# Structure, Biology, and Therapeutic Application of Toxin-Antitoxin Systems in Pathogenic Bacteria
## 4 Structural Characteristics of TA Proteins from Pathogenic Bacteria
### YefM-YoeB  
Crystallographic studies demonstrated that the YefM antitoxin of the YefM-YoeB system from _M. tuberculosis_ shows a well-defined structure with different conformations of the \(C\)-terminal part, but not the intrinsically unfolded structure that is commonly adopted by other many antiotoxins (Figure 6A) [177]. Multiple-sequence alignments suggest that the polar residues Ser4, Ser6, Arg8, Asp22, Thr29, and Ser39 of the YefM antitoxin are conserved, which might be necessary for DNAbinding. On the basis of this conservation, the putative DNA binding site could be mapped onto the YefM structure (Figure 6A). However, the _C_-terminal region of YefM that recognizes its cognate toxin is evolutionarily less conserved. In spite of this, the antitoxin was experimentally proved to neutralize different cognate toxin YoeB from _E. coli_. The structure of _M. tuberculosis_ YefM alone was determined as a homotetramer (Figure 6A), but the structure of _E. coli_ YefM was determined in a complex form with YoeB, which showed a heterotrim with a YefM:YoeB stoichiometry of 2:1 (Figure 6B). The structures of two YefM antiotoxins from _M. tuberculosis_ and _E. coli_ are well overlaid with an RMSD value of 1.08 A for 93 C\(\alpha\) atoms. They show structural differences in the central helices and the distal C-terminal region. It is possible that the conformational change of _E. coli_ YefM is induced by its complex formation with the cognate YoeB. Cell viability experiments combined with site-directed mutagenesis demonstrated that four residues of _E. coli_ YoeB, Glu46, Arg65, His83, and Tyr84, are required for its RNase activity and cell toxicity [124]. In consistent with this, the three C-terminal amino acids of _E. coli_ YoeB, Tyr82, His83, and Tyr84, form a canonical fold like a microbial RNase active site. Interestingly, some hydrophobic residues of _M. tuberculosis_ YefM for the formation of the tetramer correspond to the residues of _E. coli_ YefM that participate in the complex formation with YoeB. Figure 3: Structures of the CcdB–gyrase complexes from _V. fischeri_ and _E. coli_. (**A**) Structure of the CcdB–gyrase complex from _V. fischeri_ (left panel) and the gyrase binding site of the CcdB dimer (right panel). Gyrase binding segments include the residues Asp27–Asn31 and Glu91–Ile105, which are marked in purple and cyan, respectively. Two residues, Asp99 and Arg462, forming the hydrogen bond between CcdB and gyrase are indicated. (**B**) Structure of the CcdB–gyrase complex from _E. coli_ (left panel) and the gyrase binding site of the CcdB dimer (right panel). Gyrase binding segments include the residues Asp23–Thr27 and Glu87–Ile101, which are marked in red and dark yellow, respectively. Trp99, which is involved in gyrase binding, is marked in cyan. Three residues, Asn92, Asn95, and Arg462, forming the hydrogen bonds between CcdB and gyrase are indicated. Hydrogen bonds are depicted as dotted lines. Figure 4: Structural comparisons of VapBC proteins from _M. tuberculosis._ (**A**) Structure of the VapBC complex (Rv0626–Rv0627). Four conserved residues of VapC are clustered to form an acidic pocket. (**B**) Structure of the VapBC complex (Rv0623–Rv0624). Three conserved acidic residues of each VapC subunit are positioned in the dimeric interface of VapC. (**C**) Structure of the VapBC complex (Rv2009–Rv2010). VapC dimers bind to one and two VapB with a stoichiometry of 2:1 and 2:2, respectively. (**D**) Structure of the VapBC complex (Rv0300–Rv0301). Four conserved residues of each VapC monomer are clustered to form an acidic pocket. Figure 5: Structural comparisons of FitAB and VapBC proteins from _N. gonorrhoeae_, _R. felis_, and _S. flexneri_. (**A**) Structure of the FitAB-DNA complex from _N. gonorrhoeae_. The conserved acidic residues that form an acidic pocket of the FitB toxin are indicated on the right side. (**B**) Structure of the VapBC–DNA complex from _R. felis_; (**C**) structure of the VapBC complex from _S. flexneri_. Figure 6: Structures of (**A**) _M. tuberculosis_ YefM and (**B**) _E.