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A conserved face of the Jagged/Serrate DSL domain is involved in Notch trans-activation and cis-inhibition

Nature Structural & Molecular Biology volume 15, pages 849–857 (2008)Cite this article

Abstract

The Notch receptor and its ligands are key components in a core metazoan signaling pathway that regulates the spatial patterning, timing and outcome of many cell-fate decisions. Ligands contain a disulfide-rich Delta/Serrate/LAG-2 (DSL) domain required for Notch trans-activation or cis-inhibition. Here we report the X-ray structure of a receptor binding region of a Notch ligand, the DSL-EGF3 domains of human Jagged-1 (J-1DSL-EGF3). The structure reveals a highly conserved face of the DSL domain, and we show, by functional analysis of Drosophila melanogster ligand mutants, that this surface is required for both cis- and trans-regulatory interactions with Notch. We also identify, using NMR, a surface of Notch-1 involved in J-1DSL-EGF3 binding. Our data imply that cis- and trans-regulation may occur through the formation of structurally distinct complexes that, unexpectedly, involve the same surfaces on both ligand and receptor.

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Figure 1: Specific recognition of N-111–13 by J-1DSL-EGF3.
Figure 2: J-1DSL-EGF3 and N-111–13 architecture.
Figure 3: Predicting surfaces involved in binding and recognition.
Figure 4: Functional analysis of Serrate (Ser) DSL mutants in D. melanogaster wing disc and S2 cells reveals residues important for trans-activation and cis-inhibition.
Figure 5: Quantification of mutant phenotypes and mapping onto structures.

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References

  1. Artavanis-Tsakonas, S., Rand, M.D. & Lake, R.J. Notch signaling: cell fate control and signal integration in development. Science 284, 770–776 (1999).

    Article  CAS  PubMed  Google Scholar 

  2. Lai, E.C. Notch signaling: control of cell communication and cell fate. Development 131, 965–973 (2004).

    Article  CAS  PubMed  Google Scholar 

  3. Milner, L.A., Kopan, R., Martin, D.I. & Bernstein, I.D. A human homologue of the Drosophila developmental gene, Notch, is expressed in CD34+ hematopoietic precursors. Blood 83, 2057–2062 (1994).

    CAS  PubMed  Google Scholar 

  4. Nyfeler, Y. et al. Jagged1 signals in the postnatal subventricular zone are required for neural stem cell self-renewal. EMBO J. 24, 3504–3515 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. McKenzie, G.J. et al. Notch signalling in the regulation of peripheral T-cell function. Semin. Cell Dev. Biol. 14, 127–134 (2003).

    Article  CAS  PubMed  Google Scholar 

  6. Hansson, E.M., Lendahl, U. & Chapman, G. Notch signaling in development and disease. Semin. Cancer Biol. 14, 320–328 (2004).

    Article  CAS  PubMed  Google Scholar 

  7. 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  PubMed  Google Scholar 

  8. 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  PubMed  PubMed Central  Google Scholar 

  9. 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  PubMed  PubMed Central  Google Scholar 

  10. Gordon, W.R. et al. Structural basis for autoinhibition of Notch. Nat. Struct. Mol. Biol. 14, 295–300 (2007).

    Article  CAS  PubMed  Google Scholar 

  11. Nichols, J.T. et al. DSL ligand endocytosis physically dissociates Notch1 heterodimers before activating proteolysis can occur. J. Cell Biol. 176, 445–458 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. 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 

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

    Article  CAS  PubMed  Google Scholar 

  14. 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  PubMed  Google Scholar 

  15. Jarriault, S. et al. Signalling downstream of activated mammalian Notch. Nature 377, 355–358 (1995).

    Article  CAS  PubMed  Google Scholar 

  16. de Celis, J.F. & Bray, S. Feed-back mechanisms affecting Notch activation at the dorsoventral boundary in the Drosophila wing. Development 124, 3241–3251 (1997).

