Comparative Proteomics Identifies the Cell-Associated Lethality of M. tuberculosis ReIBE-like Toxin-Antitoxin Complexes

The Mycobacterium tuberculosis (Mtb) genome encodes approximately 90 toxin-antitoxin protein complexes, including three ReIBE family members, which are believed to play a major role in bacterial fitness and pathogenicity. We have determined the crystal structures of Mtb ReIBE-2 and ReIBE-3, and the structures reveal homologous heterotetramers. Our structures suggest ReIE-2, and by extension the closely related ReIE-1, use a different catalytic mechanism than ReIE-3, because our analysis of the ReIE-2 structure predicts additional amino acid residues that are likely to be functionally significant and are missing from analogous positions in the ReIE-3 structure. Toxicity assays corroborate our structural findings; overexpression of ReIE-3, whose active site is more similar to Escherichia coli YoeB, has limited consequences on bacterial growth, whereas ReIE-1 and ReIE-2 overexpression results in acute toxicity. Moreover, ReIE-2 overexpression results in an elongated cell phenotype in Mycobacterium smegmatis and protects M. tuberculosis against antibiotics, suggesting a different functional role for ReIE-2.