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Structural basis for autoinhibition of Notch

Nature Structural & Molecular Biology volume 14, pages 295–300 (2007)Cite this article

A Corrigendum to this article was published on 01 May 2007

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Abstract

Notch receptors transmit signals between adjacent cells. Signaling is initiated when ligand binding induces metalloprotease cleavage of Notch within an extracellular negative regulatory region (NRR). We present here the X-ray structure of the human NOTCH2 NRR, which adopts an autoinhibited conformation. Extensive interdomain interactions within the NRR bury the metalloprotease site, showing that a substantial conformational movement is necessary to expose this site during activation by ligand. Leukemia-associated mutations in NOTCH1 probably release autoinhibition by destabilizing the conserved hydrophobic core of the NRR.

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Figure 1: Notch domain organization and overall views of the structure.
Figure 2: Interface between the LNR and HD domains.
Figure 3: Cell-based reporter gene assays showing that stepwise removal of LNR domain elements causes ligand-independent activation of NOTCH.

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  • 01 April 2007

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Notes

  1. *NOTE: In the version of this article initially published, three references were missing from the reference list. The corrected reference appears above and references have been renumbered accordingly. The error has been corrected in the HTML and PDF versions of the article.

References

  1. Bray, S.J. Notch signalling: a simple pathway becomes complex. Nat. Rev. Mol. Cell Biol. 7, 678–689 (2006).

    Article  CAS  Google Scholar 

  2. Weng, A.P. et al. Activating mutations of NOTCH1 in human T cell acute lymphoblastic leukemia. Science 306, 269–271 (2004).

    Article  CAS  Google Scholar 

  3. Blaumueller, C.M., Qi, H., Zagouras, P. & Artavanis-Tsakonas, S. Intracellular cleavage of Notch leads to a heterodimeric receptor on the plasma membrane. Cell 90, 281–291 (1997).

    Article  CAS  Google Scholar 

  4. Logeat, F. et al. The Notch1 receptor is cleaved constitutively by a furin-like convertase. Proc. Natl. Acad. Sci. USA 95, 8108–8112 (1998).

    Article  CAS  Google Scholar 

  5. Sanchez-Irizarry, C. et al. Notch subunit heterodimerization and prevention of ligand-independent proteolytic activation depend, respectively, on a novel domain and the LNR repeats. Mol. Cell. Biol. 24, 9265–9273 (2004).

    Article  CAS  Google Scholar 

  6. Fehon, R.G. et al. Molecular interactions between the protein products of the neurogenic loci Notch and Delta, two EGF-homologous genes in Drosophila. Cell 61, 523–534 (1990).

    Article  CAS  Google Scholar 

  7. Weinmaster, G. The ins and outs of notch signaling. Mol. Cell. Neurosci. 9, 91–102 (1997).

    Article  CAS  Google Scholar 

  8. Brou, C. et al. A novel proteolytic cleavage involved in Notch signaling: the role of the disintegrin-metalloprotease TACE. Mol. Cell 5, 207–216 (2000).

    Article  CAS  Google Scholar 

  9. Mumm, J.S. et al. A ligand-induced extracellular cleavage regulates gamma-secretase-like proteolytic activation of Notch1. Mol. Cell 5, 197–206 (2000).

    Article  CAS  Google Scholar 

  10. Kopan, R. & Goate, A. A common enzyme connects notch signaling and Alzheimer's disease. Genes Dev. 14, 2799–2806 (2000).

    Article  CAS  Google Scholar 

  11. Schroeter, E.H., Kisslinger, J.A. & Kopan, R. Notch-1 signalling requires ligand-induced proteolytic release of intracellular domain. Nature 393, 382–386 (1998).

    Article  CAS  Google Scholar 

  12. Struhl, G. & Adachi, A. Nuclear access and action of notch in vivo. Cell 93, 649–660 (1998).

    Article  CAS  Google Scholar 

  13. Struhl, G., Fitzgerald, K. & Greenwald, I. Intrinsic activity of the Lin-12 and Notch intracellular domains in vivo. Cell 74, 331–345 (1993).

    Article  CAS  Google Scholar 

  14. Kopan, R., Schroeter, E.H., Weintraub, H. & Nye, J.S. Signal transduction by activated mNotch: importance of proteolytic processing and its regulation by the extracellular domain. Proc. Natl. Acad. Sci. USA 93, 1683–1688 (1996).

