# Structure, Biology, and Therapeutic Application of Toxin-Antitoxin Systems in Pathogenic Bacteria
## 4 Structural Characteristics of TA Proteins from Pathogenic Bacteria
### VapBC  
The VapC toxins belong to the piT \(N\)-terminal domain (PIN-domain) family, which share an \(\alpha/\beta/\alpha\) sandwich structures and four conserved catalytic acidic residues although sequence identity within them is low [181]. The structures are highly similar to those of the T4 RNase H nuclease domain [17]. The structure of _M. tuberculosis_ VapC toxin (Rv0627) in complex with the \(C\)-terminal region of its cognate VapB antitoxin (Rv0626) was determined [173]. The structure of Rv0627 shows a compact \(\alpha/\beta/\alpha\) core domain and a protruding clip structure of two \(\alpha\)-helices (Figure 4A). The core domain is composed of a four-stranded parallel \(\beta\)-sheet that is surrounded by five \(\alpha\)-helices. The structure of the bound Rv0626 antitoxin shows 33 out of 86 residues. The missing residues are located in the \(N\)-terminal DNA binding region of the antitoxin. The TA complex is achieved mainly by hydrophobic interactions in a groove formed between the core domain and the clip structure of the toxin. The VapC toxin has four highly conserved acidic residues, Asp26, Glu57, Asp115, and Asp135, which are clustered to form a negatively charged cavity (Figure 4A). The crystal structure of the VapBC complex (Rv0623-Rv0624) from _M. tuberculosis_ revealed distinct structural differences between the Rv0624 toxin and PIN domain proteins, although they have similar overall architectures [176] (Figure 4B). The \(C\)-terminal fragment of Rv0624 does not form the secondary structure, whereas the corresponding region of other VapC members commonly forms an \(\alpha\)-helix followed by one \(\beta\)-strand. In addition, Rv0623, except for the \(N\)-terminal \(\alpha\)-helix, is structurally different from other VapB members, and its interaction with Rv0624 shows a characteristic structural feature. Three conserved residues (Asp4, Glu40 and Asp99) of the active site of the toxin are positioned in a concave surface of the homodimeric interface of the toxin (Figure 4B). These residues coordinate a Mg\({}^{2+}\) ion by a hydrogen bond network. The crystal structure of the VapBC complex (Rv2009-Rv2010) from _M. tuberculosis_revealed one tetramer of VapBC heterodimers and two heterotrimers (VapBC\({}_{2}\)) in each asymmetric unit of the crystal (Figure 4C) [175]. This observation indicates that the VapB antitoxin can bind to the VapC toxin in both 1:1 and 1:2 ratios. The catalytic site of the toxin consists of spatially close acidic residues and two divalent metal ions. High-resolution crystal structure of the VapBC complex (Rv0300-Rv0301) from _M. tuberculosis_ shows Mg\({}^{2+}\) ions that are bound and unbound to the active site of the Rv0301 toxin, which forms a tightly packed globular protein with the characteristics of a typical PIN-domain (Figure 4D) [174]. The conserved acidic residues of the toxin are Asp9, Glu43, Asp99, and Asp117. The Rv0300 antitoxin adopts significantly different conformations due to its different hinges, called open and closed antioxins, which are wrapped around the Mg\({}^{2+}\) ion-bound and -unbound toxins, respectively. The dimeric structure of the \(N\)-terminal \(\beta\)1-\(\alpha\)1-\(\alpha\)2 of the antitoxin can be annotated as a RHH domain family, which is commonly found in transcription factors that bind to DNA in a sequence-specific manner. A structural homology search using the program DALI showed that Rv0300 is structurally the closest to the antitoxin FitA from _Neisseria gonorrhoeae (N. gonorrhoeae)_. The overall octameric structure of the Rv0300-Rv0301 complex is analogous to those of the FitAB complex from _N. gonorrhoeae_[162], the VapBC complex from _Shigella flexneri_ (_S. flexneri_) [164], and the VapBC2 complex from _Rickettsia felis_ (_R. felis_) [163]. The structure of the toxin shows that its active site is coordinated with one Mg\({}^{2+}\) ion, suggesting that the catalysis requires at least a single divalent metal ion. In _N.