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.

Autoinhibition and activation mechanisms of the Wiskott–Aldrich syndrome protein

Abstract

The Rho-family GTPase, Cdc42, can regulate the actin cytoskeleton through activation of Wiskott–Aldrich syndrome protein (WASP) family members. Activation relieves an autoinhibitory contact between the GTPase-binding domain and the carboxy-terminal region of WASP proteins. Here we report the autoinhibited structure of the GTPase-binding domain of WASP, which can be induced by the C-terminal region or by organic co-solvents. In the autoinhibited complex, intramolecular interactions with the GTPase-binding domain occlude residues of the C terminus that regulate the Arp2/3 actin-nucleating complex. Binding of Cdc42 to the GTPase-binding domain causes a dramatic conformational change, resulting in disruption of the hydrophobic core and release of the C terminus, enabling its interaction with the actin regulatory machinery. These data show that ‘intrinsically unstructured’ peptides such as the GTPase-binding domain of WASP can be induced into distinct structural and functional states depending on context.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Amino-acid sequences of human WASP and N-WASP with consensus secondary structure of the GBD constructs of WASP shown above.
Figure 2: Biochemical characterization of the WASP GBD.
Figure 3: Stereoviews of the best-fit superpositions of the backbone (N, Ca, C) atoms of the 20 final NMR structures of GBD2 in 5% TFE (a) and GBD4–C (b).
Figure 4: Autoinhibited and activated structures of the WASP GBD.
Figure 5: Steric incompatibility of the autoinhibited and activated structures.

References

  1. 1

    Bourne, H. R., Sanders, D. A. & McCormick, F. The GTPase superfamily: a conserved switch for diverse cell functions. Nature 348, 125– 131 (1990).

    ADS  CAS  Article  Google Scholar 

  2. 2

    Hall, A. Rho GTPases and the actin cytoskeleton. Science 279, 509–514 (1998).

    ADS  CAS  Article  Google Scholar 

  3. 3

    Machesky, L. M. & Insall, R. H. Signaling to actin dynamics. J. Cell Biol. 146, 267– 272 (1999).

    CAS  Article  Google Scholar 

  4. 4

    Derry, J. M. J., Ochs, H. D. & Francke, U. Isolation of a novel gene mutated in the Wiskott–Aldrich syndrome. Cell 78, 635– 644 (1994).

    CAS  Article  Google Scholar 

  5. 5

    Burbelo, P. D., Drechsel, D. & Hall, A. A conserved binding motif defines numerous candidate target proteins for both Cdc42 and Rac GTPases. J. Biol. Chem. 270, 29071–29074 ( 1995).

    CAS  Article  Google Scholar 

  6. 6

    Abdul-Manan, N. et al. Structure of Cdc42 in complex with the GTPase binding domain of the Wiskott–Aldrich syndrome protein. Nature 399, 379–383 (1999).

    ADS  CAS  Article  Google Scholar 

  7. 7

    Miki, H. et al. Induction of filopodium formation by a WASP-related actin-depolymerizing protein N-WASP. Nature 391, 93– 96 (1998).

    ADS  CAS  Article  Google Scholar 

  8. 8

    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).

    CAS  Article  Google Scholar 

  9. 9

    Rudolph, M. et al. The Cdc42/Rac interactive binding region motif of the Wiskott Aldrich syndrome protein (WASP) is necessary but not sufficient for tight binding to Cdc42 and structure formation. J. Biol. Chem. 273, 18067–18076 (1998).

    CAS  Article  Google Scholar 

  10. 10

    Buck, M. Trifluoroethanol and colleagues: cosolvents come of age. Recent studies with peptides and proteins. Quart. Rev. Biophys. 31, 297–355 (1998).

    CAS  Article  Google Scholar 

  11. 11

    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).

    CAS  Article  Google Scholar 

  12. 12

    Rohl, C. A., Chakrabartty, A. & Baldwin, R. L. Helix propagation and N-cap propensities of the amino acids measured in alanine-based peptides in 40 volume precent trifluoroethanol. Protein Sci. 5, 2623–2637 (1996).

