The Cytoplasmic Tail of the Notch Ligand Jagged-1: Intrinsic Disorder, Induced Order and Molecular Interactions

Notch signaling plays a key role in cell differentiation in all metazoans. As both receptors and ligands are cell-surface proteins, Notch signaling is restricted to nearby interacting cells. Notch ligands are membrane-spanning proteins made of a large extracellular region, a transmembrane segment, and a 100–200 residue cytoplasmic tail. The sequence of the intracellular region of Jagged-1, one of the five ligands to Notch receptors in man, is very well conserved throughout evolution but does not encode any globular domain. The cytoplasmic tail of Jagged-1 mediates protein–protein interactions through the C-terminal PDZ binding motif, is involved in ligand endocytosis triggered by mono-ubiquitination, and, as a consequence of regulated intramembrane proteolysis, can be released into the cytosol as a signaling fragment. The intracellular region of Jagged-1 may then exist in at least two forms: as a membrane-tethered protein located at the interface between the membrane and the cytoplasm, and as a soluble nucleocytoplasmic protein. To investigate its structural properties, a recombinant protein corresponding to the human Jagged-1 intracellular region (J1_tmic) was expressed, purified, and characterized in different environments using various biophysical methods such as circular dichroism, tryptophan fluorescence, size-exclusion chromatography, and NMR. In solution, J1_tmic behaves as an intrinsically disordered protein, but displays a significant helical propensity. In the presence of SDS micelles or negatively charged phospholipid micelles and vesicles, used to mimick the interface between the plasma membrane and the cytosol, J1_tmic gains partial helical structure. The partial folding and association of the intracellular region of Jagged-1 with the membrane is expected to reduce its “capture radius” towards target proteins and to make selected residues unavailable for post-translational modifications or binding. Binding of Jagged-1 intracellular region to the PDZ domain of afadin, a protein located at cell-cell adherens junctions, couples Notch signaling with the adhesion system and the cytoskeleton. The interaction between the PDZ domain of afadin (AF6_PDZ) and a series of polypeptides comprising the Jagged-1 PDZ-binding motif (EYIV) was investigated using NMR chemical shift perturbation and surface plasmon resonance. It was shown that binding of Jagged-1 intracellular region to AF6_PDZ is strictly local, involving only the last six residues of the binding motif and the PDZ binding groove, and that it does not trigger global folding of J1_tmic. In the C-terminal region of Jagged-1 cytoplasmic tail, four potential phosphorylation sites can be identified, one of them (Y1216) located in the PDZbinding motif. It was found that, while phosphorylation at any of these sites disrupts binding of the C-terminal peptides to lipid micelles, phosphorylation at (Y1216) also affects the interaction with AF6_PDZ, with a reduction in the binding affinity. Phosphorylation thus provides a potential way to modulate the interaction of Jagged-1 C-terminal region not only with the membrane but also with the partner PDZ. It was also shown that the R1213Q mutation in the PDZ binding motif associated with a congenital obstruction of the bile ducts, increases the affinity for AF6_PDZ. In summary, this work presents the first biochemical and structural characterization of Jagged-1 cytoplasmic tail in solution and in environments that mimic the membrane/cytoplasm interface, and the first biophysical study on its interaction with the afadin PDZ domain.