title: Structural Analysis of the 14-3-3ζ/Chibby Interaction Involved in Wnt/β-Catenin Signaling.
abstract: The partially disordered Chibby (Cby) is a conserved nuclear protein that antagonizes the Wnt/β-catenin signaling pathway. By competing with the Tcf/Lef family proteins for binding to β-catenin, Cby abrogates the β-catenin-mediated transcription of Wnt signaling genes. Additionally, upon phosphorylation on S20 by the kinase Akt, Cby forms a complex with 14-3-3 to facilitate the nuclear export of β-catenin, which represents another crucial mechanism for the regulation of Wnt signaling. To obtain a mechanistic understanding of the 14-3-3/Cby interaction, we have extensively characterized the complex using X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and isothermal titration calorimetry (ITC). The crystal structure of the human 14-3-3ζ/Cby protein-peptide complex reveals a canonical binding mode; however the residue at the +2 position from the phosphorylated serine is shown to be uniquely oriented relative to other solved structures of 14-3-3 complexes. Our ITC results illustrate that although the phosphorylation of S20 is essential for Cby to recognize 14-3-3, residues flanking the phosphorylation site also contribute to the binding affinity. However, as is commonly observed in other 14-3-3/phosphopeptide crystal structures, residues of Cby flanking the 14-3-3 binding motif lack observable electron density. To obtain a more detailed binding interface, we have completed the backbone NMR resonance assignment of 14-3-3ζ. NMR titration experiments reveal that residues outside of the 14-3-3 conserved binding cleft, namely a flexible loop consisting of residues 203-210, are also involved in binding Cby. By using a combined X-ray and NMR approach, we have dissected the molecular basis of the 14-3-3/Cby interaction.
authors.affiliation: Department of Biochemistry, The University of Western Ontario, London, Ontario N6A 5C1, Canada.
authors.forename: Ryan C
authors.initials: RC
authors.lastname: Killoran
authors.affiliation: Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore, 117543, Singapore.
authors.forename: Jingsong
authors.initials: J
authors.lastname: Fan
authors.affiliation: Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore, 117543, Singapore.
authors.forename: Daiwen
authors.initials: D
authors.lastname: Yang
authors.affiliation: Department of Biochemistry, The University of Western Ontario, London, Ontario N6A 5C1, Canada.
authors.forename: Brian H
authors.initials: BH
authors.lastname: Shilton
authors.affiliation: Department of Biochemistry, The University of Western Ontario, London, Ontario N6A 5C1, Canada.
authors.forename: Wing-Yiu
authors.initials: WY
authors.lastname: Choy
mesh_terms: D048948:14-3-3 Proteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D059372:Nucleotide Motifs; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D060449:Wnt Signaling Pathway
chemical_list: D048948:14-3-3 Proteins; C473750:CBY1 protein, human; D002352:Carrier Proteins; D009687:Nuclear Proteins; D010455:Peptides