    CAS  Article  Google Scholar 

  13. 13

    Machesky, L. M. et al. Scar, a WASp-related protein, activates nucleation of actin filaments by the Arp2/3 complex. Proc. Natl Acad. Sci. USA 96, 3739–3744 (1999).

    ADS  CAS  Article  Google Scholar 

  14. 14

    Winter, D., Lechler, T. & Li, R. Activation of the yeast Arp2/3 complex by Bee1p, a WASP-family protein. Curr. Biol. 9, 501–504 ( 1999).

    CAS  Article  Google Scholar 

  15. 15

    Yarar, D. et al. The Wiskott–Aldrich syndrome protein directs actin-based motility by stimulating actin nucleation with the Arp2/3 complex. Curr. Biol. 9, 555–558 ( 1999).

    CAS  Article  Google Scholar 

  16. 16

    Bi, E. & Zigmond, S. H. Actin polymerization: where the WASP stings. Curr. Biol. 9, R160– R163 (1999).

    CAS  Article  Google Scholar 

  17. 17

    Sharman, G. J. & Searle, M. S. Cooperative interaction between the three strands of a designed antiparallel β-sheet. J. Am. Chem. Soc. 120, 5291– 5300 (1998).

    CAS  Article  Google Scholar 

  18. 18

    Oda, A. et al. Collagen induces tyrosine phosphorylation of Wiskott–Aldrich syndrome protein in human platelets. Blood 92, 1852–1858 (1998).

    CAS  PubMed  Google Scholar 

  19. 19

    Baba, Y. et al. Involvement of Wiskott–Aldrich syndrome protein in B-cell cytoplasmic tyrosine kinase pathway. Blood 93, 2003–2012 (1999).

    CAS  Google Scholar 

  20. 20

    Guinamard, R. et al. Tyrosine phosphorylation of the Wiskott–Aldrich syndrome protein by Lyn and Btk regulated by CDC42. FEBS Lett. 434, 431–436 (1998).

    CAS  Article  Google Scholar 

  21. 21

    Kwan, S. et al. Scanning of the Wiskott–Aldrich syndrome (WAS) gene: identification of 18 novel alternations including a possible mutation hotspot at ARG86 resulting in thrombocytopenia, a mild WAS phenotype. Hum. Mol. Genet. 4, 1995–1998 (1995).

    CAS  Article  Google Scholar 

  22. 22

    Villa, A. et al. X-Linked thrombocytopenia and Wiskott–Aldrich syndrome are allelic diseases with mutations in the WASP gene. Nature Genet. 9, 414–417 ( 1995).

    CAS  Article  Google Scholar 

  23. 23

    Shcherbina, A., Rosen, F. S. & Remold-O'Donnell, E. WASP levels in platelets and lymphocytes of Wiskott–Aldrich syndrome patients correlate with cell dysfunction. J. Immunol. 163, 6314–6320 ( 1999).

    CAS  PubMed  Google Scholar 

  24. 24

    Derry, J. M. J. et al. WASP gene mutations in Wiskott–Aldrich syndrome and X-linked thrombocytopenia. Hum. Mol. Genet. 4, 1127–1135 (1995).

    CAS  Article  Google Scholar 

  25. 25

    Kolluri, R. et al. Identification of WASP mutations in patients with Wiskott–Aldrich syndrome and isolated thrombocytopenia reveals allelic heterogeneity at the WAS locus. Hum. Mol. Genet. 4, 1119– 1126 (1995).

    CAS  Article  Google Scholar 

  26. 26

    Block, C. et al. Quantitative structure-activity analysis correlation Ras/Raf interaction in vitro to Raf activation in vivo. Nature Struct. Biol. 3, 244–251 ( 1996).

    CAS  Article  Google Scholar 

  27. 27

    Campbell, S. et al. Increasing complexity of Ras signaling. Oncogene 17, 1395–1413 ( 1998).

    CAS  Article  Google Scholar 

  28. 28

    Zhao, Z. S. et al. A conserved negative regulatory region in alphaPAK: inhibition of PAK kinases reveals their morphological roles downstream of Cdc42 and Rac1. Mol. Cell. Biol. 18, 2153– 2163 (1998).

