Structural analysis uncovers lipid-binding properties of Notch ligands

Abstract

The Notch pathway is a core cell-cell signaling system in metazoan organisms with key roles in cell-fate determination, stem cell maintenance, immune system activation, and angiogenesis. Signals are initiated by extracellular interactions of the Notch receptor with Delta/Serrate/Lag-2 (DSL) ligands, whose structure is highly conserved throughout evolution. To date, no structure or activity has been associated with the extreme N termini of the ligands, even though numerous mutations in this region of Jagged-1 ligand lead to human disease. Here, we demonstrate that the N terminus of human Jagged-1 is a C2 phospholipid recognition domain that binds phospholipid bilayers in a calcium-dependent fashion. Furthermore, we show that this activity is shared by a member of the other class of Notch ligands, human Delta-like-1, and the evolutionary distant Drosophila Serrate. Targeted mutagenesis of Jagged-1 C2 domain residues implicated in calcium-dependent phospholipid binding leaves Notch interactions intact but can reduce Notch activation. These results reveal an important and previously unsuspected role for phospholipid recognition in control of this key signaling system.

Graphical abstract

Figure 1

The Structure of Jagged1_N-EGF3 Reveals a C2 Domain N Terminal to the DSL Motif (A) The structure of J-1_N-EGF3 is shown in ribbon representation, with colors ranging from blue at the N terminus to red at the C terminus. Disulphide bonds are shown as sticks within semitransparent spheres, and the two glycans are shown as blue sticks. (B) Overlay of the N-terminal C2 domain of J-1_N-EGF3 (blue) on the Munc13-1 C2b domain (gray). (C) Alagille-syndrome-associated missense mutations that affect the C2 domain (red) are mapped onto the structure of J-1_N-EGF3 (gray cartoon and solid surface) and are located within the core of the domain (inset panel shows section at the level indicated by dotted line). (D) Structure-based sequence alignment of the Munc13-1 C2b domain and the N-terminal domains of Notch ligands. Most sequences associated with β strands contain some regions of absolute (red) or high (blue) sequence homology, whereas loop regions are more variable. Residues that coordinate the Ca²⁺ ion within the Munc13 domain are indicated, as are candidate Ca²⁺ ligand residues in J-1 mutated in this study (^∗). See also Figures S1 and S2.

Figure 2

Notch Ligands Contain a Functional Ca²⁺-Dependent Phospholipid Domain at Their N Termini (A) Representative Thermofluor thermal denaturation curves reveal that WT J-1_N-EGF3-Fc shows a shift in T_m to a higher temperature in the presence of 2 mM CaCl₂ (blue), but not 2 mM MgCl₂ (pink). This shift is not seen in the D140A/D144A double mutant. Error bars are ± SD. (B) Notch-ligand proteins containing a WT C2 domain show a calcium-dependent shift in T_m, whereas the D140A/D144A J-1 mutant or ΔC2 domain ligands do not. T_m values were extracted from thermal denaturation data using a Boltzmann sigmoidal fit. Means and SDs for five (J-1) or three (Dll-1) independent repeats are reported. (C) Plate-based assays (see Experimental Procedures) show that N-EGF3 constructs of three diverse Notch ligands (J-1, Dll-1, and Drosophila Serrate) all bind liposomes made from a PC/PS/PE mixture, whereas ΔC2 domain ligands do not. (D) Using the same assay, all three ligands are Ca²⁺ dependent in liposome binding, but they differ in their affinity for liposomes of a constant composition. See also Figures S3–S5.

Figure 3

Mutation of Residues within the β5-β6 Loop of the Jagged-1 C2 Domain Leaves Notch Binding Intact but Disturbs Phospholipid Binding and Notch Activation (A and B) Notch-1 binding of WT J-1_N-EGF3 and mutants assessed by ELISA. Substitution F207A (within the Notch binding face of the DSL domain; Cordle et al., 2008a) reduces binding to background levels, whereas D140A/D144A double substitution in the β5-β6 loop leaves binding intact. (C) Liposome binding by WT J-1_N-EGF3 and mutants assessed using fluorescently labeled liposomes (PC/PE/PS). (D) The ability of WT J-1_N-EGF3 F207A control and the D140A/D144A mutant to activate receptor in Notch1-transfected cells was assessed using a split luciferase reporter system. (E) Structure of WT J-1_N-EGF3 at pH 7.5 in the presence of 7.5 mM Ca²⁺ reveals a single calcium coordinated between the β5-β6 and β1-β2 loops. J-1 is shown as a cartoon representation (cyan, apo structure; teal, Ca²⁺-bound form copy A) with key residues highlighted as stick representations and the bound Ca²⁺ (pink) and a water molecule (teal) involved in coordination shown as spheres. Note the bidentate coordination by D140, which explains the loss of Ca²⁺ dependence associated with the D140A/D144A double mutant. All error bars are ± SD. See also Figures S1 and S5.