# Structure, Biology, and Therapeutic Application of Toxin-Antitoxin Systems in Pathogenic Bacteria
## 4 Structural Characteristics of TA Proteins from Pathogenic Bacteria
### Atypical TA Modules  
The EcPaaA2-EcParE2 complex from _E. coli_ O157 forms a unique hetero-hexadecamer both in the crystal and in solution [166] (Figure 8A). EcParE2 is structurally most similar to the RelE/ParE toxin family, which folds as two \(N\)-terminal \(\alpha\)-helices followed by a four-stranded antiparallel \(\beta\)-sheet. However, EcParE2 shows a low sequence identity with well-known _E. coli_ RelE and YoeB. EcParE2 does not bind to DNA gyrase and also not inhibits protein synthesis, suggesting that the RelE/ParE family has a third, distinct, yet unidentified function. A combination of small-angle X-ray scattering (SAXS) and NMR studies described the conformational ensemble of the EcPaaA2 antitoxin [165], which is an atypical member of the RelB antitoxin family. In solution, the ensemble exhibits an anisotropy in its coverage of the available conformational space compared to initially selected structures. The EcPaaA2 antitoxin is highly flexible and adopts two \(\alpha\)-helices connected by a flexible linker. TacT toxin from _S. typhimurium_ is a unique type of acetyltransferases in that it adopts an extension of \(\alpha\)3 helix and a bending of \(\beta\)2 and \(\beta\)3 strands with an insertion of short helices between them, compared to other acetyltransferases (Figure 8B). This protein forms a homodimer with an extensive dimeric interface and a positively charged groove for tRNA binding (Figure 8B). The structural model suggested that TacT-mediated acetylation of primary amine group of aminoacyl-tRNA is unable to form a peptide bond with the carboxyl group of the polypeptide chain located in the P site of ribosome. A novel type II TA system from _B. abortus_ was identified as the BrnAT system [172]; it consists of the toxin BrnT and the antitoxin BrnA, which are co-expressed and form a 2:2 tetrameric complex. As shown in typical type II TA systems, the tetrameric BrnAT complex binds to its own promoter and downregulates its expression. BrnT has a central scaffold that is composed of a four-stranded antiparallel \(\beta\)-sheet with a \(\beta\)2-\(\beta\)3-\(\beta\)4-\(\beta\)5 topology, in which a hydrophobic face binds to two short \(\alpha\)-helices (Figure 8C) [172]. The BrnT toxin shows a similar secondary topology to the RelE toxin family, but overall tertiary structural similarity between them is low. Despite the structural differences between BrnT and other RelE family members, they are functionally conserved as ribonucleases that inhibit protein synthesis in vivo. The most conserved residues of the BrnT toxin are situated in the solvent-exposed surfaces of the \(\beta\)-scaffold and its interacting \(\alpha\)-helix. A combination of cell toxicity assay and site-directed mutagenesis revealed that eight of the conserved residues are involved in cell toxicity and that five (Asp6, Glu7, Lys9, Arg41, and Glu78) of these eight residues are important for a ribonuclease activity [172]. These residues, except for Glu78 (not shown in the structure), are colour-mapped onto the BrnT structure in Figure 8C. A combined approach of NMR spectroscopy and molecular dynamics simulation revealed that the Fst toxin from _E. faecalis_ has the transmembrane segment spanning residues Asp3-Asp26, which form a stable helical structure and are completely immersed in the membrane (Figure 8D) [145]. An intrinsically disordered stretch near the C-terminus, including acidic residues (Asp26-Glu27-Glu28-Asp29-Asp30), is located outside the membrane (Figure 8D). The protruding unstructured C-terminal region and transmembrane orientation of the Fst toxin is distinct from those of common antimicrobial peptides. This suggests that they have different mechanisms of action, although both perturb membrane integrity and topology. SpoIISA TA system from _B. subtilis_ has a higher molecular weight, with 248 amino acid residues, compared to other TA system toxins. This protein is predicted to have transmembrane and cytoplasmic domains.