    Article  CAS  Google Scholar 

  15. Lieber, T., Kidd, S., Alcamo, E., Corbin, V. & Young, M.W. Antineurogenic phenotypes induced by truncated Notch proteins indicate a role in signal transduction and may point to a novel function for Notch in nuclei. Genes Dev. 7, 1949–1965 (1993).

    Article  CAS  Google Scholar 

  16. Rebay, I., Fehon, R.G. & Artavanis-Tsakonas, S. Specific truncations of Drosophila Notch define dominant activated and dominant negative forms of the receptor. Cell 74, 319–329 (1993).

    Article  CAS  Google Scholar 

  17. Berry, L.W., Westlund, B. & Schedl, T. Germ-line tumor formation caused by activation of glp-1, a Caenorhabditis elegans member of the Notch family of receptors. Development 124, 925–936 (1997).

    CAS  PubMed  Google Scholar 

  18. Greenwald, I. & Seydoux, G. Analysis of gain-of-function mutations of the lin-12 gene of Caenorhabditis elegans. Nature 346, 197–199 (1990).

    Article  CAS  Google Scholar 

  19. Vardar, D., North, C.L., Sanchez-Irizarry, C., Aster, J.C. & Blacklow, S.C. Nuclear magnetic resonance structure of a prototype Lin12-Notch repeat module from human Notch1. Biochemistry 42, 7061–7067 (2003).

    Article  CAS  Google Scholar 

  20. Holm, L. & Sander, C. Mapping the protein universe. Science 273, 595–603 (1996).

    Article  CAS  Google Scholar 

  21. Maeda, T. et al. Solution structure of the SEA Domain from the murine homologue of ovarian cancer antigen CA125 (MUC16). J. Biol. Chem. 279, 13174–13182 (2004).

    Article  CAS  Google Scholar 

  22. Macao, B., Johansson, D.G.A., Hansson, G.C. & Hard, T. Autoproteolysis coupled to protein folding in the SEA domain of the membrane-bound MUC1 mucin. Nat. Struct. Mol. Biol. 13, 71–76 (2006).

    Article  CAS  Google Scholar 

  23. Mumm, J.S. & Kopan, R. Notch signaling: from the outside in. Dev. Biol. 228, 151–165 (2000).

    Article  CAS  Google Scholar 

  24. Itoh, M. et al. Mind bomb is a ubiquitin ligase that is essential for efficient activation of Notch signaling by Delta. Dev. Cell 4, 67–82 (2003).

    Article  CAS  Google Scholar 

  25. Le Borgne, R., Bardin, A. & Schweisguth, F. The roles of receptor and ligand endocytosis in regulating Notch signaling. Development 132, 1751–1762 (2005).

    Article  CAS  Google Scholar 

  26. Wang, W. & Struhl, G. Drosophila Epsin mediates a select endocytic pathway that DSL ligands must enter to activate Notch. Development 131, 5367–5380 (2004).

    Article  CAS  Google Scholar 

  27. Wang, W. & Struhl, G. Distinct roles for Mind bomb, Neuralized and Epsin in mediating DSL endocytosis and signaling in Drosophila. Development 132, 2883–2894 (2005).

    Article  CAS  Google Scholar 

  28. Bingham, S. et al. Neurogenic phenotype of mind bomb mutants leads to severe patterning defects in the zebrafish hindbrain. Dev. Dyn. 228, 451–463 (2003).

    Article  CAS  Google Scholar 

  29. Lai, E.C., Roegiers, F., Qin, X., Jan, Y.N. & Rubin, G.M. The ubiquitin ligase Drosophila Mind bomb promotes Notch signaling by regulating the localization and activity of Serrate and Delta. Development 132, 2319–2332 (2005).

    Article  CAS  Google Scholar 

  30. Le Borgne, R., Remaud, S., Hamel, S. & Schweisguth, F. Two distinct E3 ubiquitin ligases have complementary functions in the regulation of delta and serrate signaling in Drosophila. PLoS Biol. 3, 688–696 (2005).

    Article  CAS  Google Scholar 

  31. Le Borgne, R. & Schweisguth, F. Unequal segregation of Neuralized biases Notch activation during asymmetric cell division. Dev. Cell 5, 139–148 (2003).

