Biophysical determinants of Notch signaling

Notch signaling is involved in many biological contexts such as cancer, stem cell development, and neural cell development. Because of the importance of Notch function in health and disease, the Notch signaling pathway has emerged as a potential therapeutic target.;Mammalian Notch receptors are single-pass transmembrane glycoprotein receptors, which contain 29-36 EGF like repeats. The fucosyltransferase termed Pofut1 transfers fucose to the serine or threonine residue of the O-fucosylation consensus sequence on some EGF domains of Notch receptors. The glycosyltransferases termed Fringe can elongate O-fucose moieties by adding N-acetylglucosamine, which may be subsequently modified by galactose and sialic acid. These O-fucosylated glycans play key roles in modulating Notch-mediated signal transduction events.;Here, we have observed how O-fucosylated glycan modifications modulate Notch receptor-ligand interactions using surface plasmon resonance techniques. A biphasic binding and dissociation pattern was observed, suggesting a two-state receptor-ligand interaction model characterized by initial formation of a transient receptor-ligand complex followed by a conformational change that leads to a more stable receptor-ligand complex. Primary and secondary on and off-rates for the four binding-competent Notch1-Notch ligand pairs were observed to be distinct and characteristic for each Notch ligand. The overall association constants observed when Dll-1 or Dll-4 interacted with Fringe-modified Notch1 were significantly greater than when these ligands interacted with unmodified Notch1, with enhancement likely due to Fringe modifications of fucose moieties within EGF domains 16-36. By contrast, Fringe modification of Notch1 did not significantly modulate interactions with Jag-1 or Jag-2. Mutational analyses confirm prior observations that the O-fucosylation site within EGF repeat 12 dictates much, if not all of the binding between Notch1 and its ligands. Finally, we observe that Fringe modification of Dll-4 enhances its ability to bind to Notch1.;Our data reveal that the molecular basis of glycan-dependent Notch-Notch ligand binding. We propose a two-state binding model with triple stranded structure for Notch-Notch ligand complex arrangement. Here, O-fucosylation and Fringe modification of Notch receptors play a key role in both the binding and the conformational change.