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:

Molecular mechanism of NPF recognition by EH domains

Nature Structural Biology volume 7, pages 1018–1022 (2000)Cite this article

Abstract

Eps15 homology (EH) domains are protein interaction modules that recognize Asn-Pro-Phe (NPF) motifs in their biological ligands to mediate critical events during endocytosis and signal transduction. To elucidate the structural basis of the EH–NPF interaction, the solution structures of two EH–NPF complexes were solved using NMR spectroscopy. The first complex contains a peptide representing the Hrb C-terminal NPFL motif; the second contains a peptide in which an Arg residue substitutes the C-terminal Leu. The NPF residues are almost completely embedded in a hydrophobic pocket on the EH domain surface and the backbone of NPFX adopts a conformation reminiscent of the Asx-Pro type I β-turn motif. The residue directly following NPF is crucial for recognition and is required to complete the β-turn. Five amino acids on the EH surface mediate specific recognition of this residue through hydrophobic and electrostatic contacts. The complexes explain the selectivity of the second EH domain of Eps15 for NPF over DPF motifs and reveal a critical aromatic interaction that provides a conserved anchor for the recognition of FW, WW, SWG and HTF ligands by other EH domains.

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: Structures of EH2 in complex with NPFHrb and with NPFHrb(L→R).
Figure 2: Intermolecular NOEs between EH2 and NPFHrb(L→R).
Figure 3: Residues of the EH domain interacting with NPFHrb(L→R).

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

References

  1. Santolini, E., Salcini, A.E., Kay, B.K., Yamabhai, M. & Di Fiore, P.P. Exp. Cell Res. 253 , 186–209 (1999).

    Article  CAS  Google Scholar 

  2. Yamabhai, M., et al. J. Biol. Chem. 273, 31401– 31407 (1998).

    Article  CAS  Google Scholar 

  3. Chen, H. et al. Nature 394, 793–797 (1998).

    Article  CAS  Google Scholar 

  4. Haffner, C. et al. FEBS Lett. 419, 175– 180 (1997).

    Article  CAS  Google Scholar 

  5. Wendland, B. & Emr, S.D. J. Cell Biol. 141, 71–84 (1998).

    Article  CAS  Google Scholar 

  6. Salcini, A.E. et al. Genes Dev. 11, 2239– 2249 (1997).

    Article  CAS  Google Scholar 

  7. Doria, M. et al. J. Cell Biol. 147, 1379– 1384 (1999).

    Article  CAS  Google Scholar 

  8. de Beer, T., Carter, R.E., Lobel-Rice, K.E., Sorkin, A. & Overduin, M. Science 281, 1357–1360 (1998).

    Article  CAS  Google Scholar 

  9. Whitehead, B., Tessari, M., Carotenuto, A., van Bergen en Henegouwen, P.M. & Vuister, G.W. Biochemistry 38, 11271–11277 ( 1999).

