Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

The assembly of a GTPase–kinase signalling complex by a bacterial catalytic scaffold

Abstract

The fidelity and specificity of information flow within a cell is controlled by scaffolding proteins that assemble and link enzymes into signalling circuits1,2. These circuits can be inhibited by bacterial effector proteins that post-translationally modify individual pathway components3,4,5,6. However, there is emerging evidence that pathogens directly organize higher-order signalling networks through enzyme scaffolding7,8, and the identity of the effectors and their mechanisms of action are poorly understood. Here we identify the enterohaemorrhagic Escherichia coli O157:H7 type III effector EspG as a regulator of endomembrane trafficking using a functional screen, and report ADP-ribosylation factor (ARF) GTPases and p21-activated kinases (PAKs) as its relevant host substrates. The 2.5 Å crystal structure of EspG in complex with ARF6 shows how EspG blocks GTPase-activating-protein-assisted GTP hydrolysis, revealing a potent mechanism of GTPase signalling inhibition at organelle membranes. In addition, the 2.8 Å crystal structure of EspG in complex with the autoinhibitory Iα3-helix of PAK2 defines a previously unknown catalytic site in EspG and provides an allosteric mechanism of kinase activation by a bacterial effector. Unexpectedly, ARF and PAKs are organized on adjacent surfaces of EspG, indicating its role as a ‘catalytic scaffold’ that effectively reprograms cellular events through the functional assembly of GTPase-kinase signalling complex.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: EspG inhibits endomembrane trafficking and disrupts Golgi architecture.
Figure 2: The structure of EspG in complex with GTP-bound ARF6.
Figure 3: The structure of EspG in complex with PAK2 Iα3 peptide.
Figure 4: EspG functions as a catalytic scaffold at membrane organelles.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors for the reported crystal structures have been deposited with the Protein Data Bank under accession codes 3PCR and 3PCS.

References

  1. Scott, J. D. & Pawson, T. Cell signaling in space and time: where proteins come together and when they’re apart. Science 326, 1220–1224 (2009)

    Article  ADS  CAS  Google Scholar 

  2. Lim, W. A. Designing customized cell signalling circuits. Nature Rev. Mol. Cell Biol. 11, 393–403 (2010)

    Article  CAS  Google Scholar 

  3. Duesbery, N. S. et al. Proteolytic inactivation of MAP-kinase-kinase by anthrax lethal factor. Science 280, 734–737 (1998)

    Article  ADS  CAS  Google Scholar 

  4. Yarbrough, M. L. et al. AMPylation of Rho GTPases by Vibrio VopS disrupts effector binding and downstream signaling. Science 323, 269–272 (2009)

    Article  CAS  Google Scholar 

  5. Schmidt, G. et al. Gln 63 of Rho is deamidated by Escherichia coli cytotoxic necrotizing factor-1. Nature 387, 725–729 (1997)

    Article  ADS  CAS  Google Scholar 

  6. Li, H. et al. The phosphothreonine lyase activity of a bacterial type III effector family. Science 315, 1000–1003 (2007)

    Article  ADS  CAS  Google Scholar 

  7. Alto, N. M. et al. The type III effector EspF coordinates membrane trafficking by the spatiotemporal activation of two eukaryotic signaling pathways. J. Cell Biol. 178, 1265–1278 (2007)

    Article  CAS  Google Scholar 

  8. Vingadassalom, D. et al. Insulin receptor tyrosine kinase substrate links the E. coli O157:H7 actin assembly effectors Tir and EspFU during pedestal formation. Proc. Natl Acad. Sci. USA 106, 6754–6759 (2009)

    Article  ADS  CAS  Google Scholar 

  9. Rivera, V. M. et al. Regulation of protein secretion through controlled aggregation in the endoplasmic reticulum. Science 287, 826–830 (2000)

    Article  ADS  CAS  Google Scholar 

  10. Winnen, B. et al. Hierarchical effector protein transport by the Salmonella Typhimurium SPI-1 type III secretion system. PLoS ONE 3, e2178 (2008)

