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The dynamic mechanism of 4E-BP1 recognition and phosphorylation by mTORC1
Journal
Molecular Cell
Volume
81
Number
11
Pages / Article-Number
2403-2416.e5
Keywords
NMR spectroscopy; atypical kinase; hierarchical phosphorylation; intrinsically disordered protein; kinetic modelling; mTOR signaling; multi‑site binding; protein dynamics; target of rapamycin; translational control
Mesh terms
Adaptor Proteins, Signal Transducing, metabolism; Binding Sites; Cell Cycle Proteins, metabolism; Chaetomium, genetics; Cloning, Molecular; Crystallography, X-Ray; Escherichia coli, metabolism; Eukaryotic Initiation Factor-4E, metabolism; Gene Expression; Genetic Vectors, metabolism; Humans; Kinetics; Mechanistic Target of Rapamycin Complex 1, metabolism; Models, Molecular; Phosphorylation; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Protein Processing, Post-Translational; Recombinant Proteins, metabolism; Regulatory-Associated Protein of mTOR, metabolism; Signal Transduction; Structural Homology, Protein; Substrate Specificity; TOR Serine-Threonine Kinases, metabolism
Abstract
The activation of cap-dependent translation in eukaryotes requires multisite, hierarchical phosphorylation of 4E-BP by the 1 MDa kinase mammalian target of rapamycin complex 1 (mTORC1). To resolve the mechanism of this hierarchical phosphorylation at the atomic level, we monitored by NMR spectroscopy the interaction of intrinsically disordered 4E binding protein isoform 1 (4E-BP1) with the mTORC1 subunit regulatory-associated protein of mTOR (Raptor). The N-terminal RAIP motif and the C-terminal TOR signaling (TOS) motif of 4E-BP1 bind separate sites in Raptor, resulting in avidity-based tethering of 4E-BP1. This tethering orients the flexible central region of 4E-BP1 toward the mTORC1 kinase site for phosphorylation. The structural constraints imposed by the two tethering interactions, combined with phosphorylation-induced conformational switching of 4E-BP1, explain the hierarchy of 4E-BP1 phosphorylation by mTORC1. Furthermore, we demonstrate that mTORC1 recognizes both free and eIF4E-bound 4E-BP1, allowing rapid phosphorylation of the entire 4E-BP1 pool and efficient activation of translation. Finally, our findings provide a mechanistic explanation for the differential rapamycin sensitivity of the 4E-BP1 phosphorylation sites.
