The dynamic mechanism of 4E-BP1 recognition and phosphorylation by mTORC1

•Binding of 4E-BP1 by mTORC1 subunit Raptor observed NMR•Raptor recognizes two interaction sites in intrinsically disordered 4E-BP1•Two-site binding orients for hierarchical multisite phosphorylation•The ternary eIF4E:4E-BP1:mTORC1 assembly mediates rapid signaling The activation cap-dependent translation eukaryotes requires multisite, phosphorylation 4E-BP the 1 MDa kinase mammalian target rapamycin complex (mTORC1). To resolve mechanism this at atomic level, we monitored NMR spectroscopy 4E protein isoform (4E-BP1) with regulatory-associated mTOR (Raptor). N-terminal RAIP motif and C-terminal TOR (TOS) bind separate Raptor, resulting avidity-based tethering 4E-BP1. This flexible central region toward site phosphorylation. structural constraints imposed interactions, combined phosphorylation-induced conformational switching 4E-BP1, explain hierarchy mTORC1. Furthermore, demonstrate that both free eIF4E-bound allowing entire pool efficient translation. 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An example part studied solution viral nucleocapsids reach sizes several megadaltons (Communie 2013Communie Habchi Yabukarski Blocquel Schneider Tarbouriech Papageorgiou Ruigrok R.W. Jamin al.Atomic description nucleoprotein phosphoprotein Hendra virus.PLoS Pathog. 2013; 9: e1003631Crossref (58) 2011Jensen Communie Ribeiro E.A. Martinez Desfosses Salmon Mollica Gabel Longhi al.Intrinsic disorder measles virus nucleocapsids.Proc. 2011; 108: 9839-9844Crossref (157) Here, investigate interplay among phosphorylation, binding. We employ monitor level study influenced presence follow changes peak intensities times. Notably, effective experiments increases 13 kDa 163 Using representatives different states, map relevant configuration space obtain full mTORC1-mediated, 4E-BP1-based control. optimized X-ray crystal Chaetomium thermophilum (CtRaptor, 44% identity Raptor), after limited proteolysis, 3.5 (Table 1). Compared previous crystallographic model 4.3 allowed representation backbone poly-alanine chain, current data enabled side-chain modeling, thus significantly improved model. CtRaptor adopts Z-shaped conformation (RNC) WD40 positioned opposite ends armadillo repeat-containing (ARM) (Figures 1A–1C). Three extended stretches (termed S1, S2, S3) remain stably associated core proteolytic digestion (Figure S1A). S1 (residues 212–255; 18–56 Raptor) attached N-terminally RNC runs along ARM contact blades two, three, four domain. S2 S3 linker 939–991; 806–840 terminal ?-helical repeat unit flanged side 1090–1171; 950–1009 domain, cleft formed concave surface connects aligns Arabidopsis thaliana (AtRaptor) S1B; present all orthologs may beyond contributing integrity. identify TOS-motif site, soaked crystals synthetic selenomethionine-labeled peptide (FEMDI) derived sequence. identified positive difference density anomalous pocket transition domains, indicating S1C). These good agreement those extracted mTORC1-4E-BP1 cryoelectron microscopy (cryo-EM) AtRaptor structures peptides Scholar).Table 1Data collection refinement statisticsDatasetCtRaptor (PDB: 6ZPN)Data collectionSpace groupP41212Unit cell: a, b, c (Å)183.30, 183.30, 271.39Unit ?, ?, ? (°)90, 90, 90Resolution (Å)49.97–3.5 (3.6–3.5)R means (%)36.7 (395.5)CC1/298.9 (36.7)I/?I5 (0.8)No. reflections: total752,603 (60,129)No. unique58,884 (4,557)Completeness (%)99.9 (100)Redundancy12.8 (13.2)RefinementResolution (Å)47.46–3.5No. reflections58,659Rwork/Rfree22.8/24.5No. atoms: protein34,649No. non-protein0No. solvent0Average B factor: protein142.8Average non-proteinN/AAverage solventN/ABond lengths (root mean square deviation (RMSD), Å)0.005Bond angles (RMSD, °)0.877Ramachandran plot: % favored92.3Ramachandran allowed7.5Ramachandran outliers0.2 Open table new tab aqueous features intense peaks narrow amide 1H chemical shift dispersion, expected IDP 1D S1D). 93% non-proline amides could assigned pH 6.0, assignment was transferred titration 8.0 S1E). value biochemical stability full-length were then mapped 2D [15N,1H]-TROSY spectra obtained during Raptor. Chemical perturbations marginal, individual decreased concentrations, interaction. following 15N-labeled unlabeled S1) displayed bar graphs showing ratio I/I0 intensity Raptor-bound (I) (I0). Residues below detection limit reference spectrum (I0 ? 0) excluded analysis, shown residues. non-zero initial > disappear (I short negative 1E S2A–S2C). distinguish scenarios residue-specific manner. substantial reduction intensity, disappearance saturating conditions, fully protonated. Additional occur intermediate within (Lukhele 2013Lukhele bipartite interface.Structure. 2186-2196Abstract (63) contrast, preserve loc