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How bacterial ADP-ribosylating toxins recognize substrates

Nature Structural & Molecular Biology volume 11, pages 868–876 (2004)Cite this article

Abstract

ExoS and ExoT are bifunctional type III cytotoxins of Pseudomonas aeruginosa that contain an N-terminal RhoGAP domain and a C-terminal ADP-ribosylation domain. Although they share 76% amino acid identity, ExoS and ExoT ADP-ribosylate different substrates. Using protein modeling and site-directed mutagenesis, the regions of ExoS and ExoT that define substrate specificity were determined. Regions B (active site loop), C (ARTT motif) and E (PN loop) on ExoS are necessary and sufficient to recognize ExoS targets, whereas regions B, C and E on ExoT are necessary but not sufficient to recognize ExoT targets, such as the Crk proteins. A specific Crk recognition motif on ExoT was defined as region A (helix α1). The electrostatic properties of regions A, B, C and E define the substrate specificity of ExoS and ExoT and these interactions can explain how other bacterial ADP-ribosylating toxins recognize their unique substrates.

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Figure 1: Protein modeling of the ADP-ribosylation domains of ExoS and ExoT.
Figure 2: Regions B, C and E are required for substrate recognition of ExoS and ExoT.
Figure 3: Regions B, C and E constitute the substrate recognition sites for ExoS, but not for ExoT.
Figure 4: ExoT(S:BCE) showed a target profile similar to that of ExoS.
Figure 5: Region A of ExoT is the specific Crk recognition motif.
Figure 6: In vivo substrate specificity of ExoS and ExoT.
Figure 7: Effect of NaCl on substrate recognition of ExoS and ExoT.
Figure 8: The ADP-ribosylating toxins recognize protein substrates through electrostatic interaction.

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Acknowledgements

This study was supported by a grant from the US National Institutes of Health (AI-30162) to J.T.B.

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Authors and Affiliations

  1. Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, 53226, Wisconsin, USA

    Jianjun Sun, Anthony W Maresso & Joseph T Barbieri

  2. Department of Biochemistry, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, 53226, Wisconsin, USA

    Jung-Ja P Kim

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Correspondence to Joseph T Barbieri.

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Supplementary information

Supplementary Fig. 1

Indicated toxins were incubated with GST-Crk-I or moesin with the presence of NAD/P32-NAD and FAS, and reaction rates (V) were calculated as described in Methods. The data from at least three independent experiments were fit to Michaelis-Menten equation in both SigmaPlot and EnzFitter, where the values of Km and Vmax were calculated. (PDF 84 kb)

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Sun, J., Maresso, A., Kim, JJ. et al. How bacterial ADP-ribosylating toxins recognize substrates. Nat Struct Mol Biol 11, 868–876 (2004). https://doi.org/10.1038/nsmb818

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