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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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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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