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Chemical inhibition of N-WASP by stabilization of a native autoinhibited conformation

Nature Structural & Molecular Biology volume 11, pages 747–755 (2004)Cite this article

Abstract

Current drug discovery efforts focus primarily on proteins with defined enzymatic or small molecule binding sites. Autoregulatory domains represent attractive alternative targets for small molecule inhibitors because they also occur in noncatalytic proteins and because allosteric inhibitors may avoid specificity problems inherent in active site–directed inhibitors. We report here the identification of wiskostatin, a chemical inhibitor of the neural Wiskott-Aldrich syndrome protein (N-WASP). Wiskostatin interacts with a cleft in the regulatory GTPase-binding domain (GBD) of WASP in the solution structure of the complex. Wiskostatin induces folding of the isolated, unstructured GBD into its autoinhibited conformation, suggesting that wiskostatin functions by stabilizing N-WASP in its autoinhibited state. The use of small molecules to bias conformational equilibria represents a potentially general strategy for chemical inhibition of autoinhibited proteins, even in cases where such sites have not been naturally evolved in a target.

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Figure 1: Structure and regulation of the Wiskott-Aldrich syndrome protein family.
Figure 2: Structure and potency of wiskostatin and derivatives.
Figure 3: Wiskostatin targets N-WASP.
Figure 4: The solution structure of wiskostatin bound to mini-WASP.
Figure 5: Wiskostatin stabilizes the autoinhibited fold of the WASP GBD.

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References

  1. DeDecker, B.S. Allosteric drugs: thinking outside the active-site box. Chem. Biol. 7, R103–R107 (2000).

    Article  CAS  Google Scholar 

  2. Specht, K.M. & Shokat, K.M. The emerging power of chemical genetics. Curr. Opin. Cell Biol. 14, 155–159 (2002).

    Article  CAS  Google Scholar 

  3. Higgs, H.N. & Pollard, T.D. Activation by Cdc42 and PIP(2) of Wiskott-Aldrich syndrome protein (WASP) stimulates actin nucleation by Arp2/3 complex. J. Cell Biol. 150, 1311–1320 (2000).

    Article  CAS  Google Scholar 

  4. Ma, L., Rohatgi, R. & Kirschner, M.W. The Arp2/3 complex mediates actin polymerization induced by the small GTP-binding protein Cdc42. Proc. Natl. Acad. Sci. USA 95, 15362–15367 (1998).

    Article  CAS  Google Scholar 

  5. Ma, L., Cantley, L.C., Janmey, P.A. & Kirschner, M.W. Corequirement of specific phosphoinositides and small GTP-binding protein Cdc42 in inducing actin assembly in Xenopus egg extracts. J. Cell Biol. 140, 1125–1136 (1998).

    Article  CAS  Google Scholar 

  6. Peterson, J.R., Lokey, R.S., Mitchison, T.J. & Kirschner, M.W. A chemical inhibitor of N-WASP reveals a new mechanism for targeting protein interactions. Proc. Natl. Acad. Sci. USA 98, 10624–10629 (2001).

    Article  CAS  Google Scholar 

  7. Rohatgi, R. et al. The interaction between N-WASP and the Arp2/3 complex links Cdc42-dependent signals to actin assembly. Cell 97, 221–231 (1999).

    Article  CAS  Google Scholar 

  8. Marchand, J.B., Kaiser, D.A., Pollard, T.D. & Higgs, H.N. Interaction of WASP/Scar proteins with actin and vertebrate Arp2/3 complex. Nat. Cell Biol. 3, 76–82 (2001).

    Article  CAS  Google Scholar 

  9. Panchal, S.C., Kaiser, D.A., Torres, E., Pollard, T.D. & Rosen, M.K. A conserved amphipathic helix in WASP/Scar proteins is essential for activation of Arp2/3 complex. Nat. Struct. Biol. 10, 591–598 (2003).

    Article  CAS  Google Scholar 

  10. Rohatgi, R., Ho, H.Y. & Kirschner, M.W. Mechanism of N-WASP activation by CDC42 and phosphatidylinositol 4, 5-bisphosphate. J. Cell Biol. 150, 1299–1310 (2000).

