Molecular Basis for Jagged-1/Serrate Ligand Recognition by the Notch Receptor

Abstract

Background: The site of Jagged/Serrate ligand recognition by Notch is unknown.Results: Two critical residues involved in an intramolecular hydrophobic interaction across the central β-sheet of EGF12 form a ligand-binding platform.Conclusion: The ligand-binding region is adjacent to a Fringe-sensitive residue involved in modulating Notch activity.Significance: The results have implications for understanding receptor/ligand recognition, Notch regulation by O-glycosylation, and the development of paralogue-specific antibodies.

Fringe-mediated extension of O -linked fucose in the ligand-binding region of Notch1 increases binding to mammalian Notch ligands

“…S9) and can be seen to extend the ligand-binding surface comprising residues L468, E473, Q475, and I477 identified in a previous mutagenesis study ( Fig. 4C) (32). Additional contacts are formed between the GlcNAc sugar and residues D464 and M479.…”

“…S6). Yields of this Notch construct are very low, and, because it contains multiple possible glycosylation sites in addition to containing both the subsidiary ligand binding site for Serrate/Jagged around EGF8 (34) as well as the major site in EGF12 (32), it cannot be used to dissect the effects of Fringe modifications within the primary ligand-binding site. Coupling the shorter, prokaryotic hN1 [11][12][13] to the chip surface did not allow ligand binding to be detected-presumably due to steric interference of the chip matrix with the binding site.…”

“…Thr 466 is highlighted in cyan. (C ) hN1 12 unmodified and disaccharide X-ray structures highlighting residues shown to contribute to Jagged1 binding in a previous mutagenesis study (red); those residues that did not contribute to binding are also shown (blue) (32). T466 is highlighted in yellow, together with the disaccharide; the sugar may extend the known ligand-binding surface or act indirectly to promote complex formation.…”

“…Purification and refolding of prokaryotic hN1 11-13 variants were performed as described (32). Sequences corresponding to hJagged1 NE3 -Fc, hDll-1 NE3 -Fc, hDll-4 NE3 -Fc, and hN1 1-14 -Fc were cloned into a modified pLEXm eukaryotic expression plasmid (45).…”

“…The core ligand-binding site in hN1 is located on the central β-sheet of EGF12 (32), directly adjacent to the threonine residue that forms part of the O-fucosylation consensus sequence within this domain (T466). Sugar modifications at this position therefore have the potential to directly or indirectly affect formation of the Notch ligand complex through intermolecular or intramolecular effects.…”