The crystal structure of the Rv0301‐Rv0300 VapBC‐3 toxin—antitoxin complex from M. tuberculosis reveals a Mg²⁺ ion in the active site and a putative RNA‐binding site

Abstract

VapBC pairs account for 45 out of 88 identified toxin-antitoxin (TA) pairs in the Mycobacterium tuberculosis (Mtb) H37Rv genome. A working model suggests that under times of stress, antitoxin molecules are degraded, releasing the toxins to slow the metabolism of the cell, which in the case of VapC toxins is via their RNase activity. Otherwise the TA pairs remain bound to their promoters, autoinhibiting transcription. The crystal structure of Rv0301-Rv0300, an Mtb VapBC TA complex determined at 1.49 Å resolution, suggests a mechanism for these three functions: RNase activity, its inhibition by antitoxin, and its ability to bind promoter DNA. The Rv0301 toxin consists of a core of five parallel beta strands flanked by alpha helices. Three proximal aspartates coordinate a Mg 21 ion forming the putative RNase active site. The Rv0300 antitoxin monomer is extended in structure, consisting of an N-terminal beta strand followed by four helices. The last two helices wrap around the toxin and terminate near the putative RNase active site, but with different conformations. In one conformation, the C-terminal arginine interferes with Mg 21 ion coordination, suggesting a mechanism by which the antitoxin can inhibit toxin activity. At the N-terminus of the antitoxin, two pairs of Ribbon-Helix-Helix (RHH) motifs are related by crystallographic twofold symmetry. The resulting hetero-octameric complex is similar to the FitAB system, but the two RHH motifs are about 30 Å closer together in the Rv0301-Rv0300 complex, suggesting either a different span of the DNA recognition sequence or a conformational change.

Structure-Function Analysis of VapB4 Antitoxin Identifies Critical Features of a Minimal VapC4 Toxin-Binding Module

“…The existing crystal structures of VapBC pairs show that the C-terminal portions of the VapB antitoxins wrap around and bind in grooves on the surface of their VapC partners (33,(35)(36)(37)(38). Secondary structure prediction indicates the presence of two ␣ helices (␣2 and ␣3) in the C terminus of VapB4 at positions which match the positions of the two helices that grip VapC5 in the VapBC5 structure (37) (Fig.…”

“…However, the fact that inclusion of the N-terminal domain suppresses the defect caused by some of the mutations in the C-terminal domain suggests that features in the DNAbinding module might function to stabilize the interaction between the toxin and antitoxin. Indeed, crystal structures of VapBC pairs indicate that dimerization of VapB toxin-antitoxin pairs involves interactions between the DNA-binding modules of the antitoxins (35)(36)(37)(38). These contacts certainly contribute to the formation of heteromeric complexes seen in the crystal structures and may help stabilize the interaction of some of the VapB4 mutants with VapC4.…”

“…The analysis of the crystal structure of the VapBC TA complex from Shigella flexneri suggests that 4 aromatic residues in the C-terminal domain of VapB (Trp47, Trp50, Phe51, and Phe60) contact the hydrophobic core of VapC, and 2 residues (Arg64 and Gln66) interact with the conserved negatively charged amino acid residues of the PIN domain (33). Similarly, the crystal structures of VapBC complexes from M. tuberculosis, Neisseria gonorrhoeae, and Rickettsia felis suggest that multiple contacts govern the interactions between the VapB antitoxins and their cognate VapC toxins (34)(35)(36)(37)(38). These structures raise the question of how many protein-protein contacts are required for stable VapBC interaction and whether binding is likely to be sensitive to smallmolecule protein-protein interaction inhibitors.…”

Preliminary crystallographic analysis of recombinant VapBC-15 toxin–antitoxin complex fromMycobacterium tuberculosis

“…asymmetric unit (Matthews, 1968). The M. tuberculosis VapBC-3 (Rv0300-Rv0301) TA complex (Min et al, 2012; PDB entry 3h87), which shares 39% sequence identity with M. tuberculosis VapBC-15, was used as a template for molecular-replacement studies. PDB entry 3h87 has two heterodimers of toxin (chains A and B) and antitoxin (chains C and D) in the asymmetric unit.…”

“…Presently, the crystal structures of only two VapBC complexes have been determined from M. tuberculosis (Miallau et al, 2009;Min et al, 2012) out of 47 putative VapBC family members. The function of these Vaps is still unknown in M. tuberculosis and therefore structural and functional elucidations of these protein complexes are necessary in order to understand the pathogenicity of tuberculosis.…”