Crystal Structure of the VapBC-15 Complex from Mycobacterium Tuberculosis Reveals a Two-Metal Ion dependent PIN-Domain Ribonuclease and a Variable Mode of Toxin-Antitoxin Assemblypetea

Although PIN (PilT N-terminal)-domain proteins are known to have ribonuclease activity, their specific mechanism of action remains unknown. VapCs form a family of ribonucleases that possess a PIN-domain assembly and are known as toxins. The activities of VapCs are impaired by VapB antitoxins. Here we present the crystal structure of the VapBC-15 toxin–antitoxin complex from Mycobacterium tuberculosis determined to 2.1 Å resolution. The VapB-15 and VapC-15 components assemble into one heterotetramer (VapB$_2$C$_2$) and two heterotrimers (VapBC$_2$) in each asymmetric unit of the crystal. The active site of VapC-15 toxin consists of a cluster of acidic amino acid residues and two divalent metal ions, forming a well organised ribonuclease active site. The distribution of the catalytic-site residues of the VapC-15 toxin is similar to that of T4 RNase H and of Methanococcus jannaschii FEN-1, providing strong evidence that these three proteins share a similar mechanism of activity. The presence of both VapB$_2$C$_2$ and VapBC$_2$ emphasizes the fact that the same antitoxin can bind the toxin in 1:1 and 1:2 ratios. The crystal structure determination of the VapBC-15 complex reveals for the first time a PIN-domain ribonuclease protein that shows two metal ions at the active site and a variable mode of toxin–antitoxin assembly. The structure further shows that VapB-15 antitoxin binds to the same groove meant for the binding of putative substrate (RNA), resulting in the inhibition of VapC-15’s toxicity