    CAS  Article  Google Scholar 

  29. 29

    Manser, E. et al. A brain serine/threonine protein kinase activated by Cdc42 and Rac1. Nature 367, 40– 46 (1994).

    ADS  CAS  Article  Google Scholar 

  30. 30

    Stokoe, D. et al. Activation of Raf as a result of recruitment to the plasma membrane. Science 264, 1463– 1467 (1994).

    ADS  CAS  Article  Google Scholar 

  31. 31

    Spolar, R. S. & Record, M. T. Jr Coupling of local folding to site-specific binding of proteins to DNA. Science 263, 777–784 (1994).

    ADS  CAS  Article  Google Scholar 

  32. 32

    Wright, P. E. & Dyson, H. J. Intrinsically unstructured proteins: re-assessing the protein structure- function paradigm. J. Mol. Biol. 293, 321–331 ( 1999).

    CAS  Article  Google Scholar 

  33. 33

    Yamazaki, T. et al. 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).

    CAS  Article  Google Scholar 

  34. 34

    Logan, T. M. et al. A general method for assigning NMR spectra of denatured proteins using 3D HC(CO)NH-TOCSY triple resonance experiments. J. Biomol. NMR 3, 225–231 ( 1993).

    CAS  Article  Google Scholar 

  35. 35

    Kay, L. E. et al. 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).

    ADS  CAS  Article  Google Scholar 

  36. 36

    Kuboniwa, H. et al. Measurement of HN-H alpha J couplings in calcium-free calmodulin using new 2D and 3D water-flip-back methods. J. Biomol. NMR 4, 871–878 (1994).

    CAS  Article  Google Scholar 

  37. 37

    Neri, D. et al. Stereospecific nuclear magnetic resonance assignments of the methyl groups of valine and leucine in the DNA-binding domain of the 434 repressor by biosynthetically directed fractional 13C labeling. Biochemistry 28, 7510–7516 (1989).

    CAS  Article  Google Scholar 

  38. 38

    Farrow, N. A. et al. Backbone dynamics of a free and phosphopeptide-complexed Src homology 2 domain studied by 15N NMR relaxation. Biochemistry 33, 5984–6003 (1994).

    CAS  Article  Google Scholar 

  39. 39

    Brunger, A. T. et al. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998).

    CAS  Article  Google Scholar 

  40. 40

    Nilges, M. et al. 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).

    CAS  Article  Google Scholar 

  41. 41

    Brünger, A. T. X-PLOR manual. (Yale Univ. Press, New Haven, 1993).

    Google Scholar 

  42. 42

    Laskowski, R. A. et al. AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J. Biomol. NMR 8, 477–486 (1996).

    CAS  Article  Google Scholar 

  43. 43

    Carson, M. J. Ribbons 2. 0. J. Appl. Crystallogr. 24, 958–961 (1991).

    Article  Google Scholar 

  44. 44

    Nicholls, A., Sharp, K. A. & Honig, B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins Struct. Funct. Genet. 11, 281–296 (1991).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank M. Lu for analytical ultracentrifugation experiments; W. Hu for communicating results before publication; M. Buck, Y.M. Chook, J. Goldberg, B. Schulman, B. W. Trotter and M. R. Wood for discussion and critical reading of the manuscript; R. Cerione and K. Siminovitch for providing cDNAs for Cdc42 and WASP, respectively; L. Kay for providing many of the NMR pulse sequences used in this study; F. Delaglio for providing nmrPipe and TALOS software; A. Majumdar, W. Hu and B. Aghazadeh for assistance in NMR data acquisition; J. Hubbard for computer system support and M. Fiore for expert administrative assistance. A.S.K. is supported by a fellowship from the Damon Runyon-Walter Winchell Foundation, and L.T.K. by the Charles H. Revson Foundation. M.K.R. acknowledges support from the NIH (PECASE program), Arnold and Mabel Beckman Foundation, and Sidney Kimmel Foundation for Cancer Research.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Michael K. Rosen.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Kim, A., Kakalis, L., Abdul-Manan, N. et al. Autoinhibition and activation mechanisms of the Wiskott–Aldrich syndrome protein. Nature 404, 151–158 (2000). https://doi.org/10.1038/35004513

Download citation

Further reading

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