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:

C-terminal binding domain of Rho GDP-dissociation inhibitor directs N-terminal inhibitory peptide to GTPases

Nature volume 387, pages 814–819 (1997)Cite this article

Abstract

The Rho GDP-dissociation inhibitors (GDIs) negatively regulate Rho-family GTPases1,2. The inhibitory activity of GDI derives both from an ability to bind the carboxy-terminal isoprene of Rho family members and extract them from membranes3,4, and from inhibition of GTPase cycling between the GTP- and GDP-bound states4,5. Here we demonstrate that these binding and inhibitory functions of rhoGDI can be attributed to two structurally distinct regions of the protein. A carboxy-terminal folded domain of relative molecular mass 16,000 (Mr 16K) binds strongly to the Rho-family member Cdc42, yet has little effect on the rate of nucleotide dissociation from the GTPase. The solution structure of this domain shows a β-sandwich motif with a narrow hydrophobic cleft that binds isoprenes, and an exposed surface that interacts with the protein portion of Cdc42. The amino-terminal region of rhoGDI is unstructured in the absence of target and contributes little to binding, but is necessary to inhibit nucleotide dissociation from Cdc42. These results lead to a model of rhoGDI function in which the carboxy-terminal binding domain targets the amino-terminal inhibitory region to GTPases, resulting in membrane extraction and inhibition of nucleotide cycling.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: a, b, 1H/15N HSQC spectra of GDIΔ22 (a) and GDIΔ59(b).
Figure 2: a, b, 1H/15N HSQC spectra of GDIΔ22 (a) and GDIΔ59(b).
Figure 3: a, b, 1H/15N HSQC spectra of GDIΔ22 (a) and GDIΔ59(b).
Figure 4: a, Normalized change in fluorescence (F) of the (Mant-GDP)–Cdc42 complex on addition of fluorescein-conjugate.
Figure 5: a, Normalized change in fluorescence (F) of the (Mant-GDP)–Cdc42 complex on addition of fluorescein-conjugate.
Figure 6: a, Backbone overlay (residues 69–202) of the 20 final NMR structures of GDIΔ59.
Figure 7: a, Backbone overlay (residues 69–202) of the 20 final NMR structures of GDIΔ59.
Figure 8: a, Backbone overlay (residues 69–202) of the 20 final NMR structures of GDIΔ59.
Figure 9: a, Backbone overlay (residues 69–202) of the 20 final NMR structures of GDIΔ59.

Similar content being viewed by others

References

  1. Ueda, T., Kikuchi, A., Ohga, N., Yamamoto, J. & Takai, Y. Purification and characterization from bovine brain cytosol of a novel regulatory protein inhibiting the dissociation of GDP from and the subsequent binding of GTP to rhoB p20, a ras p21-like GTP-binding protein. J. Biol. Chem. 265, 9373–9380 (1990).

    CAS  PubMed  Google Scholar 

  2. Fukumoto, Y. et al. Molecular cloning and characterization of a novel type of regulatory protein (GDI) for the rho proteins, ras p21-like small GTP-binding proteins. Oncogene 5, 1321–1328 (1990).

    CAS  Google Scholar 

  3. Isomura, M., Kikuchi, A., Ohga, N. & Takai, Y. Regulation of binding of RhoB p20 to membranes by its specific regulatory protein, GDP dissociation inhibitor. Oncogene 6, 119–124 (1991).

    CAS  PubMed  Google Scholar 

  4. Bokoch, G. M. & Der, C. J. Emerging concepts in the Ras superfamily of GTP-binding proteins. FASEB J. 7, 750–759 (1993).

    Article  CAS  Google Scholar 

  5. Hancock, J. F. & Hall, A. Anovel role for RhoGDI as an inhibitor of GAP proteins. EMBO J. 12, 1915–1921 (1993).

    Article  CAS  Google Scholar 

  6. Platko, J. V. et al. Asingle residue can modify target-binding affinity and activity of the functional domain of the Rho-subfamily GDP dissociation inhibitors. Proc. Natl Acad. Sci. USA 92, 2974–2978 (1995).

