Structural complementation of the catalytic domain of pseudomonas exotoxin A

Abstract

The catalytic moiety of Pseudomonas exotoxin A (domain III or PE3) inhibits protein synthesis by ADP-ribosylation of eukaryotic elongation factor 2. PE3 is widely used as a cytocidal payload in receptor-targeted protein toxin conjugates. We have designed and characterized catalytically inactive fragments of PE3 that are capable of structural complementation. We dissected PE3 at an extended loop and fused each fragment to one subunit of a heterospecific coiled coil. In vitro ADP-ribosylation and protein translation assays demonstrate that the resulting fusions-supplied exogenously as genetic elements or purified protein fragments-had no significant catalytic activity or effect on protein synthesis individually but, in combination, catalyzed the ADP-ribosylation of eukaryotic elongation factor 2 and inhibited protein synthesis. Although complementing PE3 fragments are catalytically less efficient than intact PE3 in cell-free systems, co-expression in live cells transfected with transgenes encoding the toxin fusions inhibits protein synthesis and causes cell death comparably as intact PE3. Complementation of split PE3 offers a direct extension of the immunotoxin approach to generate bispecific agents that may be useful to target complex phenotypes.

Keywords: ADP-ribosylation; ADP-ribosyltransferase; ADPRT; DT; MBP; RRL; SEM; cytotoxicity; diphtheria toxin; eEF2; elongation factor 2; eukaryotic elongation factor 2; exotoxin A; maltose-binding protein; rabbit reticulocyte lysate; standard error of the mean; structural complementation.

Figure 1. Design of split PE3

A, Backbone trace of PE3 (residues 400 to 613) taken from the refined structure of the wildtype exotoxin A (PDB: 1IKQ), colored by B-factor. The four mobile loops identified are labeled L1 to L4. The site of dissection, Asp⁴⁸⁸/Ala⁴⁸⁹, is indicated by the solid triangle. The catalytically essential Glu553 residue is shown in gold. B, Schematic and homology model (templated to PDB co-ordinates from 1DMA and 1R48) of the fusion constructs PEα and PEβ generated by linking the split PE3 fragments to a heterodimeric coiled coil by Oakley and Kim . NAD⁺ is shown as black sticks.

Figure 2. Structural complementation of split PE3 restores enzymatic activity

ADP-ribosyltransferase activity was measured by incorporation of 6-carboxyfluorescein-17-NAD⁺ into yeast eEF2 as described in Materials and Methods. Background intensity refers to the fluorescence of a mock sample containing fluorescein-NAD⁺ alone. PE3 constructs were present at 20 µM (complex in the case of split PE3 fragments). The split PE3 fragments harboring a heterospecific coiled-coil are designated α and β, and their coiled-free counterparts A and B, following the nomenclature as described in the main text. Bars represent means ± SEM from duplicate measurements. Asterisks indicate statistically significant differences (p < 0.05) from background. Note the lack of ADPRT activity in coil-free PE3-A and PE3-B.

Figure 3. Comparative enzymatic activities of split and intact PE3

ADPRT activity was measured by incorporation of 6-carboxyfluorescein-17-NAD⁺ into yeast eEF2 as described in Materials and Methods. Concentrations for split PE3 refer to the PEα:PEβ complex (harboring a heterodimeric coiled coil) or PE-A+PE-B (coil-free). The coil-free fragments do not heterodimerize as judged by pull-down experiments (Figure S2, Supplementary Data). Relative fluorescence incorporation was normalized against the fluorescence of a mock sample containing fluorescein-NAD⁺ alone. Symbols represent means ± SEM from duplicate measurements. Lines are best fits to the Hill equation.

Figure 4. Split PE3 inhibits protein synthesis following structural complementation in a dose-dependent manner

Rabbit reticulocyte lysate (RRL) was treated with various purified constructs as indicated for 30 min. prior to the addition of 400 ng of luciferase mRNA. Residual translational activity was measured as [³⁵S]-labeled luciferase or by luciferin chemiluminescence. A, Lysate were treated with the indicated protein constructs at 300 nM in the presence or absence of 50 µM NAD⁺. Densitometric measurements of [³⁵S] incorporation for all samples, except for intact PE3 and the α+β heterodimer in the presence of NAD⁺, were randomly ±15%. B, RRL was titrated with intact PE3 (solid squares) or TEV-activated PE3 α+β heterodimer (open squares) in the presence of 50 µM NAD⁺. C, Titration with NAD⁺ in the presence of 300 nM intact PE3 (solid) or α+β heterodimer (open). Symbols represent means of four experiments ± SEM. Lines are best fits to the Hill equation.

Figure 5. Complementation of genetically encoded split PE3 inhibits protein synthesis in vitro and in live cells

A, Rabbit reticulocyte lysate was treated with purified mRNA encoding the indicated constructs in the presence of 50 µM NAD⁺. Two different total mRNA doses (800 and 400 ng) were used. To account for the depletion of amino acid precursors prior to addition of luciferase mRNA, 800 ng of a control mRNA encoding for EGFP was also tested. Densitometric comparison of [³⁵S] incorporation with a no-mRNA sample showed ~50% lower intensity for EGFP and other samples (except for those treated with intact PE3 and the α+β heterodimer, which are undetectable above background). B, HEK293 cells were transfected with expression plasmids encoding intact PE3 or various combinations of PEα and/or PEβ fragments. After 24 and 48 h, protein synthesis was measured by [³H]-leucine incorporation. Bars represent means ± SEM relative to vector control from triplicate experiments. Asterisks without brackets indicate statistically significant differences (p < 0.05) from vector control for the corresponding time point. Brackets indicate pairwise comparisons as shown. C, HEK293T cells stably expressing a destabilized EGFP (dEGFP) were transfected with plasmids as for the [³H]-leucine uptake assay. Cellular dEGFP was measured by flow cytometry at 24 h after transfection. Bars represent means ± SEM relative to vector control from four experiments.

Figure 6. Co-transfection of split PE3 causes cell death

HEK293 cells were transiently transfected with expression plasmids encoding the indicated constructs and incubated for 48 or 72 h. A constant amount of total DNA (500 ng per 10⁵ cells) was used in each treatment. A, Cytotoxicity relative to intact PE3 was measured by XTT reduction. Bars represent means of viability relative to vector ± SEM from four experiments. Asterisks without brackets indicate statistically significant differences (p < 0.05) from vector control for the corresponding time point. Brackets indicate pairwise comparisons as shown. B. Relative dose dependence of cytotoxicity as measured by resazurin reduction. The dose of plasmids encoding each construct was diluted with vector as indicated while maintaining the total DNA at 500 ng. Symbols represent means of four experiments ± SEM. Lines are best fits to the Hill equation.