title: An allosteric conduit facilitates dynamic multisite substrate recognition by the SCFCdc4ubiquitin ligase.
abstract: The ubiquitin ligase SCFCdc4mediates phosphorylation-dependent elimination of numerous substrates by binding one or more Cdc4 phosphodegrons (CPDs). Methyl-based NMR analysis of the Cdc4 WD40 domain demonstrates that Cyclin E, Sic1 and Ash1 degrons have variable effects on the primary Cdc4WD40binding pocket. Unexpectedly, a Sic1-derived multi-CPD substrate (pSic1) perturbs methyls around a previously documented allosteric binding site for the chemical inhibitor SCF-I2. NMR cross-saturation experiments confirm direct contact between pSic1 and the allosteric pocket. Phosphopeptide affinity measurements reveal negative allosteric communication between the primary CPD and allosteric pockets. Mathematical modelling indicates that the allosteric pocket may enhance ultrasensitivity by tethering pSic1 to Cdc4. These results suggest negative allosteric interaction between two distinct binding pockets on the Cdc4WD40domain may facilitate dynamic exchange of multiple CPD sites to confer ultrasensitive dependence on substrate phosphorylation.
authors.affiliation: Molecular Structure &Function, The Hospital for Sick Children, Toronto, Ontario, Canada M5G 0A4.
authors.forename: Veronika
authors.initials: V
authors.lastname: Csizmok
authors.affiliation: Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada M5G 1X5.
authors.forename: Stephen
authors.initials: S
authors.lastname: Orlicky
authors.affiliation: Institute for Research in Immunology and Cancer, Université de Montréal, Montréal, Quebec, Canada H3C 3J7.
authors.forename: Jing
authors.initials: J
authors.lastname: Cheng
authors.affiliation: Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada M5S 1A8.|Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada M5S 1A8.|School of Polymer Science and Engineering, Qingdao University of Science and Technology, Shandong 266042, China.
authors.forename: Jianhui
authors.initials: J
authors.lastname: Song
authors.affiliation: Molecular Structure &Function, The Hospital for Sick Children, Toronto, Ontario, Canada M5G 0A4.
authors.forename: Alaji
authors.initials: A
authors.lastname: Bah
authors.affiliation: Institute for Research in Immunology and Cancer, Université de Montréal, Montréal, Quebec, Canada H3C 3J7.
authors.forename: Neda
authors.initials: N
authors.lastname: Delgoshaie
authors.affiliation: Molecular Structure &Function, The Hospital for Sick Children, Toronto, Ontario, Canada M5G 0A4.
authors.forename: Hong
authors.initials: H
authors.lastname: Lin
authors.affiliation: Molecular Structure &Function, The Hospital for Sick Children, Toronto, Ontario, Canada M5G 0A4.
authors.forename: Tanja
authors.identifier: 0000-0002-1827-3811
authors.initials: T
authors.lastname: Mittag
authors.affiliation: Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada M5G 1X5.|Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada M5S 1A8.|Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada M5S 1A8.
authors.forename: Frank
authors.initials: F
authors.lastname: Sicheri
authors.affiliation: Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada M5S 1A8.|Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada M5S 1A8.
authors.forename: Hue Sun
authors.initials: HS
authors.lastname: Chan
authors.affiliation: Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada M5G 1X5.|Institute for Research in Immunology and Cancer, Université de Montréal, Montréal, Quebec, Canada H3C 3J7.
authors.forename: Mike
authors.initials: M
authors.lastname: Tyers
authors.affiliation: Molecular Structure &Function, The Hospital for Sick Children, Toronto, Ontario, Canada M5G 0A4.|Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada M5S 1A8.
authors.forename: Julie D
authors.initials: JD
authors.lastname: Forman-Kay
mesh_terms: D000494:Allosteric Regulation; D000495:Allosteric Site; D001665:Binding Sites; D003001:Cloning, Molecular; D019927:Cyclin E / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D050756:Cyclin-Dependent Kinase Inhibitor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D010748:Phosphopeptides; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D000072757:Protein Conformation, beta-Strand; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D044843:SKP Cullin F-Box Protein Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: C098733:ASH1 protein, S cerevisiae; D019927:Cyclin E; D050756:Cyclin-Dependent Kinase Inhibitor Proteins; D010748:Phosphopeptides; D011994:Recombinant Proteins; D012097:Repressor Proteins; C086926:SIC1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D044843:SKP Cullin F-Box Protein Ligases