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.

  • Article
  • Published:

Crystal structure and mutational analysis of a perlecan-binding fragment of nidogen-1

Nature Structural Biology volume 8, pages 634–640 (2001)Cite this article

Abstract

Nidogen, an invariant component of basement membranes, is a multifunctional protein that interacts with most other major basement membrane proteins. Here, we report the crystal structure of the mouse nidogen-1 G2 fragment, which contains binding sites for collagen IV and perlecan. The structure is composed of an EGF-like domain and an 11-stranded β-barrel with a central helix. The β-barrel domain has unexpected similarity to green fluorescent protein. A large surface patch on the β-barrel is strikingly conserved in all metazoan nidogens. Site-directed mutagenesis demonstrates that the conserved residues are involved in perlecan binding.

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: The nidogen G2 structure.
Figure 2: Comparison of the β-barrels of nidogen G2 and Aequora victoria GFP22.
Figure 3: Alignment of nidogen G2 sequences.
Figure 4: The conserved surface patch.

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

References

  1. Timpl, R. & Brown, J.C. Supramolecular assembly of basement membranes. Bioessays 18, 123–132 (1996).

    Article  CAS  Google Scholar 

  2. Erickson, A.C. & Couchman, J.R. Still more complexity in mammalian basement membranes. J. Histochem. Cytochem. 48, 1291–1306 (2000).

    Article  CAS  Google Scholar 

  3. Kimura, N., Toyoshima, T., Kojima, T. & Shimane, M. Entactin-2: a new member of basement membrane protein with high homology to entactin/nidogen. Exp. Cell Res. 241, 36–45 (1998).

    Article  CAS  Google Scholar 

  4. Kohfeldt, E., Sasaki, T., Göhring, W. & Timpl, R. Nidogen-2: a new basement membrane protein with diverse binding properties. J. Mol. Biol. 282, 99–109 (1998).

    Article  CAS  Google Scholar 

  5. Hutter, H. et al. Conservation and novelty in the evolution of cell adhesion and extracellular matrix genes. Science 287, 989–994 (2000).

    Article  CAS  Google Scholar 

  6. Rubin, G.M. et al. Comparative genomics of the eukaryotes. Science 287, 2204–2215 (2000).

    Article  CAS  Google Scholar 

  7. Durkin, M.E. et al. Amino acid sequence and domain structure of entactin. Homology with epidermal growth factor precursor and low density lipoprotein receptor. J. Cell Biol. 107, 2749–2756 (1988).

    Article  CAS  Google Scholar 

  8. Mann, K. et al. Amino acid sequence of mouse nidogen, a multidomain basement membrane protein with binding activity for laminin, collagen IV and cells. EMBO J. 8, 65–72 (1989).

    Article  CAS  Google Scholar 

  9. Fox, J.W. et al. Recombinant nidogen consists of three globular domains and mediates binding of laminin to collagen IV. EMBO J. 10, 3137–3146 (1991).

    Article  CAS  Google Scholar 

  10. Reinhardt, D. et al. Mapping of nidogen binding sites for collagen type IV, heparan sulfate proteoglycan, and zinc. J. Biol. Chem. 268, 10881–10887 (1993).

    CAS  PubMed  Google Scholar 

  11. Ekblom, P. et al. Role of mesenchymal nidogen for epithelial morphogenesis in vitro. Development 120, 2003–2014 (1994).

    CAS  PubMed  Google Scholar 

  12. Kadoya, Y. et al. Importance of nidogen binding to laminin γ1 for branching epithelial morphogenesis of the submandibular gland. Development 124, 683–691 (1997).

    CAS  PubMed  Google Scholar 

  13. Murshed, M. et al. The absence of nidogen 1 does not affect murine basement membrane formation. Mol. Cell. Biol. 20, 7007–7012 (2000).

    Article  CAS  Google Scholar 

  14. Kim, S. & Wadsworth, W.G. Positioning of longitudinal nerves in C. elegans by nidogen. Science 288, 150–154 (2000).

    Article  CAS  Google Scholar 

  15. Kang, S.H. & Kramer, J.M. Nidogen is nonessential and not required for normal type IV collagen localization in Caenorhabditis elegans. Mol. Biol. Cell 11, 3911–3923 (2000).

    Article  CAS  Google Scholar 

  16. Mayer, U., Kohfeldt, E. & Timpl, R. Structural and genetic analysis of the laminin–nidogen interaction. Ann. N.Y. Acad. Sci. 857, 130–142 (1998).

    Article  CAS  Google Scholar 

  17. Pöschl, E. et al. Site-directed mutagenesis and structural interpretation of the nidogen binding site of the laminin γ1 chain. EMBO J. 15, 5154–5159 (1996).