    Article  CAS  Google Scholar 

  32. Pavlopoulos, E. et al. neuralized encodes a peripheral membrane protein involved in delta signaling and endocytosis. Dev. Cell 1, 807–816 (2001).

    Article  CAS  Google Scholar 

  33. Seugnet, L., Simpson, P. & Haenlin, M. Requirement for dynamin during Notch signaling in Drosophila neurogenesis. Dev. Biol. 192, 585–598 (1997).

    Article  CAS  Google Scholar 

  34. Parks, A.L., Klueg, K.M., Stout, J.R. & Muskavitch, M.A. Ligand endocytosis drives receptor dissociation and activation in the Notch pathway. Development 127, 1373–1385 (2000).

    CAS  PubMed  Google Scholar 

  35. Ahimou, F., Mok, L.P., Bardot, B. & Wesley, C. The adhesion force of Notch with Delta and the rate of Notch signaling. J. Cell Biol. 167, 1217–1229 (2004).

    Article  CAS  Google Scholar 

  36. Maskos, K. et al. Crystal structure of the catalytic domain of human tumor necrosis factor-alpha-converting enzyme. Proc. Natl. Acad. Sci. USA 95, 3408–3412 (1998).

    Article  CAS  Google Scholar 

  37. Sun, X. & Artavanis-Tsakonas, S. Secreted forms of DELTA and SERRATE define antagonists of Notch signaling in Drosophila. Development 124, 3439–3448 (1997).

    CAS  PubMed  Google Scholar 

  38. Varnum-Finney, B. et al. Immobilization of Notch ligand, Delta-1, is required for induction of notch signaling. J. Cell Sci. 113, 4313–4318 (2000).

    CAS  PubMed  Google Scholar 

  39. Chen, N. & Greenwald, I. The lateral signal for LIN-12/Notch in C. elegans vulval development comprises redundant secreted and transmembrane DSL proteins. Dev. Cell. 6, 183–192 (2004).

    Article  CAS  Google Scholar 

  40. Malecki, M.J. et al. Leukemia-associated mutations within the NOTCH1 heterodimerization domain fall into at least two distinct mechanistic classes. Mol. Cell. Biol. 26, 4642–4651 (2006).

    Article  CAS  Google Scholar 

  41. Otwinowsk, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

    Article  Google Scholar 

  42. Perrakis, A., Morris, R. & Lazmin, V. Automated protein model building combined with iterative structure refinement. Nat. Struct. Biol. 6, 458–463 (1999).

    Article  CAS  Google Scholar 

  43. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  Google Scholar 

  44. Brunger, A.T., Adams, P. & Clore, G. Crystallography and NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr D Biol. Crystallogr. 54, 905–921 (1998).

    Article  CAS  Google Scholar 

  45. Murshudov, G., Vagin, A. & Dodson, E. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240–245 (1997).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank Mike Eck and Angela Toms for crystallographic suggestions and Kelly Arnett and Mike Malecki for critical reading of the manuscript. This work was supported by American Cancer Society Postdoctoral Fellowships (WRG and DVU), a Leukemia and Lymphoma Society Fellowship (WRG), and NIH grants to SCB and JCA.

Author information

Author notes

  1. Didem Vardar-Ulu

    Present address: Present address: Department of Chemistry, Wellesley College, 106 Central Street, Wellesley, Massachusetts 02481, USA.,

  2. Wendy R Gordon and Didem Vardar-Ulu: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, 77 Ave. Louis Pasteur, Boston, 02115, Massachusetts, USA

    Wendy R Gordon, Didem Vardar-Ulu, Gavin Histen, Cheryll Sanchez-Irizarry, Jon C Aster & Stephen C Blacklow

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  1. Wendy R Gordon
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Corresponding author

Correspondence to Stephen C Blacklow.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Sequence alignment of the NRR region of various Notch receptors (PDF 2309 kb)

Supplementary Fig. 2

Representative electron density (PDF 370 kb)

Supplementary Fig. 3

Comparison of human NOTCH2 HD domain with SEA domains from mucins (PDF 175 kb)

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Gordon, W., Vardar-Ulu, D., Histen, G. et al. Structural basis for autoinhibition of Notch. Nat Struct Mol Biol 14, 295–300 (2007). https://doi.org/10.1038/nsmb1227

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