    Article  CAS  Google Scholar 

  11. Kim, A.S., Kakalis, L.T., Abdul-Manan, N., Liu, G.A. & Rosen, M.K. Autoinhibition and activation mechanisms of the Wiskott-Aldrich syndrome protein. Nature 404, 151–158 (2000).

    Article  CAS  Google Scholar 

  12. Blanchoin, L. et al. Direct observation of dendritic actin filament networks nucleated by Arp2/3 complex and WASP/Scar proteins. Nature 404, 1007–1011 (2000).

    Article  CAS  Google Scholar 

  13. Mullins, R.D. & Machesky, L.M. Actin assembly mediated by Arp2/3 complex and WASP family proteins. Methods Enzymol. 325, 214–237 (2000).

    Article  CAS  Google Scholar 

  14. Torres, E. Biochemical Characterization of the Activation and Signal Integration Mechanisms of WASP/N-WASP. Thesis, Graduate School of Cornell Medical College (2003).

  15. Torres, E. & Rosen, M.K. Contingent phosphorylation/dephosphorylation provides a mechanism of molecular memory in WASP. Mol. Cell 11, 1215–1227 (2003).

    Article  CAS  Google Scholar 

  16. Caron, E. Regulation of Wiskott-Aldrich syndrome protein and related molecules. Curr. Opin. Cell Biol. 14, 82–87 (2002).

    Article  CAS  Google Scholar 

  17. Buck, M., Xu, W. & Rosen, M.K. Global disruption of the WASP autoinhibited structure on Cdc42 binding. Ligand displacement as a novel method for monitoring amide hydrogen exchange. Biochemistry 40, 14115–14122 (2001).

    Article  CAS  Google Scholar 

  18. Buck, M., Xu, W. & Rosen, M.K. A two-state allosteric model for autoinhibition rationalizes WASP signal integration and targeting. J. Mol. Biol. 338, 271–285 (2004).

    Article  CAS  Google Scholar 

  19. Panchal, S.C., Kaiser, D.A., Torres, E., Pollard, T.D. & Rosen, M.K. A conserved amphipathic helix in WASP/Scar proteins is essential for activation of Arp2/3 complex. Nat. Struct. Biol. 10, 591–598 (2003).

    Article  CAS  Google Scholar 

  20. Prehoda, K.E., Scott, J.A., Mullins, R.D. & Lim, W.A. Integration of multiple signals through cooperative regulation of the N-WASP-Arp2/3 complex. Science 290, 801–806 (2000).

    Article  CAS  Google Scholar 

  21. Rohatgi, R., Nollau, P., Ho, H.Y., Kirschner, M.W. & Mayer, B.J. Nck and phosphatidylinositol 4,5-bisphosphate synergistically activate actin polymerization through the N-WASP-Arp2/3 pathway. J. Biol. Chem. 276, 26448–26452 (2001).

    Article  CAS  Google Scholar 

  22. Fukuoka, M. et al. A novel neural Wiskott-Aldrich syndrome protein (N-WASP) binding protein, WISH, induces Arp2/3 complex activation independent of Cdc42. J. Cell Biol. 152, 471–482 (2001).

    Article  CAS  Google Scholar 

  23. Carlier, M.F. et al. GRB2 links signaling to actin assembly by enhancing interaction of neural Wiskott-Aldrich syndrome protein (N-WASP) with actin-related protein (ARP2/3) complex. J. Biol. Chem. 275, 21946–21952 (2000).

    Article  CAS  Google Scholar 

  24. Cory, G.O., Cramer, R., Blanchoin, L. & Ridley, A.J. Phosphorylation of the WASP-VCA domain increases its affinity for the Arp2/3 complex and enhances actin polymerization by WASP. Mol. Cell 11, 1229–1239 (2003).

    Article  CAS  Google Scholar 

  25. Suetsugu, S. et al. Sustained activation of N-WASP through phosphorylation is essential for neurite extension. Dev. Cell 3, 645–658 (2002).

    Article  CAS  Google Scholar 

  26. Prehoda, K.E. & Lim, W.A. How signaling proteins integrate multiple inputs: a comparison of N-WASP and Cdk2. Curr. Opin. Cell Biol. 14, 149–154 (2002).