    CAS  PubMed  Google Scholar 

  17. Franklin, J.L. et al. Autonomous and non-autonomous regulation of mammalian neurite development by Notch1 and Delta1. Curr. Biol. 9, 1448–1457 (1999).

    Article  CAS  PubMed  Google Scholar 

  18. Glittenberg, M., Pitsouli, C., Garvey, C., Delidakis, C. & Bray, S. Role of conserved intracellular motifs in Serrate signalling, cis-inhibition and endocytosis. EMBO J. 25, 4697–4706 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Micchelli, C.A., Rulifson, E.J. & Blair, S.S. The function and regulation of cut expression on the wing margin of Drosophila: Notch, Wingless and a dominant negative role for Delta and Serrate. Development 124, 1485–1495 (1997).

    CAS  PubMed  Google Scholar 

  20. Sakamoto, K., Ohara, O., Takagi, M., Takeda, S. & Katsube, K. Intracellular cell-autonomous association of Notch and its ligands: a novel mechanism of Notch signal modification. Dev. Biol. 241, 313–326 (2002).

    Article  CAS  PubMed  Google Scholar 

  21. Fleming, R.J. Structural conservation of Notch receptors and ligands. Semin. Cell Dev. Biol. 9, 599–607 (1998).

    Article  CAS  PubMed  Google Scholar 

  22. Rebay, I. et al. Specific EGF repeats of Notch mediate interactions with Delta and Serrate: implications for Notch as a multifunctional receptor. Cell 67, 687–699 (1991).

    Article  CAS  PubMed  Google Scholar 

  23. Hambleton, S. et al. Structural and functional properties of the human notch-1 ligand binding region. Structure 12, 2173–2183 (2004).

    Article  CAS  PubMed  Google Scholar 

  24. Abbott, R.J. et al. Structural and functional characterization of a novel T cell receptor co-regulatory protein complex, CD97–CD55. J. Biol. Chem. 282, 22023–22032 (2007).

    Article  CAS  PubMed  Google Scholar 

  25. Henderson, S.T., Gao, D., Christensen, S. & Kimble, J. Functional domains of LAG-2, a putative signaling ligand for LIN-12 and GLP-1 receptors in Caenorhabditis elegans. Mol. Biol. Cell 8, 1751–1762 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Shimizu, K. et al. Mouse Jagged1 physically interacts with Notch2 and other notch receptors. Assessment by quantitative methods. J. Biol. Chem. 274, 32961–32969 (1999).

    Article  CAS  PubMed  Google Scholar 

  27. Tax, F.E., Yeargers, J.J. & Thomas, J.H. Sequence of C. elegans lag-2 reveals a cell-signalling domain shared with Delta and Serrate of Drosophila. Nature 368, 150–154 (1994).

    Article  CAS  PubMed  Google Scholar 

  28. Salmon, D. et al. Solution structure and backbone dynamics of the Trypanosoma cruzi cysteine protease inhibitor chagasin. J. Mol. Biol. 357, 1511–1521 (2006).

    Article  CAS  PubMed  Google Scholar 

  29. Cordle, J. et al. Localization of the Delta-like-1-binding site in human Notch-1 and its modulation by calcium affinity. J. Biol. Chem. 283, 11785–11793 (2008).

    Article  CAS  PubMed  Google Scholar 

  30. Panin, V.M., Papayannopoulos, V., Wilson, R. & Irvine, K.D. Fringe modulates Notch-ligand interactions. Nature 387, 908–912 (1997).

    Article  CAS  PubMed  Google Scholar 

  31. Kohsaka, T. et al. The significance of human jagged 1 mutations detected in severe cases of extrahepatic biliary atresia. Hepatology 36, 904–912 (2002).

    Article  CAS  PubMed  Google Scholar 

  32. Warthen, D.M. et al. Jagged1 (JAG1) mutations in Alagille syndrome: increasing the mutation detection rate. Hum. Mutat. 27, 436–443 (2006).

    Article  CAS  PubMed  Google Scholar 

  33. Bruckner, K., Perez, L., Clausen, H. & Cohen, S. Glycosyltransferase activity of Fringe modulates Notch-Delta interactions. Nature 406, 411–415 (2000).

