Functional Diversity of Cytotoxic tRNase/Immunity Protein Complexes from Burkholderia pseudomallei

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.

Keywords: Burkholderia pseudomallei; crystal structure; protein complex; ribonuclease; small-angle x-ray scattering (SAXS); tRNase; toxin; toxin/immunity complexes.

FIGURE 1.

Structure of the CdiA-CT/CdiI^E479 toxin/immunity protein complex. A, CdiA-CT/CdiI^E479 complex is shown as a schematic with secondary structure elements of CdiI indicated with prime symbols. B, CdiA-CT/CdiI^E479 complex formation is mediated by electrostatic interactions. Interacting residues are in stick representation with nitrogen and oxygen atoms colored in blue and red (respectively), and bonds are shown as black dashed lines. The view is rotated 180° about the x axis relative to A.

FIGURE 2.

Biolayer interferometry of the CdiA-CT/CdiI^E479 binding interaction. Immobilized CdiI^E479-His₆ was exposed to varying concentrations (1.5–5 μm) of CdiA-CT^E479, and the binding interaction and dissociation monitored a wavelength shift (nm). Representative association and dissociation curves are presented with the overall correlation coefficient (R²) shown for the fit.

FIGURE 3.

Sequence and structure alignment of CdiA-CT^E479 and CdiA-CT^1026b nuclease domains. A, sequence alignment of CdiA-CT^E479 and CdiA-CT^1026b toxins with proposed active-site residues highlighted in red and conserved residues in blue. Secondary structure elements are colored gold and green for CdiA-CT^E479 and CdiA-CT^1026b, respectively. B, superimposition of CdiA-CT^E479 and CdiA-CT^1026b nuclease domains. Secondary structure elements that superimpose are color-coded in gold (CdiA-CT^E479) and green (CdiA-CT^1026b), and those that do not align rendered in white (CdiA-CT^E479) and gray (CdiA-CT^1026b). C, active site of CdiA-CT^E479 and CdiA-CT^1026b nuclease domain. Predicted active-site residues are shown in stick representation (nitrogen and oxygen atoms are colored blue and red, respectively).

FIGURE 4.

CdiA-CT^E479 growth inhibition and tRNase activities. A, growth inhibition activity of CdiA-CT^E479 variants. The indicated toxins were expressed in E. coli cells from a rhamnose-inducible promoter as described under “Experimental Procedures.” Expression was induced at 0 min, and cell growth was monitored by measuring the optical density at 600 nm (OD₆₀₀). The curve labeled repressed corresponds to un-induced cells carrying the wild-type CdiA-CT^E479 construct. The average ± S.E. from three independent biological replicates is presented. B, in vitro nuclease assays. The indicated CdiA-CT^E479 variants were purified and incubated with total E. coli RNA. Reactions were run on denaturing 6% polyacrylamide gels and stained with ethidium bromide. C, mutant CdiA-CT^E479 domains bind to CdiI^E479 immunity protein. Isolated toxin domains were mixed with purified CdiI^E479-His₆ and then subjected to Ni²⁺-affinity chromatography. Lanes labeled input show the protein mixtures loaded onto the column; free lanes show proteins that failed to bind the column, and bound indicates proteins eluted from the column with imidazole. Prior work has shown that CdiA-CT^E479 does not bind to Ni²⁺-NTA-agarose resin (16). D, CdiA-CT^E479 cleaves unmodified tRNAs produced by in vitro transcription. E. coli tRNA^Gln and tRNA^Asp transcripts were incubated with purified CdiA-CT^E479 and CdiI^E479, and reactions were analyzed on denaturing 6% polyacrylamide gels stained with ethidium bromide. Experiments in B–D were repeated twice with essentially identical results. Representative data are shown for each experiment.

FIGURE 5.

Sequence and structure comparison of CdiI^E479 and CdiI^1026b immunity proteins. A, structure-based sequence alignment of CdiI^E479 (blue) and CdiI^1026b (cyan) with secondary structure elements indicated above and below the sequence alignment. Conserved residues are highlighted in blue. B, superimposition of CdiI^E479 and CdiI^1026b structures. Secondary elements that partially or fully superimpose are labeled.

FIGURE 6.

CdiA-CT/CdiI^E479 and CdiA-CT/CdiI^1026b complexes interact through distinct electrostatic surfaces. A, electrostatic surface map of the CdiA-CT/CdiI^E479 complex interface. Negative, positive, and neutral surfaces regions are shown in red, blue, and white, respectively. B, electrostatic surface map of the CdiA-CT/CdiI^1026b complex interface. Yellow arrows indicate the minimal width of each active-site pocket.

FIGURE 7.

Computational modeling and SAXS analysis of tRNA/CdiA-CT complexes. A, CdiA-CT^1026b (green schematic) binding with its active-site residues adjacent to the backbone of the tRNA^Cys (PDB code 1B23) amino acceptor stem loop with active-site residues shown as spheres (oxygen and nitrogen atoms colored red and blue, respectively). B, SAXS electron density envelope (white mesh) fitted with the docking solution showing the monomeric CdiA-CT^1026b toxin (red schematic) bound to tRNA^Cys (green and blue). C, CdiA-CT^E479 (green schematic) binding with its active-site residues adjacent to the backbone of the tRNA^Cys T-loop with active-site residues shown as spheres (oxygen and nitrogen atoms colored red and blue, respectively). D, SAXS electron density envelope (white mesh) fitted with the docking solution showing the tetrameric CdiA-CT^E479 toxin bound to four molecules of tRNA^Cys (green and blue).

FIGURE 8.

Inactive CdiA-CT^E479 and CdiA-CT^1026b toxin domains bind to endogenous tRNA. A, agarose gel analysis of catalytically inactive CdiA-CT^E479 and CdiA-CT^1026b toxins purified under non-denaturing conditions. Control RNA is from yeast (Sigma). B, size-exclusion chromatography of the purified tRNA/CdiA-CT(D214A)^1026b complex. C, size-exclusion chromatography of the purified tRNA/CdiA-CT(D243A)^E479 complex. Chromatography migration standards are as follows: bovine thyroglobulin (670 kDa), bovine γ-globulin (158 kDa), chicken ovalbumin (44 kDa), horse myoglobin (17 kDa), and vitamin B₁₂ (1.3 kDa). Each experiment was carried out in triplicate with similar results. Representative data are shown for each experiment.

FIGURE 9.

SAXS analyses of tRNA/CdiA-CT complexes. Plots for tRNA/CdiA-CT^1026b (I) and tRNA/CdiA-CT^E479 (II) SAXS data. A, log I(q) versus q plot with experimental SAXS profile shown in blue and the corresponding structural model fitted data via Crysol (41) shown in green. B, Guinier plots. C, Kratky plots. D, P(r) plots.