    Article  ADS  CAS  Google Scholar 

  7. Kay, L. E. Field gradient techniques in NMR spectroscopy. Curr. Opin. Struct. Biol. 5, 674–681 (1995).

    Article  CAS  Google Scholar 

  8. Schalk, I. et al. Structure and mutational analysis of Rab GDP-dissociation inhibitor. Nature 381, 42–48 (1996).

    Article  ADS  CAS  Google Scholar 

  9. Zalcman, G. et al. RhoGDI-3 is a new GDP dissociation inhibitor (GDI). J. Biol. Chem. 271, 30366–30374 (1996).

    Article  CAS  Google Scholar 

  10. Storch, J. Diversity of fatty acid-binding protein structure and function: studies with fluorescent ligands. Mol. Cell. Biochem. 123, 45–53 (1993).

    Article  CAS  Google Scholar 

  11. Hoffmann, R. W. Allylic 1,3-strain as a controlling factor in stereoselective transformations. Chem. Rev. 89, 1841–1860 (1989).

    Article  CAS  Google Scholar 

  12. Wiberg, K. B. & Murko, M. A. Rotational barriers. 2. Energies of alkane rotamers. An examination of gauche interactions. J. Am. Chem. Soc. 110, 8029–8038 (1988).

    Article  CAS  Google Scholar 

  13. Silvius, J. R. & l'Heureux, F. Fluorimetric evaluation of the affinities of isoprenylated peptides for lipid bilayers. Biochemistry 33, 3014–3022 (1994).

    Article  CAS  Google Scholar 

  14. Philips, M. R. et al. Carboxyl methylation of Ras-related proteins during signal transduction in neutrophils. Science 259, 977–980 (1993).

    Article  ADS  CAS  Google Scholar 

  15. Ghomashchi, R., Zhang, X., Liu, L. & Gelb, M. H. Binding of prenylated and polybasic peptides to membranes: affinities and intervesicle exchange. Biochemistry 34, 11910–11918 (1995).

    Article  CAS  Google Scholar 

  16. Na, S. et al. D4-GDI, a substate of CPP32, is proteolyzed during Fas-induced apoptosis. J. Biol. Chem. 271, 11209–11213 (1996).

    Article  CAS  Google Scholar 

  17. Danley, D. E., Chuang, T.-H. & Bokach, G. M. Defective Rho GTPase regulation by IL-1β-converting enzyme-mediated cleavage of D4 GDP dissociation inhibitor. J. Immunol. 157, 500–503 (1996).

    CAS  PubMed  Google Scholar 

  18. Nomanbhoy, T. K. & Cerione, R. A. Characterization of the interaction between RhoGDI and Cdc42Hs using fluorescence spectroscopy. J. Biol. Chem. 271, 10004–10009 (1996).

    Article  CAS  Google Scholar 

  19. Wu, J. W., Leonard, D. A., Cerione, R. A. & Manor, D. Interaction between Cdc42Hs and Rho-GDI is mediated through the Rho-insert region. J. Biol. Chem.(in the press).

  20. Clore, G. M. & Gronenborn, A. M. Multidimensional heteronuclear nuclear magnetic resonance of proteins. Methods Enzymol. 239, 349–363 (1994).

    Article  CAS  Google Scholar 

  21. Edison, A. S., Abildgaard, F., Westler, W. M., Mooberry, E. S. & Markley, J. L. Practical introduction to theory and implementation of multinuclear, multidimensional nuclear magnetic resonance experiments. Methods Enzymol. 239, 3–79 (1994).

    Article  CAS  Google Scholar 

  22. Neri, D., Szyperski, T., Otting, G., Senn, H. & Wuthrich, K. 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).

    Article  CAS  Google Scholar 

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

    Google Scholar 

  24. Kuboniwa, H., Grzesiek, S., Delaglio, F. & Bax, A. Measurement of HN-Hα J couplings in calcium-free calmodulin using new 2D and 3D water-flip-back methods. J. Biomol. NMR 4, 871–878 (1994).

