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

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-1(DSL-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-1(DSL-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.

Figure 1. Specific recognition of N-1_11-13 by J-1_DSL-EGF3

(a) Western blot analysis to demonstrate the pull down of N-1_11-13 with His-tagged J-1_DSL-EGF3 on capture with Ni-NTA Magnetic Agarose Beads. Anti-Notch antibody was used for detection. (b) The calcium dependence of the interaction was demonstrated with biotinylated N-1_11-13 immobilised on Dynabeads M-270 Streptavidin (lanes 1-4). Lanes 5 and 6 were control beads without N-1_11-13. Detection of His-tagged J-1_DSL-EGF3 by anti-RGS.His HRP conjugate showed the interaction of N-1_11-13 with J-1_DSL-EGF3 occurred in the presence of calcium (Ca) and was inhibited by EDTA (E). Anti-Notch antibody confirmed equivalent amounts of N-1_11-13 were eluted from the beads (lower panel). (c) SPR measurements were taken with 7085RU of J-1_DSL-EGF3 coupled to the chip surface and N-1_11-13 (without the BirA-tag) injected over the chip surface at the concentrations indicated in Tris buffered saline supplemented with 1mM Ca²⁺. (d) Trace from (c) corrected relative to reference cell. (e) To demonstrate the dependence on Ca²⁺ of the specific interaction three sequential injections of N-1_11-13 at 110μM were made over the same J-1_DSL-EGF3 surface. The first and last injections were carried out in the buffer described above, the second injection contained an additional 10mM EGTA to chelate available Ca²⁺. Interaction of N-1_11-13 with the J-1_DSL-EGF3 coupled surface and with the reference surface are shown by the red and blue traces respectively. (f) Addition of unlabelled J-1_DSL-EGF3 (0.15mM) to ¹⁵N-labelled N-1_11-13 (0.5mM) leads to a loss of intensity of NMR peaks following formation of the large complex between the two proteins. The red line is a visual aid to highlight the greater intensity changes for V453 and G472.

Figure 2. J-1_DSL-EGF3 & N-1_11-13 architecture

(a) The overall structure of J-1_DSL-EGF3 is shown in a cartoon representation coloured from blue at the N-terminus (residue 187) to red at the C-terminus (residue 335). Disulphide bonds are shown in yellow stick representations and two views differing by a rotation of 90° about the long axis are shown. (b) Stereo view of the DSL domain fold. (c) Sequences of the four J-1_DSL-EGF3 domains with disulphide bond pairings indicated. (d) Crystallographic structure of N-1_11-13 shown as in panel (a). The bound Ca²⁺ in each EGF domain is shown in a space-filling representation. All structural figures were generated with PyMol (http://www.pymol.org) .

Figure 3. Predicting surfaces involved in binding and recognition

Analysis of (a) an alignment of Jagged/Delta family DSL domains representing a variety of species (H. sapiens Jagged-1, residues 187-229; D. melanogaster Serrate, residues 237-279; H. sapiens Jagged-2, residues 198-240; H. sapiens Delta-like 1, residues 179-221; D. melanogaster Delta, residues 184-226; H. sapiens Delta-like 4, residues 175-217; C. elegans LAG-2, residues 124-166; H. sapiens Delta-like 3, residues 178-215) and (b) the DSL structure, reveals a series of highly-conserved, but surface-exposed residues. Residues which are conserved and predicted to form a Notch binding face are coloured red, cysteines are coloured yellow, while a non-conserved residue on the opposite face is coloured blue. (c) Electrostatic surface potential of J-1_DSL-EGF3 and N-1_11-13 plotted at +/- 4 kT/e using APBS . Note the positively charged patch (blue) within the DSL domain of Jagged-1 and the negatively charged surface (red) of Notch. Highlighted by green bands are the surfaces predicted by sequence/structure analysis (J-1_DSL-EGF3) and NMR studies (N-1_11-13) to be involved in binding.

Figure 4. Functional analysis of Serrate DSL mutants in Drosophila wing disc and S2 cells reveals residues important for trans-activation and cis-inhibition

(a) Left panel, wingless mRNA expression in wild-type 3rd instar wing disc, which on the dorsal (d)-ventral (v) boundary (arrow) acts as a reporter for Notch activity. Central panel shows wing disc over-expressing a Serrate construct along the anterior (a)-posterior (p) compartment boundary; cell nuclei (blue), and anti-Serrate (green). Right panel, wild-type adult wing. (b) Over-expression of wild type and mutant Serrate constructs illustrating four different phenotypic classes. Far left panels, wingless mRNA expression in imaginal wing discs. Ectopic expression is marked with an arrow and suppression of endogenous D-V boundary expression is marked with an arrowhead. Centre left panels, immunofluorescence staining of Serrate (green) and Wingless (purple). Centre right panels, localisation of expressed Serrate construct (green) with actin (purple) at the adherens junction of wing disc epithelial cells. Far right panels, adult wing phenotypes. Ectopic margins are indicated by arrows and notching of endogenous wing margin by asterisks. The results presented are from fly cultures maintained at 18°C, except for the data representative of Class IV which were obtained from a 29°C fly culture. (c) Cell aggregation assay of the Notch-binding potential of different full-length Serrate constructs. The Serrate construct used is labelled in each panel. Panels show merged immunofluoresence images of Serrate (green) and Notch (purple) expression. Cell junctions with clustered Serrate and Notch are indicated by arrowheads.

Figure 5. Quantification of mutant phenotypes and mapping onto structures

(a) Graph shows % of total Notch expressing cells bound to cells that express different Serrate constructs. Error bars represent standard deviation from quadruplicate experiments. (b) Western blot analyses of representative experiments to demonstrate the pull down of J-1_DSL-EGF3 wild-type and mutant constructs by biotinylated N-1_11-13 immobilised on Dynabeads M-270 Streptavidin. Detection by anti-RGS.His HRP conjugate (upper panels) demonstrated interaction of N-1_11-13 with J-1_DSL-EGF3 wild-type, F199A, R201A and N215A but not F207A. A faint upper band (indicated by an asterisk) is attributed to a trace of His-tagged N-1_11-13. Streptavidin-HRP conjugate was used to show equivalent amounts of N-1_11-13 were eluted from the beads for each mutant (lower panels). (c) Surface representations of both J-1_DSL-EGF3 and N-1_11-13 are shown in two orientations related by 180 degree rotation about the long axis. The J-1_DSL-EGF3 is coloured by mutant phenotype class (class 1 - green, class II - yellow, class III - red, class IV - orange) whilst the residues implicated in J-1_DSL-EGF3 binding by NMR are coloured red on the surface of N-1_11-13. We have termed the faces implicated in binding the “front” view on each molecule.