ABSTRACT Many Gram‐negative bacterial species use contact‐dependent growth inhibition (CDI) systems to deliver toxic proteins into neighboring competitors. CDI + strains deploy CdiA effector proteins, which translocate their C‐terminal toxin (CT) domains into target bacteria through a receptor‐mediated delivery pathway. To protect against auto‐intoxication, CDI + bacteria also produce CdiI immunity proteins that neutralize CT toxin activity. Here, we present the crystal structure of the CT·CdiI O32:H37 complex from Escherichia coli O32:H37. CT O32:H37 adopts the same fold as the tRNase domain of colicin D, and the nucleases share similar catalytic centers. However, unlike colicin D, which cleaves the anticodon loops of tRNA Arg isoacceptors, CT O32:H37 exhibits nonspecific RNase activity. Notably, we find that endogenous elongation factor Tu (EF‐Tu) co‐purifies with the over‐produced CT·CdiI O32:H37 complex. Although EF‐Tu does not bind stably to CT O32:H37 in the absence of CdiI O32:H37 , the translation factor is required for toxic RNase activity in vitro. AlphaFold 3 modeling and site‐directed mutagenesis indicate that CT O32:H37 interacts with the N‐terminal GTPase domain of EF‐Tu. EF‐Tu appears to stabilize residue Trp52 within the hydrophobic core of the toxin, which in turn supports the RNase active site through an unusual hydrogen‐bonding interaction with the catalytic His67 residue. Thus, EF‐Tu is hijacked as an essential co‐factor to organize the toxin's catalytic center.
Nhan et al. (Tue,) studied this question.