    Article  ADS  Google Scholar 

  11. Shaw, R. K. et al. Enteropathogenic Escherichia coli type III effectors EspG and EspG2 disrupt the microtubule network of intestinal epithelial cells. Infect. Immun. 73, 4385–4390 (2005)

    Article  CAS  Google Scholar 

  12. Tomson, F. L. et al. Enteropathogenic Escherichia coli EspG disrupts microtubules and in conjunction with Orf3 enhances perturbation of the tight junction barrier. Mol. Microbiol. 56, 447–464 (2005)

    Article  CAS  Google Scholar 

  13. Yoshida, S. et al. Microtubule-severing activity of Shigella is pivotal for intercellular spreading. Science 314, 985–989 (2006)

    Article  ADS  CAS  Google Scholar 

  14. Germane, K. L., Ohi, R., Goldberg, M. B. & Spiller, B. W. Structural and functional studies indicate that Shigella VirA is not a protease and does not directly destabilize microtubules. Biochemistry 47, 10241–10243 (2008)

    Article  CAS  Google Scholar 

  15. Davis, J. et al. Novel fold of VirA, a type III secretion system effector protein from Shigella flexneri . Protein Sci. 17, 2167–2173 (2008)

    Article  CAS  Google Scholar 

  16. Kahn, R. A. Toward a model for Arf GTPases as regulators of traffic at the Golgi. FEBS Lett. 583, 3872–3879 (2009)

    Article  CAS  Google Scholar 

  17. D’Souza-Schorey, C. & Chavrier, P. ARF proteins: roles in membrane traffic and beyond. Nature Rev. Mol. Cell Biol. 7, 347–358 (2006)

    Article  Google Scholar 

  18. Bokoch, G. M. Biology of the p21-activated kinases. Annu. Rev. Biochem. 72, 743–781 (2003)

    Article  CAS  Google Scholar 

  19. Borthakur, A. et al. Enteropathogenic Escherichia coli inhibits butyrate uptake in Caco-2 cells by altering the apical membrane MCT1 level. Am. J. Physiol. Gastrointest. Liver Physiol. 290, G30–G35 (2006)

    Article  CAS  Google Scholar 

  20. Guttman, J. A. et al. Aquaporins contribute to diarrhoea caused by attaching and effacing bacterial pathogens. Cell. Microbiol. 9, 131–141 (2007)

    Article  CAS  Google Scholar 

  21. Pasqualato, S., Menetrey, J., Franco, M. & Cherfils, J. The structural GDP/GTP cycle of human Arf6. EMBO Rep. 2, 234–238 (2001)

    Article  CAS  Google Scholar 

  22. Hanzal-Bayer, M., Renault, L., Roversi, P., Wittinghofer, A. & Hillig, R. C. The complex of Arl2-GTP and PDE delta: from structure to function. EMBO J. 21, 2095–2106 (2002)

    Article  CAS  Google Scholar 

  23. Isabet, T. et al. The structural basis of Arf effector specificity: the crystal structure of ARF6 in a complex with JIP4. EMBO J. 28, 2835–2845 (2009)

    Article  CAS  Google Scholar 

  24. O’Neal, C. J., Jobling, M. G., Holmes, R. K. & Hol, W. G. Structural basis for the activation of cholera toxin by human ARF6-GTP. Science 309, 1093–1096 (2005)

    Article  ADS  Google Scholar 

  25. Shiba, T. et al. Molecular mechanism of membrane recruitment of GGA by ARF in lysosomal protein transport. Nature Struct. Biol. 10, 386–393 (2003)

    Article  CAS  Google Scholar 

  26. Zhao, L. et al. Direct and GTP-dependent interaction of ADP ribosylation factor 1 with coatomer subunit beta. Proc. Natl Acad. Sci. USA 94, 4418–4423 (1997)

