Contact-dependent growth inhibition (CDI) is a mechanism of inter-cellular competition in which Gram-negative bacteria exchange polymorphic toxins using type V secretion systems. Here, we present structures of the CDI toxin from Escherichia coli NC101 in ternary complex with its cognate immunity protein and elongation factor Tu (EF-Tu). The toxin binds exclusively to domain 2 of EF-Tu, partially overlapping the site that interacts with the 3'-end of aminoacyl-tRNA (aa-tRNA). The toxin exerts a unique ribonuclease activity that cleaves the single-stranded 3'-end from tRNAs that contain guanine discriminator nucleotides. EF-Tu is required to support this tRNase activity in vitro, suggesting the toxin specifically cleaves substrate in the context of GTP·EF-Tu·aa-tRNA complexes. However, superimposition of the toxin domain onto previously solved GTP·EF-Tu·aa-tRNA structures reveals potential steric clashes with both aa-tRNA and the switch I region of EF-Tu. Further, the toxin induces conformational changes in EF-Tu, displacing a β-hairpin loop that forms a critical salt-bridge contact with the 3'-terminal adenylate of aa-tRNA. Together, these observations suggest that the toxin remodels GTP·EF-Tu·aa-tRNA complexes to free the 3'-end of aa-tRNA for entry into the nuclease active site.

Bacteria use several different secretion systems to deliver toxic EndoU ribonucleases into neighboring cells. Here, we present the first structure of a prokaryotic EndoU toxin in complex with its cognate immunity protein. The contact-dependent growth inhibition toxin CdiA-CT^STECO31 from Escherichia coli STEC_O31 adopts the eukaryotic EndoU fold and shares greatest structural homology with the nuclease domain of coronavirus Nsp15. The toxin contains a canonical His-His-Lys catalytic triad in the same arrangement as eukaryotic EndoU domains, but lacks the uridylate-specific ribonuclease activity that characterizes the superfamily. Comparative sequence analysis indicates that bacterial EndoU domains segregate into at least three major clades based on structural variations in the N-terminal subdomain. Representative EndoU nucleases from clades I and II degrade tRNA molecules with little specificity. In contrast, CdiA-CT^STECO31 and other clade III toxins are specific anticodon nucleases that cleave tRNA^Glu between nucleotides C37 and m² A38. These findings suggest that the EndoU fold is a versatile scaffold for the evolution of novel substrate specificities. Such functional plasticity may account for the widespread use of EndoU effectors by diverse inter-bacterial toxin delivery systems.

Many Gram-negative bacteria use CdiA effector proteins to inhibit the growth of neighboring competitors. CdiA transfers its toxic CdiA-CT region into the periplasm of target cells, where it is released through proteolytic cleavage. The N-terminal cytoplasm-entry domain of the CdiA-CT then mediates translocation across the inner membrane to deliver the C-terminal toxin domain into the cytosol. Here, we show that proteolysis not only liberates the CdiA-CT for delivery, but is also required to activate the entry domain for membrane translocation. Translocation function depends on precise cleavage after a conserved VENN peptide sequence, and the processed ∆VENN entry domain exhibits distinct biophysical and thermodynamic properties. By contrast, imprecisely processed CdiA-CT fragments do not undergo this transition and fail to translocate to the cytoplasm. These findings suggest that CdiA-CT processing induces a critical structural switch that converts the entry domain into a membrane-translocation competent conformation.