    Article  CAS  PubMed  Google Scholar 

  34. Moloney, D.J. et al. Fringe is a glycosyltransferase that modifies Notch. Nature 406, 369–375 (2000).

    Article  CAS  PubMed  Google Scholar 

  35. Xu, A. et al. In vitro reconstitution of the modulation of Drosophila notch-ligand binding by fringe. J. Biol. Chem. 282, 35153–35162 (2007).

    Article  CAS  PubMed  Google Scholar 

  36. Acar, M. et al. Rumi is a CAP10 Domain glycosyltransferase that modifies Notch and is required for Notch signaling. Cell 132, 247–258 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Muranyi, A. et al. 1H, 13C, and 15N resonance assignments of human Notch-1 calcium binding EGF domains 11–13. J. Biomol. NMR 29, 443–444 (2004).

    Article  CAS  PubMed  Google Scholar 

  38. Hutchinson, S. et al. Molecular effects of homocysteine on cbEGF domain structure: insights into the pathogenesis of homocystinuria. J. Mol. Biol. 346, 833–844 (2005).

    Article  CAS  PubMed  Google Scholar 

  39. Vonrhein, C., Blanc, E., Roversi, P. & Bricogne, G. Automated structure solution with autoSHARP. Methods Mol. Biol. 364, 215–230 (2007).

    CAS  PubMed  Google Scholar 

  40. de La Fortelle, E. & Bricogne, G. Maximum-likelihood heavy-atom parameter refinement for multiple isomorphous replacement and multiwavelength anomalous diffraction methods. Methods Enzymol. 276, 472–494 (1997).

    Article  CAS  PubMed  Google Scholar 

  41. McRee, D.E. XtalView/Xfit—a versatile program for manipulating atomic coordinates and electron density. J. Struct. Biol. 125, 156–165 (1999).

    Article  CAS  PubMed  Google Scholar 

  42. Blanc, E. et al. Refinement of severely incomplete structures with maximum likelihood in BUSTER-TNT. Acta Crystallogr. D Biol. Crystallogr. 60, 2210–2221 (2004).

    Article  CAS  PubMed  Google Scholar 

  43. Vagin, A. & Teplyakov, A. MOLREP: an automated program for molecular replacement. J. Appl. Crystallogr. 30, 1022–1025 (1997).

    Article  CAS  Google Scholar 

  44. Rao, Z. et al. The structure of a Ca2+-binding epidermal growth factor-like domain: its role in protein-protein interactions. Cell 82, 131–141 (1995).

    Article  CAS  PubMed  Google Scholar 

  45. Fleming, R.J., Scottgale, T.N., Diederich, R.J. & Artavanis-Tsakonas, S. The gene Serrate encodes a putative EGF-like trans membrane protein essential for proper ectodermal development in Drosophila melanogaster. Genes Dev. 4, 2188–2201 (1990).

    Article  CAS  PubMed  Google Scholar 

  46. Brand, A.G., Manoukian, A.S. & Perrimon, N. Ectopic expression in Drosophila. Methods Cell Biol. 44, 635–654 (1994).

    Article  CAS  PubMed  Google Scholar 

  47. Speicher, S.A., Thomas, U., Hinz, U. & Knust, E. The Serrate locus of Drosophila and its role in morphogenesis of the wing imaginal discs: control of cell proliferation. Development 120, 535–544 (1994).

    CAS  PubMed  Google Scholar 

  48. Mazaleyrat, S.L. et al. Down-regulation of Notch target gene expression by Suppressor of deltex. Dev. Biol. 255, 363–372 (2003).

    Article  CAS  PubMed  Google Scholar 

  49. Thomas, U., Jonsson, F., Speicher, S.A. & Knust, E. Phenotypic and molecular characterization of SerD, a dominant allele of the Drosophila gene Serrate. Genetics 139, 203–213 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 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  PubMed  Google Scholar 

  51. Klueg, K.M., Alvarado, D., Muskavitch, M.A. & Duffy, J.B. Creation of a GAL4/UAS-coupled inducible gene expression system for use in Drosophila cultured cell lines. Genesis 34, 119–122 (2002).