    Article  ADS  CAS  Google Scholar 

  27. Chardin, P. & McCormick, F. Brefeldin A: the advantage of being uncompetitive. Cell 97, 153–155 (1999)

    Article  CAS  Google Scholar 

  28. Lei, M. et al. Structure of PAK1 in an autoinhibited conformation reveals a multistage activation switch. Cell 102, 387–397 (2000)

    Article  CAS  Google Scholar 

  29. Bremser, M. et al. Coupling of coat assembly and vesicle budding to packaging of putative cargo receptors. Cell 96, 495–506 (1999)

    Article  CAS  Google Scholar 

  30. Goldberg, J. Structural and functional analysis of the ARF1-ARFGAP complex reveals a role for coatomer in GTP hydrolysis. Cell 96, 893–902 (1999)

    Article  CAS  Google Scholar 

  31. Minor, W., Cymborowski, M., Otwinowski, Z. & Chruszcz, M. HKL-3000: the integration of data reduction and structure solution–from diffraction images to an initial model in minutes. Acta Crystallogr. D 62, 859–866 (2006)

    Article  Google Scholar 

  32. Schneider, T. R. & Sheldrick, G. M. Substructure solution with SHELXD. Acta Crystallogr. D 58, 1772–1779 (2002)

    Article  Google Scholar 

  33. Cowtan, K. & Main, P. Miscellaneous algorithms for density modification. Acta Crystallogr. D 54, 487–493 (1998)

    Article  CAS  Google Scholar 

  34. Terwilliger, T. SOLVE and RESOLVE: automated structure solution, density modification and model building. J. Synchrotron Radiat. 11, 49–52 (2004)

    Article  CAS  Google Scholar 

  35. Cowtan, K. The Buccaneer software for automated model building. 1. Tracing protein chains. Acta Crystallogr. D 62, 1002–1011 (2006)

    Article  Google Scholar 

  36. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007)

    Article  CAS  Google Scholar 

  37. Jones, T. A. Zou, J.-Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991)

  38. Adams, P. D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D 58, 1948–1954 (2002)

    Article  Google Scholar 

  39. Davis, I. W. et al. MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res. 35, W375–W383 (2007)

    Article  ADS  Google Scholar 

Download references

Acknowledgements

We would like to thank our colleagues, specifically K. Orth, M. Rosen, M. Cobb, J. Seeman, C. Brautigam and T. Fox, for helpful discussions in preparation of this manuscript, and we are particularly indebted to members of the Structural Biology Lab and mass spectrometry facilities for their efforts on this project. We would also like to thank J. Goldberg and G. Bokoch for providing valuable reagents. We would particularly like to thank J. Cherfils for providing preliminary insights into this work and for key reagents. The structure shown in this report is derived from work performed on beamlines 19-BM and 19-ID at the Structural Biology Center, Advanced Photon Source, Argonne National Laboratory. Argonne National Laboratory is operated by UChicago Argonne, LLC, for the US Department of Energy, Office of Biological and Environmental Research, under contract DE-AC02-06CH11357. R.C.O. was supported by a NIH Molecular Microbiology training grant (5T32AI007520-12). This work was supported by the Welch Foundation (#I-1704) and a grant from the NIH (NIAID; 1RO1AI083359-01) to N.M.A.

Author information

Authors and Affiliations

Authors

Contributions

N.M.A. and A.S.S. had the general ideas for this manuscript. A.S.S., N.M.A. and S.M.B. crystallized the protein complexes and D.R.T. solved the complex structures. N.M.A., A.S.S., S.E.S., B.A.W., L.E.R. and R.C.O. planned, performed and interpreted the experiments. N.M.A. and A.S.S. wrote the manuscript and all authors provided editorial input.

Corresponding author

Correspondence to Neal M. Alto.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

The file contains Supplementary Table 1, Supplementary Figures 1-12 with legends and additional references. (PDF 13364 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Selyunin, A., Sutton, S., Weigele, B. et al. The assembly of a GTPase–kinase signalling complex by a bacterial catalytic scaffold. Nature 469, 107–111 (2011). https://doi.org/10.1038/nature09593

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature09593

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing