaR (AtaR) were also crystallized. The crystals of the AtaT-AcCoA complex diffracted to 2.5 Å resolution and the crystals of AtaR 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 P622 and P321, 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 C222, with unit-cell parameters a = 75.6, b = 87.9, c = 190.5 Å. These crystals diffracted to 3.5 Å resolution.

Mechanism of regulation and neutralization of the AtaR–AtaT toxin–antitoxin system

“…Protein production and purification. Expression and purification of the AtaR-AtaT complex from the pKK223.3 vector as well as AtaR, AtaT Y144F , the AtaT Y144F -AtaR A44-M88 complex and the AtaR-AtaT-Opr 22 complex was done as described 10 . Mutated AtaT protein versions were purified using the same principle.…”

“…AtaT modulates the interaction of AtaR with its operator. The operator of ataRT consist of two inverted repeats upstream its promoter, each containing an AtaR-binding box 10 (Supplementary Table 2). We used EMSAs to monitor, in vitro, the interaction of AtaR and AtaT-AtaR complexes with a DNA fragment of 47 base pair ( Supplementary Table 2) containing its operator region (Opr 47 ).…”

“…In vivo, AtaR A44-M88 neutralizes AtaT 10 . Figure 2b,c shows the 3.2 kDa fragment AtaR S60-M88 also neutralized AtaT and rescued translation in vitro (however, this fragment failed to neutralize AtaT in vivo, Supplementary Fig.…”

Small-Molecule Acetylation by GCN5-Related N -Acetyltransferases in Bacteria

“…The Y140F variant is inactive (156). When the corresponding residue of AtaT (Y144) was replaced by a phenylalanine, this variant was also inactive (159). As with TacT-type toxins, AtaT was shown to have a positive patch formed by the dimers that interacts with methionine-tRNA fMet (160).…”