title: Folding of an intrinsically disordered protein by phosphorylation as a regulatory switch.
abstract: Intrinsically disordered proteins play important roles in cell signalling, transcription, translation and cell cycle regulation. Although they lack stable tertiary structure, many intrinsically disordered proteins undergo disorder-to-order transitions upon binding to partners. Similarly, several folded proteins use regulated order-to-disorder transitions to mediate biological function. In principle, the function of intrinsically disordered proteins may be controlled by post-translational modifications that lead to structural changes such as folding, although this has not been observed. Here we show that multisite phosphorylation induces folding of the intrinsically disordered 4E-BP2, the major neural isoform of the family of three mammalian proteins that bind eIF4E and suppress cap-dependent translation initiation. In its non-phosphorylated state, 4E-BP2 interacts tightly with eIF4E using both a canonical YXXXXLΦ motif (starting at Y54) that undergoes a disorder-to-helix transition upon binding and a dynamic secondary binding site. We demonstrate that phosphorylation at T37 and T46 induces folding of residues P18-R62 of 4E-BP2 into a four-stranded β-domain that sequesters the helical YXXXXLΦ motif into a partly buried β-strand, blocking its accessibility to eIF4E. The folded state of pT37pT46 4E-BP2 is weakly stable, decreasing affinity by 100-fold and leading to an order-to-disorder transition upon binding to eIF4E, whereas fully phosphorylated 4E-BP2 is more stable, decreasing affinity by a factor of approximately 4,000. These results highlight stabilization of a phosphorylation-induced fold as the essential mechanism for phospho-regulation of the 4E-BP:eIF4E interaction and exemplify a new mode of biological regulation mediated by intrinsically disordered proteins.
authors.affiliation: 1] Molecular Structure and Function Program, Hospital for Sick Children, Toronto, Ontario M5G 0A4, Canada [2] Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Alaji
authors.initials: A
authors.lastname: Bah
authors.affiliation: 1] Molecular Structure and Function Program, Hospital for Sick Children, Toronto, Ontario M5G 0A4, Canada [2] Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Robert M
authors.initials: RM
authors.lastname: Vernon
authors.affiliation: Molecular Structure and Function Program, Hospital for Sick Children, Toronto, Ontario M5G 0A4, Canada.
authors.forename: Zeba
authors.initials: Z
authors.lastname: Siddiqui
authors.affiliation: 1] Molecular Structure and Function Program, Hospital for Sick Children, Toronto, Ontario M5G 0A4, Canada [2] Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Mickaël
authors.initials: M
authors.lastname: Krzeminski
authors.affiliation: 1] Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada [2] Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Ranjith
authors.initials: R
authors.lastname: Muhandiram
authors.affiliation: Molecular Structure and Function Program, Hospital for Sick Children, Toronto, Ontario M5G 0A4, Canada.
authors.forename: Charlie
authors.initials: C
authors.lastname: Zhao
authors.affiliation: Department of Biochemistry and Goodman Cancer Research Centre, McGill University, Montréal, Quebec H3G 1Y6, Canada.
authors.forename: Nahum
authors.initials: N
authors.lastname: Sonenberg
authors.affiliation: 1] Molecular Structure and Function Program, Hospital for Sick Children, Toronto, Ontario M5G 0A4, Canada [2] Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada [3] Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada [4] Department of Chemistry, University of Toronto, Toronto, Ontario M5S 3H6, Canada.
authors.forename: Lewis E
authors.initials: LE
authors.lastname: Kay
authors.affiliation: 1] Molecular Structure and Function Program, Hospital for Sick Children, Toronto, Ontario M5G 0A4, Canada [2] Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Julie D
authors.initials: JD
authors.lastname: Forman-Kay
mesh_terms: D001665:Binding Sites; D039561:Eukaryotic Initiation Factor-4E / Q000737:chemistry* / Q000378:metabolism*; D039642:Eukaryotic Initiation Factors / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D064267:Intrinsically Disordered Proteins / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D010766:Phosphorylation; D011485:Protein Binding; D017510:Protein Folding; D017433:Protein Structure, Secondary; D015398:Signal Transduction
chemical_list: C104151:EIF4EBP2 protein, human; D039561:Eukaryotic Initiation Factor-4E; D039642:Eukaryotic Initiation Factors; D064267:Intrinsically Disordered Proteins