    Article  CAS  Google Scholar 

  10. Enmon, J.L., de Beer, T. & Overduin, M. Biochemistry 39, 4309– 4319 (2000).

    Article  CAS  Google Scholar 

  11. Paoluzi, S. et al. EMBO J. 17, 6541–6550 (1998).

    Article  CAS  Google Scholar 

  12. Wilson, D.R. & Finlay, B.B. Protein Eng. 10, 519–529 (1997).

    Article  CAS  Google Scholar 

  13. Cupers, P., ter Haar, E., Boll, W. & Kirchhausen, T. J. Biol. Chem. 272, 33430–33434 ( 1997).

    Article  CAS  Google Scholar 

  14. Nakashima, S. et al. EMBO J. 18, 3629–3642 (1999).

    Article  CAS  Google Scholar 

  15. Fazioli, F., Minichiello, L., Matoskova, B., Wong, W.T. & Di Fiore, P.P. Mol. Cell Biol. 13 , 5814–5828 (1993).

    Article  CAS  Google Scholar 

  16. Aue, W.P., Bartholdi, E. & Ernst, R.R. J. Chem. Phys. 64, 2229– 2246 (1976).

    Article  CAS  Google Scholar 

  17. Braunschweiler, L. & Ernst, R.R. J. Magn. Reson. 53, 521–528 ( 1983).

    CAS  Google Scholar 

  18. Griesinger, C., Otting, G., Wüthrich, K. & Ernst, R.R. J. Am. Chem. Soc. 110, 7870–7872 (1988).

    Article  CAS  Google Scholar 

  19. Bax, A. & Davis, D.G. J. Magn. Reson. 63, 207–213 (1985).

    CAS  Google Scholar 

  20. Medvedeva, S., Simorre, J.-P., Brutscher, B., Guerlesquin, F. & Marion, D. FEBS Lett. 333, 251–256 (1993).

    Article  CAS  Google Scholar 

  21. Bodenhausen, G. & Ruben, D.J. Chem. Phys. Lett. 69, 185–189 ( 1980).

    Article  CAS  Google Scholar 

  22. Kay, L.E., Keifer, P. & Saarinen, T. J. Am. Chem. Soc. 114, 10663– 10665 (1992).

    Article  CAS  Google Scholar 

  23. Jeener, J., Meier, H., Bachmann, P. & Ernst, R.R. J. Chem. Phys. 71, 4546–4553 ( 1979).

    Article  CAS  Google Scholar 

  24. Zhang, O., Kay, L.E., Olivier, J.P. & Forman-Kay, J.D. J. Biomol. NMR 4, 845–858 (1994).

    Article  CAS  Google Scholar 

  25. Delaglio, F. et al. J. Biomol. NMR 6, 277– 293 (1995).

    Article  CAS  Google Scholar 

  26. Garrett, D.S, Powers, R., Gronenborn, A., and Clore, G.M. J. Magn Reson. 95, 214–220 (1991).

    CAS  Google Scholar 

  27. Fletcher, C.M., Jones, D.N.M., Diamond, R. & Neuhaus, D. J. Biomol. NMR 8, 292–310 (1996).

    Article  CAS  Google Scholar 

  28. Brünger, A.T, X-PLOR, version 3.1. (Yale University Press, New Haven, Connecticut; 1992).

    Google Scholar 

  29. Laskowski, R.A., Rullman, J.A., MacArthur, M.W., Kaptein, R. & Thornton, J.M. J. Biomol. NMR 8, 477–486 ( 1996).

    Article  CAS  Google Scholar 

  30. Humphrey, W.F., Dalke, A. & Schulten, K. J. Mol. Graphics 14, 33–38 (1996).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank J. Mamay for computational support and for software to manipulate VMD images and A. Sorkin, and D.N.M. Jones for insightful discussions. T.B. is a recipient of a National Institutes of Health NSRA Fellowship. A.N.H. is a recipient of an NIH training grant fellowship. Grant support to M.O. was provided by the National Institutes of Health and the Pew Scholar's program, and the NMR center is supported by the Howard Hughes Medical Institute.

Author information

Author notes

  1. Montarop Yamabhai

    Present address: Suranaree University of Technology, Nakon Ratchasima, Thailand

Authors and Affiliations

  1. Department of Pharmacology, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, 80262, Colorado, USA

    Tonny de Beer, Rebecca C. Bowers & Michael Overduin

  2. Department of Chemistry and Biochemistry, University of Colorado, Boulder, 80239, Colorado, USA

    Andrew N. Hoofnagle & Jennifer L. Enmon

  3. Department of Pharmacology, University of Wisconsin, Madison, 53706, Wisconsin, USA

    Montarop Yamabhai & Brian K. Kay

Authors
  1. Tonny de Beer
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Andrew N. Hoofnagle
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jennifer L. Enmon
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Rebecca C. Bowers
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Montarop Yamabhai
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Brian K. Kay
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Michael Overduin
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Tonny de Beer.

Rights and permissions

Reprints and permissions

About this article

Cite this article

de Beer, T., Hoofnagle, A., Enmon, J. et al. Molecular mechanism of NPF recognition by EH domains. Nat Struct Mol Biol 7, 1018–1022 (2000). https://doi.org/10.1038/80924

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/80924

This article is cited by

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