    Article  Google Scholar 

  18. Springer, T.A. An extracellular β-propeller module predicted in lipoprotein and scavenger receptors, tyrosine kinases, epidermal growth factor precursor, and extracellular matrix components. J. Mol. Biol. 283, 837–862 (1998).

    Article  CAS  Google Scholar 

  19. Hopf, M., Göhring, W., Kohfeldt, E., Yamada, Y. & Timpl, R. Recombinant domain IV of perlecan binds to nidogens, laminin–nidogen complex, fibronectin, fibulin-2 and heparin. Eur. J. Biochem. 259, 917–925 (1999).

    Article  CAS  Google Scholar 

  20. Bork, P., Downing, A.K., Kieffer, B. & Campbell, I.D. Structure and distribution of modules in extracellular proteins. Q. Rev. Biophys. 29, 119–167 (1996).

    Article  CAS  Google Scholar 

  21. Holm, L. & Sander, C. Dali/FSSP classification of three-dimensional protein folds. Nucleic Acids Res. 25, 231–234 (1997).

    Article  CAS  Google Scholar 

  22. Ormö, M. et al. Crystal structure of the Aequora victoria green fluorescent protein. Science 273, 1392–1395 (1996).

    Article  Google Scholar 

  23. Altschul, S.F. et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402 (1997).

    Article  CAS  Google Scholar 

  24. Tsien, R.Y. The green fluorescent protein. Annu. Rev. Biochem. 67, 509–544 (1998).

    Article  CAS  Google Scholar 

  25. Fujiwara, S., Shinkai, H., Mann, K. & Timpl, R. Structure and localization of O- and N-linked oligosaccharide chains on basement membrane protein nidogen. Matrix 13, 215–222 (1993).

    Article  CAS  Google Scholar 

  26. Wall, M.A., Socolich, M. & Ranganathan, R. The structural basis of red fluorescence in the tetrameric GFP homolog DsRed. Nature Struct. Biol. 7, 1133–1138 (2000).

    Article  CAS  Google Scholar 

  27. Yarbrough, D., Wachter, R.M., Kallio, K., Matz, M.V. & Remington, S.J. Refined crystal structure of DsRed, a red fluorescent protein from coral, at 2.0 Å resolution. Proc. Natl. Acad. Sci. USA 98, 462–467 (2001).

    Article  CAS  Google Scholar 

  28. Vallejo, A.N., Pogulis, R.J. & Pease, L.R. In vitro synthesis of novel genes: mutagenesis and recombination by PCR. PCR Methods Applic. 4, 123–130 (1994).

    Article  Google Scholar 

  29. Vandenberg, P. et al. Characterization of a type IV collagen major cell binding site with affinity to the α1β1 and α2β1 integrins. J. Cell Biol. 113, 1475–1483 (1991).

    Article  CAS  Google Scholar 

  30. Leslie, A.G.W. MOSFLM users guide (MRC-LMB Cambridge, UK; 1994).

    Google Scholar 

  31. Collaborative Computing Project Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994).

  32. 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).

    Article  Google Scholar 

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

    Article  Google Scholar 

  34. Laskowski, R.A., MacArthur, M.W., Moss, D.S. & Thornton, J.M. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283–291 (1993).

    Article  CAS  Google Scholar 

  35. Esnouf, R.M. An extensively modified version of MOLSCRIPT which includes greatly enhanced colouring facilities. J. Mol. Graph. 15, 132–134 (1997).

    Article  CAS  Google Scholar 

  36. Merritt, E.A. & Bacon, D.J. Raster3D: Photorealistic molecular graphics. Methods Enzymol. 277, 505–524 (1997).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank the staff at stations 9.6 and 14.2 of the SRS Daresbury for help with data collection and P. Brick for helpful discussions. This study was supported by a Wellcome Trust Senior Research Fellowship to E.H. and a grant from the Deutsche Forschungsgemeinschaft to R.T.

Author information

Authors and Affiliations

  1. Abteilung Proteinchemie, Max-Planck-Institut für Biochemie, Am Klopferspitz 18a, Martinsried, D-82152, Germany

    Michael Hopf, Walter Göhring, Albert Ries & Rupert Timpl

  2. Biophysics Section, Blackett Laboratory, Imperial College, Prince Consort Road, London, SW7 2BW, UK

    Erhard Hohenester

  3. Division of Medicine, Imperial College School of Medicine, Exhibition Road, London, SW7 2AZ, UK

    Erhard Hohenester

Authors
  1. Michael Hopf
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Walter Göhring
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Albert Ries
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Rupert Timpl
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Erhard Hohenester
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Erhard Hohenester.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hopf, M., Göhring, W., Ries, A. et al. Crystal structure and mutational analysis of a perlecan-binding fragment of nidogen-1. Nat Struct Mol Biol 8, 634–640 (2001). https://doi.org/10.1038/89683

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/89683

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