Jagged-1, one of the five Notch ligands in man, is a membrane-spanning protein made of a large extracellular region and a 125-residue cytoplasmic tail bearing a C-terminal PDZ recognition motif ((1213) RMEYIV(1218) ). 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. Using NMR chemical shift perturbation and surface plasmon resonance, we studied the interaction between the PDZ domain of afadin (AF6_PDZ) and a series of polypeptides comprising the PDZ-binding motif. Chemical shift mapping of AF6_PDZ upon binding of ligands of different length (6, 24, and 133 residues) showed that the interaction is strictly local and involves only the binding groove in the PDZ. The recombinant protein corresponding to the entire intracellular region of Jagged-1, J1_ic, is mainly disordered in solution, and chemical shift mapping of J1_ic in the presence of AF6_PDZ showed that binding is not coupled to folding. Binding studies on a series of 24-residue peptides phosphorylated at different positions showed that phosphorylation of the tyrosine at position -2 of the PDZ-binding motif decreases its affinity for AF6_PDZ, and may play a role in the modulation of this interaction. Finally, we show that the R1213Q mutation located in the PDZ-binding motif and associated with extrahepatic biliary atresia increases the affinity for AF6_PDZ, suggesting that this syndrome may arise from an imbalance in the coupling of Notch signaling to the cytoskeleton.