title: Conformational characterization of the intrinsically disordered protein Chibby: Interplay between structural elements in target recognition.
abstract: The protein Chibby (Cby) is an antagonist of the Wnt signaling pathway, where it inhibits the binding between the transcriptional coactivator β-catenin and the Tcf/Lef transcription factors. The 126 residue Cby is partially disordered; its N-terminal half is unstructured while its C-terminal half comprises a coiled-coil domain. Previous structural analyses of Cby using NMR spectroscopy suffered from severe line broadening for residues within the protein's C-terminal half, hindering detailed characterization of the coiled-coil domain. Here, we use hydrogen/deuterium exchange-mass spectrometry (HDX-MS) to examine Cby's C-terminal half. Results reveal that Cby is divided into three structural elements: a disordered N-terminal half, a coiled-coil domain, and a C-terminal unstructured extension consisting of the last ∼ 25 residues (which we term C-terminal extension). A series of truncation constructs were designed to assess the roles of individual structural elements in protein stability and Cby binding to TC-1, a positive regulator of the Wnt signaling pathway. CD and NMR data show that Cby maintains coiled-coil structure upon deletion of either disordered region. NMR and ITC binding experiments between Cby and TC-1 illustrate that the interaction is retained upon deletion of either Cby's N-terminal half or its C-terminal extension. Intriguingly, Cby's C-terminal half alone binds to TC-1 with significantly greater affinity compared to full-length Cby, implying that target binding of the coiled-coil domain is affected by the flanking disordered regions.
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 Chemistry, The University of Western Ontario, London, Ontario, N6A 5B7, Canada.
authors.forename: Modupeola A
authors.initials: MA
authors.lastname: Sowole
authors.affiliation: Department of Chemistry, The University of Western Ontario, London, Ontario, N6A 5B7, Canada.
authors.forename: Mohammad A
authors.initials: MA
authors.lastname: Halim
authors.affiliation: Department of Biochemistry, The University of Western Ontario, London, Ontario, N6A 5C1, Canada.|Department of Chemistry, The University of Western Ontario, London, Ontario, N6A 5B7, Canada.
authors.forename: Lars
authors.initials: L
authors.lastname: Konermann
authors.affiliation: Department of Biochemistry, The University of Western Ontario, London, Ontario, N6A 5C1, Canada.|Department of Chemistry, The University of Western Ontario, London, Ontario, N6A 5B7, Canada.
authors.forename: Wing-Yiu
authors.initials: WY
authors.lastname: Choy
mesh_terms: D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics; D003001:Cloning, Molecular; D017124:Conserved Sequence; D041961:Deuterium Exchange Measurement; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D064267:Intrinsically Disordered Proteins / Q000737:chemistry* / Q000235:genetics; D013058:Mass Spectrometry / Q000379:methods; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000235:genetics; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics; D011485:Protein Binding; D000072417:Protein Domains; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D060449:Wnt Signaling Pathway
chemical_list: C473750:CBY1 protein, human; D002352:Carrier Proteins; D064267:Intrinsically Disordered Proteins; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D011994:Recombinant Proteins; C418741:TCIM protein, human
keywords: Chibby; HDX-MS; ITC; NMR; Thyroid Cancer 1; coiled-coil; intrinsically disordered protein