Structural and functional insights into Escherichia coli O32:H37 contact dependent inhibition

TL;DR

This study reveals how a bacterial toxin from Escherichia coli interacts with an immunity protein and elongation factor Tu to regulate its activity.

Contribution

The paper provides structural and functional insights into a novel toxin-immunity complex and its interaction with EF-Tu.

Findings

CTO32:H37 is structurally similar to colicin D but lacks specific tRNA targeting.

CdiIO32:H37 forms a stable complex with EF-Tu, which is essential for RNase activity.

AlphaFold3 modeling suggests EF-Tu stabilizes the toxin's active site via a core tryptophan residue.

Abstract

Many gram-negative bacteria utilize contact-dependent growth inhibition (CDI) systems to introduce toxic effector proteins into neighboring cells, thereby outcompeting them. In CDI+ strains, CdiA effector proteins, carrying C-terminal toxin (CT) domains, are delivered into target cells through receptor-mediated pathways. To prevent self-intoxication, these bacteria produce CdiI immunity proteins that bind and neutralize the CT domains. Here, we present the crystal structure of the CT•CdiIO32:H37 complex from Escherichia coli O32:H37. Our findings reveal that CTO32:H37 is structurally homologous to the C-terminal tRNase domain of colicin D, and contains similar catalytic centers. However, CTO32:H37 exhibits non- specific RNase activity, unlike colicin D, which targets the anticodon loops of tRNAArg isoacceptors. The CdiIO32:H37 immunity protein features a unique fold coordinating a central Fe3+ ion with cysteine residues. Intriguingly, CT• CdiIO32:H37 complex co-purifies with endogenous elongation factor Tu (EF-Tu), forming a stable ternary complex. Although CTO32:H37 does not stably interact with EF-Tu without CdiIO32:H37, EF-Tu is essential for RNase activity in vitro. AlphaFold3 modeling suggests that CTO32:H37 binds to the GTPase domain of EF-Tu at a site overlapping the EF-Ts binding site. Our data indicate that EF-Tu supports the toxin’s active site, stabilizing a core tryptophan residue that interacts with a catalytic histidine.