    Article  CAS  Google Scholar 

  25. Spera, S. & Bax, A. Empirical correlation between protein backbone conformation and Cα and Cβ 13C nuclear magnetic resonance chemical shifts. J. Am. Chem. Soc. 117, 5491–5495 (1991).

    Google Scholar 

  26. Nilges, M., Clore, G. M. & Gronenborn, A. M. 1H-NMR stereospecific assignments by conformational data-base searches. Biopolymers 29, 813–822 (1990).

    Article  CAS  Google Scholar 

  27. Laskowski, R. A., Rullman, J. A. C., 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, 477–496 (1996).

    Article  CAS  Google Scholar 

  28. Leonard, D. A., Evans, T., Hart, M., Cerione, R. A. & Manor, D. Investigation of the GTP-binding/GTPase cycle of Cdc42Hs using fluorescence spectroscopy. Biochemistry 33, 12323–12328 (1994).

    Article  CAS  Google Scholar 

  29. Carson, M. J. Ribbons 2.0. J. Appl. Crystallogr. 24, 958–961 (1991).

    Article  Google Scholar 

  30. Nichols, 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 ((1990)).

    Article  Google Scholar 

Download references

Acknowledgements

We thank J. Kelly for participating in the sequential assignment of GDIΔ59; L. E. Kay for discussion and for providing the NMR pulse sequences; B. A. Johnson and F. Delaglio for data analysis and processing software; D. Live for assistance with NMR data acquisition; J. Hubbard for computer system support and assistance with figure preparation; and Y. M. Chook and L. E. Kay for critical reading of the manuscript. This work was supported by a grant from the Society of the Memorial Sloan-Kettering Cancer Center.

Author information

Authors and Affiliations

  1. *Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, 10021, New York, USA

    Yuying Q. Gosser & Behzad Aghazadeh

  2. †Departments of Biochemistry, and Department of Pharmacology, Molecular and Cell Biology, Cornell University, Ithaca, 14853, New York, USA

    Tyzoon K. Nomanbhoy, Danny Manor, Carolyn Combs & Richard A. Cerione

  3. ‡Graduate Program in Physiology and Biophysics, Cornell University Graduate School of Medical Sciences, New York, 10021, New York, USA

    Behzad Aghazadeh

  4. Correspondence and requests for materials should be addressed to M.K.R.,

    Michael K. Rosen

Author notes

  1. Coordinatesn of the minimized average GDIΔ59 structure and of the finalensemble of 20 structures have been deposited in the Brookhaven Protein Data Bank (accession numbers 1gdf and 1ajw, respectively).

    Authors
    1. Yuying Q. Gosser
      View author publications

      You can also search for this author in PubMed Google Scholar

    2. Tyzoon K. Nomanbhoy
      View author publications

      You can also search for this author in PubMed Google Scholar

    3. Behzad Aghazadeh
      View author publications

      You can also search for this author in PubMed Google Scholar

    4. Danny Manor
      View author publications

      You can also search for this author in PubMed Google Scholar

    5. Carolyn Combs
      View author publications

      You can also search for this author in PubMed Google Scholar

    6. Richard A. Cerione
      View author publications

      You can also search for this author in PubMed Google Scholar

    7. Michael K. Rosen
      View author publications

      You can also search for this author in PubMed Google Scholar

    Corresponding author

    Correspondence to Michael K. Rosen.

    Rights and permissions

    Reprints and permissions

    About this article

    Cite this article

    Gosser, Y., Nomanbhoy, T., Aghazadeh, B. et al. C-terminal binding domain of Rho GDP-dissociation inhibitor directs N-terminal inhibitory peptide to GTPases. Nature 387, 814–819 (1997). https://doi.org/10.1038/42961

    Download citation

    • Received:

    • Accepted:

    • Issue Date:

    • DOI: https://doi.org/10.1038/42961

    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

    Advanced 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