Structural Analysis Uncovers Lipid-Binding Properties of Notch Ligands

Abstract

SummaryThe 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.

Structural basis for Notch1 engagement of Delta-like 4

“…Crystals of a complex containing a shorter DLL4 SLP (N-EGF1) construct were obtained by the same strategy and diffracted to a resolution of 2.3 Å (table S1). We determined both structures by molecular replacement using models of unliganded Notch1(11–13) and Jagged1 (18,19). The electron density maps obtained from the high-resolution Notch1(11–13)–DLL4 SLP (N-EGF1) structure were used for analysis of atomic contacts, whereas the lower-resolution Notch1(11–13)–DLL4 SLP (N-EGF2) structure allowed us to model the additional EGF2 domain.…”

“…3A) (31). Although it has been proposed that Notch activation requires calcium-dependent phospholipid binding by ligand MNNL domains, DLL4 was not bound to any calcium ions, which would suggest that this feature is not essential for signal transduction by all ligands (19). …”

“…There are presently no high-resolution structures describing Notch-ligand complexes, although there are structures of unliganded Notch1 EGF-like repeats 11 to 13 (Notch1(11–13), amino acids 411 to 530) and an unliganded Jagged1 fragment spanning from the N terminus to EGF3 (amino acids 32 to 337) (18, 19). Mammalian NECDs consist of 29 to 36 EGF repeats followed by 3 cysteine-rich LIN repeats.…”

“…Additional regions have been implicated in Notch ligand recognition, including EGF domains 6 to 15 (21, 22). Jagged/ DLL ECDs, like those of Notch, have a modular extracellular domain architecture consisting of a region termed module at the N-terminus of Notch ligands (MNNL) domain followed by a Delta/Serrate/Lag-2 (DSL) domain, and either 6 (DLL3), 8 (DLL1 and DLL4), or 16 (Jagged1 and Jagged2) EGF repeats (19, 23). Alanine scanning mutagenesis studies have identified conserved residue Phe 207 of the Jagged1 DSL domain and residues Leu 468 , Asp 469 , and Ile 477 of Notch1 EGF12 as critical to receptor-ligand interactions (18, 24).…”

Notch, lipids, and endothelial cells

“…In fact, a C2 phospholipid recognition domain in N-terminal region of Jag1 was recently uncovered. This domain also present in Dll1 does not impact dimerization with the receptor but it regulates levels of Notch activation [86]. More recently, ligand-independent induction of Notch was observed in response to shingosine-1-phosphate (S1P) and S1P receptor 3 engagement in cancer stem cells [87].…”