Functional details of the toxin-antitoxin systems based on a structural study

Abstract

Toxin-antitoxin (TA) systems are essential for bacterial persistence under stressful conditions. In particular, M. tuberculosis express VapBC TA genes that encode the stable VapC toxin and the labile VapB antitoxin. Under normal conditions, these proteins interact to form a non-toxic TA complex, but the toxin is activated by release from the antitoxin in response to unfavorable conditions. Here, we present the crystal structure of the M. tuberculosis VapBC26 complex and show that the VapC26 toxin contains a pilus retraction protein (PilT) N-terminal (PIN) domain that is essential for ribonuclease activity and that, the VapB26 antitoxin folds into a ribbon-helix-helix DNA-binding motif at the N-terminus. The active site of VapC26 is sterically blocked by the flexible C-terminal region of VapB26. The C-terminal region of free VapB26 adopts an unfolded conformation but forms a helix upon binding to VapC26. The results of RNase activity assays show that Mg2+ and Mn2+ are essential for the ribonuclease activity of VapC26. As shown in the nuclear magnetic resonance (NMR) spectra, several residues of VapB26 participate in the specific binding to the promoter region of the VapBC26 operon. In addition, toxin-mimicking peptides were designed that inhibit TA complex formation and thereby increase toxin activity, providing a novel approach to the development of new antibiotics Pneumoniae, an inflammatory human lung disease, is caused by Streptococcus pneumoniae, which has received growing attention for its resistance to existing antibiotics. S. pneumoniae TIGR4 contains six toxin-antitoxin (TA) pairs of type II TA systems that are essential for bacterial persistence against stressful conditions such as nutrient deprivation, antibiotic treatment, and immune system attacks. In particular, S.pneumoniae express HicBA TA gene that encode the stable HicA toxin and labile HicB antitoxin. These proteins interact to form a non-toxic TA complex under normal conditions, but the toxin is activated by release from the antitoxin in response to unfavorable growth conditions. Here, we present the first crystal structure showing entire conformation of the HicBA complex from S.pneumoniae, revealing that the HicA toxin contains a double-stranded RNA binding domain (dsRBD) that is essential for RNA recognition, whereas the HicB antitoxin folds into a ribbon-helix-helix (RHH) DNA-binding motif at the C-terminus. The active site of HicA is sterically blocked by the N-terminal region of HicB. According to the RNase activity assays, His36 is essential for the ribonuclease activity of HicA. As shown in the NMR spectra, several residues of HicB participate in the specific binding to the promotor DNA of the HicBA operon. In addition, toxin-mimicking peptide was designed to inhibit TA complex formation and thereby increase toxin activity, providing a novel approach for the development of new antibiotics.