    Article  CAS  PubMed  Google Scholar 

  52. Lieber, T. et al. Single amino acid substitutions in EGF-like elements of Notch and Delta modify Drosophila development and affect cell adhesion in vitro. Neuron 9, 847–859 (1992).

    Article  CAS  PubMed  Google Scholar 

  53. Dominguez, C., Boelens, R. & Bonvin, A.M. HADDOCK: a protein-protein docking approach based on biochemical or biophysical information. J. Am. Chem. Soc. 125, 1731–1737 (2003).

    Article  CAS  PubMed  Google Scholar 

  54. Baker, N.A., Sept, D., Joseph, S., Holst, M.J. & McCammon, J.A. Electrostatics of nanosystems: application to microtubules and the ribosome. Proc. Natl. Acad. Sci. USA 98, 10037–10041 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank M. Walsh at BM14, the staff of ID29 at the European Synchrotron Radiation Facility and E. Lowe (University of Oxford) for assistance with X-ray data collection. We also thank A. McMichael, S. Hambleton & X. Xu (University of Oxford) for advice and Global Phasing for access to a beta version of Buster-TNT. We thank R. Fleming (Trinity College, Connecticut), C. Wesley (University of Vermont) and S. Artavanis-Tsakonas of Harvard Medical School for D. melanogaster cDNA constructs and fly stocks. This work was supported by the Edward Penley Abraham Cephalosporin fund (#CF065 to S.M.L.), the UK Medical Research Council (MRC; #G0400389 to S.M.L., #G000164 to P.A.H. and MRC Studentship G78/7267 to J.C.), the Wellcome Trust (#077082 to S.M.L., #078765 to P.A.H. and S.M.L. and #079440 to C.R.) and the UK Biotechnology and Biological Sciences Research Council (#C503162/1 and E002285/1 to M.B.).

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  1. Jemima Cordle and Steven Johnson: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK

    Jemima Cordle, Joyce Zi Yan Tay, Sacha Jensen, Pat Whiteman, Christina Redfield & Penny A Handford

  2. Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, UK

    Steven Johnson, Joyce Zi Yan Tay, Pietro Roversi & Susan M Lea

  3. Faculty of Life Sciences, Michael Smith Building, University of Manchester, Stopford Road, Manchester, M13 9PT, UK

    Marian B Wilkin, Beatriz Hernández de Madrid, Hideyuki Shimizu & Martin Baron

  4. Department of Immunology, Fourth Military Medical University, 17 West Changle Road, Xi'an, 710032, Shaanxi, PR China

    Boquan Jin

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Contributions

J.C. cloned, expressed and functionally characterized J-1DSL-EGF3 and N-111–13 constructs. S. Johnson, S.M.L. and P.R. solved the structure of J-1 and, with J.Z.Y.T., the structure of N-1. J.Z.Y.T. produced 15N-labeled N-1 and, with C.R., performed the NMR experiments and analyzed the data. S. Johnson, S.M.L. and P.A.H. generated functional hypotheses on the basis of the structures; S. Jensen mutagenized Serrate. P.W. produced J-1 for NMR studies, mutagenized J-1 and performed pull-down assays to characterize the mutants. B.J. generated the N-1 monoclonal antibody. M.W., B.H.-D., H.S. and M.B. performed and analyzed experiments in D. melanogaster. S. Johnson, M.B., C.R., S.M.L. and P.A.H. wrote the manuscript.

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Correspondence to Christina Redfield, Martin Baron, Susan M Lea or Penny A Handford.

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Cordle, J., Johnson, S., Zi Yan Tay, J. et al. A conserved face of the Jagged/Serrate DSL domain is involved in Notch trans-activation and cis-inhibition. Nat Struct Mol Biol 15, 849–857 (2008). https://doi.org/10.1038/nsmb.1457

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