    Article  CAS  Google Scholar 

  27. Schlessinger, J. Signal transduction. Autoinhibition control. Science 300, 750–752 (2003).

    Article  CAS  Google Scholar 

  28. Pufall, M.A. & Graves, B.J. Autoinhibitory domains: modular effectors of cellular regulation. Annu. Rev. Cell Dev. Biol. 18, 421–462 (2002).

    Article  CAS  Google Scholar 

  29. Schindler, T. et al. Structural mechanism for STI–571 inhibition of abelson tyrosine kinase. Science 289, 1938–1942 (2000).

    Article  CAS  Google Scholar 

  30. Peterson, J.R. & Golemis, E.A. Autoinhibited proteins as promising drug targets. J. Cell. Biochem. (in the press).

  31. Hammarstrom, P., Wiseman, R.L., Powers, E.T. & Kelly, J.W. Prevention of transthyretin amyloid disease by changing protein misfolding energetics. Science 299, 713–716 (2003).

    Article  Google Scholar 

  32. Foster, B.A., Coffey, H.A., Morin, M.J. & Rastinejad, F. Pharmacological rescue of mutant p53 conformation and function. Science 286, 2507–2510 (1999).

    Article  CAS  Google Scholar 

  33. Yamazaki, T., Lee, W. & Arrowsmith, C.H. A suite of triple resonance NMR experiments for the backbone assignment of 15N, 13C, 2H labeled proteins with high sensitivity. J. Am. Chem. Soc. 116, 11655–11666 (1994).

    Article  CAS  Google Scholar 

  34. Kay, L.E., Xu, G.Y., Singer, A.U., Muhandiram, D.R. & Formankay, J.D. A gradient-enhanced HCCH-TOCSY experiment for recording side-chain proton and carbon-13 correlations in water samples of proteins. J. Magn. Reson. Ser. B 101, 333–337 (1993).

    Article  CAS  Google Scholar 

  35. Cornilescu, G., Delaglio, F. & Bax, A. Protein backbone angle restraints from searching a database for chemical shift and sequence homology. J. Biomol. NMR 13, 289–302 (1999).

    Article  CAS  Google Scholar 

  36. Nilges, M., Macias, M.J., O'Donoghue, S.I. & Oschkinat, H. Automated NOESY interpretation with ambiguous distance restraints: the refined NMR solution structure of the pleckstrin homology domain from beta-spectrin. J. Mol. Biol. 269, 408–422 (1997).

    Article  CAS  Google Scholar 

  37. Brünger, A.T. X-PLOR: A System for X-ray Crystallography and NMR (Yale Univ. Press, New Haven, 1992).

    Google Scholar 

  38. Laskowski, R.A., Rullman, J.A., MacArthur, M.W., Kaptein, R. & Thornton, J.M. AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J. Biomol. NMR 8, 477–486 (1996).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank N. Ayad, S. Eden and G. Hoffmann for critical reading of the manuscript and A. Majumdar for assistance with NMR spectra. This work was supported by grants from the US National Institutes of Health (J.R.P., GM197000; L.C.B., GM07739; M.K.R., GM56322), the Welch Foundation (I–1544, M.K.R.) and the Cancer Research Institute (D.M.).

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Author notes

  1. Jeffrey R Peterson

    Present address: Tumor Cell Biology, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, Pennsylvania, 19111, USA

  2. Jeffrey R Peterson and Lincoln C Bickford: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Cell Biology and Department of Systems Biology, Harvard Medical School, 240 Longwood Avenue, Boston, 02115, Massachusetts, USA

    Jeffrey R Peterson & Marc W Kirschner

  2. Structural Biology, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, 10021, New York, USA

    Lincoln C Bickford & Ouathek Ouerfelli

  3. Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, 75390, Texas, USA

    David Morgan & Michael K Rosen

  4. Department of Pathology and Laboratory Medicine, Hospital of the University of Pennsylvania, 3400 Spruce Street, 6 Founders Pavilion, Philadelphia, 19104, Pennsylvania, USA

    Annette S Kim

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Correspondence to Marc W Kirschner or Michael K Rosen.

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Peterson, J., Bickford, L., Morgan, D. et al. Chemical inhibition of N-WASP by stabilization of a native autoinhibited conformation. Nat Struct Mol Biol 11, 747–755 (2004). https://doi.org/10.1038/nsmb796

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