Abstract
Contact-dependent growth inhibition (CDI) is a widespread mechanism of inter-bacterial competition. CDI(+) bacteria deploy large CdiA effector proteins, which carry variable C-terminal toxin domains (CdiA-CT). CDI(+) cells also produce CdiI immunity proteins that specifically neutralize cognate CdiA-CT toxins to prevent auto-inhibition. Here, we present the crystal structure of the CdiA-CT/CdiI(E479) toxin/immunity protein complex from Burkholderia pseudomallei isolate E479. The CdiA-CT(E479) tRNase domain contains a core α/β-fold that is characteristic of PD(D/E)XK superfamily nucleases. Unexpectedly, the closest structural homolog of CdiA-CT(E479) is another CDI toxin domain from B. pseudomallei 1026b. Although unrelated in sequence, the two B. pseudomallei nuclease domains share similar folds and active-site architectures. By contrast, the CdiI(E479) and CdiI(1026b) immunity proteins share no significant sequence or structural homology. CdiA-CT(E479) and CdiA-CT(1026b) are both tRNases; however, each nuclease cleaves tRNA at a distinct position. We used a molecular docking approach to model each toxin bound to tRNA substrate. The resulting models fit into electron density envelopes generated by small-angle x-ray scattering analysis of catalytically inactive toxin domains bound stably to tRNA. CdiA-CT(E479) is the third CDI toxin found to have structural homology to the PD(D/E)XK superfamily. We propose that CDI systems exploit the inherent sequence variability and active-site plasticity of PD(D/E)XK nucleases to generate toxin diversity. These findings raise the possibility that many other uncharacterized CDI toxins may belong to the PD(D/E)XK superfamily.
Highlights
Contact-dependent growth inhibition (CDI) is a widespread mechanism of inter-bacterial competition
CDIϩ bacteria are protected from toxicity by CdiI immunity proteins, which bind to the CdiA delivers its C-terminal toxin domain (CdiA-CT) toxin domain and neutralize its activity
Structure of the CdiA-CT/CdiIE479 Toxin/Immunity Protein Complex—We previously reported that overexpression of the CdiA-CT/CdiIE479/His6 complex in E. coli cells leads to tRNA degradation and concomitant growth arrest [16]
Summary
Structure of the CdiA-CT/CdiIE479 Toxin/Immunity Protein Complex—We previously reported that overexpression of the CdiA-CT/CdiIE479/His complex in E. coli cells leads to tRNA degradation and concomitant growth arrest [16]. Helices ␣1Ј, ␣4Ј, and the -sheet of CdiIE479 interact with the end of the nuclease -sheet and the extended loop connecting ␣2Ј and 3Ј (Fig. 1B) This proteinprotein interface buries 1015 Å2 of surface area, corresponding to 14% of the nuclease domain and 18% of the immunity protein total surface area. In accord with this extensive interaction surface, biolayer interferometry showed that CdiA-CTE479 and CdiIE479 form a relatively high affinity complex with an apparent equilibrium dissociation constant of 72 Ϯ 23 nM (Fig. 2). We note that helix ␣1 from CdiA-CTE479 is significantly
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