title: Flexibility of the PDZ-binding motif in the micelle-bound form of Jagged-1 cytoplasmic tail.
abstract: Human Jagged-1, one of the ligands of Notch receptors, is a transmembrane protein composed of a large extracellular region and a 125-residue cytoplasmic tail which bears a C-terminal PDZ recognition motif. To investigate the interaction between Jagged-1 cytoplasmic tail and the inner leaflet of the plasma membrane we determined, by solution NMR, the secondary structure and dynamics of the recombinant protein corresponding to the intracellular region of Jagged-1, J1_tmic, bound to negatively charged lysophospholipid micelles. NMR showed that the PDZ binding motif is preceded by four alpha-helical segments and that, despite the extensive interaction between J1_tmic and the micelle, the PDZ binding motif remains highly flexible. Binding of J1_tmic to negatively charged, but not to zwitterionic vesicles, was confirmed by surface plasmon resonance. To study the PDZ binding region in more detail, we prepared a peptide corresponding to the last 24 residues of Jagged-1, J1C24, and different phosphorylated variants of it. J1C24 displays a marked helical propensity and undergoes a coil-helix transition in the presence of negatively charged, but not zwitterionic, lysophospholipid micelles. Phosphorylation at different positions drastically decreases the helical propensity of the peptides and abolishes the coil-helix transition triggered by lysophospholipid micelles. We propose that phosphorylation of residues upstream of the PDZ binding motif may shift the equilibrium from an ordered, membrane-bound, interfacial form of Jagged-1 C-terminal region to a more disordered form with an increased accessibility of the PDZ recognition motif, thus playing an indirect role in the interaction between Jagged-1 and the PDZ-containing target protein.
authors.affiliation: International Centre for Genetic Engineering and Biotechnology, AREA Science Park Padriciano 99, 1-34149 Trieste, Italy.
authors.forename: Matija
authors.initials: M
authors.lastname: Popovic
authors.forename: Ventsislav
authors.initials: V
authors.lastname: Zlatev
authors.forename: Vesna
authors.initials: V
authors.lastname: Hodnik
authors.forename: Gregor
authors.initials: G
authors.lastname: Anderluh
authors.forename: Isabella C
authors.initials: IC
authors.lastname: Felli
authors.forename: Sándor
authors.initials: S
authors.lastname: Pongor
authors.forename: Alessandro
authors.initials: A
authors.lastname: Pintar
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002942:Circular Dichroism; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000072100:Jagged-1 Protein; D007700:Kinetics; D008246:Lysophospholipids / Q000737:chemistry / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008823:Micelles; D008969:Molecular Sequence Data; D054731:PDZ Domains; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D018583:Pliability; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D000072099:Serrate-Jagged Proteins; D020349:Surface Plasmon Resonance
chemical_list: D002135:Calcium-Binding Proteins; D036341:Intercellular Signaling Peptides and Proteins; C000606273:JAG1 protein, human; D000072100:Jagged-1 Protein; D008246:Lysophospholipids; D008565:Membrane Proteins; D008823:Micelles; D010455:Peptides; D011994:Recombinant Proteins; D000072099:Serrate-Jagged Proteins