1. Volume 57, Issue 6, Pages 1074-1087 (March 2015)
Molecular Architecture of 4E-BP Translational Inhibitors Bound to eIF4E 
Daniel Peter, Cátia Igreja, Ramona Weber, Lara Wohlbold, Catrin Weiler, Linda Ebertsch, Oliver Weichenrieder, Elisa Izaurralde 
Molecular Cell 
Volume 57, Issue 6, Pages (March 2015)
DOI: /j.molcel
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2. Molecular Cell 2015 57, 1074-1087DOI: (10.1016/j.molcel.2015.01.017)
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3. Figure 1 Overall Structures of 4E-BPs and eIF4G Bound to eIF4E
(A) Domain organization of eIF4G and the 4E-BPs analyzed in this study. The 4E-BPs contain a canonical (C) and a non-canonical (NC) 4E-BM. CUP and 4E-T contain a region with similarity to human 4E-T; eIF4G contains a PABP-interacting region and MIF4G and MA3 domains. See also Figure S1.
(B–D) Overview of the structures of eIF4E bound to the indicated 4E-BPs. Selected secondary structure elements are labeled in black for eIF4E and in color for 4E-BPs.
(E) Superposition of the structures of eIF4E bound to 4E-T, Thor, and 4E-BP1 reveals differences in the conformation of the NC loops.
(F) Schematic representation of eIF4E bound to 4E-BPs.
(G) Alignment of the eIF4G and 4E-BP structures by superposition of their canonical helices indicates that the elbow loop is a common structural feature of 4E-BPs.
(H) Overview of the structure of eIF4E bound to eIF4G and cap analog. See also Figure S2.
(I) Schematic representation of eIF4E bound to eIF4G and m7GTP.
(J) Overview of the structure of the eIF4E-CUP complex (PDB accession number 4AXG; Kinkelin et al., 2012).
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4. Figure 2
Surface Representation of the 4E-BP and eIF4G Binding Interfaces on eIF4E
(A–D) The eIF4E residues that are within 4 Å̊ of the bound 4E-BP or eIF4G peptides are shown in color. Selected residues lining the binding surface are labeled in black for eIF4E and in color for 4E-BPs and eIF4G. The 4E-BP and the eIF4G peptides are shown as sticks.
(E) Superposition of the eIF4E surfaces that bind eIF4G and 4E-BPs. The region of overlap between the eIF4G and 4E-BP binding surfaces is shown in yellow. The surface shared by 4E-BPs but not used by eIF4G is colored in cyan. Surfaces used exclusively by Thor and 4E-T are shown in magenta and green, respectively.
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5. Figure 3
Interaction of the Canonical Motifs of 4E-BPs and eIF4G with the Dorsal Surface of eIF4E
(A–H) Close-up views of the interaction between the dorsal surface of eIF4E and the canonical helices of 4E-BPs and eIF4G. Selected interface residues are shown as gray sticks for eIF4E and as colored sticks for 4E-BPs and eIF4G. See also Figures S2–S4.
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6. Figure 4 The Elbow and the Non-Canonical Loops
(A–C) Close-up views of the elbow loops and interactions within the loops. The side chains of L67Thor, K69BP1 and K234ET have been omitted for clarity.
(D–F) Close-up views of the interaction between eIF4E and the 4E-BP linker regions C-terminal to the elbow loops. Selected interface residues are shown as gray and colored sticks for eIF4E and 4E-BPs, respectively. The side chain of K69BP1 has been omitted for clarity; R254ET and Q294ET are not visible in the structure. Conserved phosphorylated residues in Thor and 4E-BP1 are boxed.
(G–I) Interaction of the non-canonical loops with the lateral surface of eIF4E. See also Figure S2J.
(J–L) Close-up views of the non-canonical loops and interactions with residues lining the lateral hydrophobic pocket of eIF4E. Selected 4E-BP and eIF4E residues are shown as sticks (C78–R81Thor, G80–S83BP1, and G40–I444ET). eIF4E secondary structure elements (β1 and β2) and the side chains of Dm I1124E (Hs I794E), C78Thor, L80Thor, T82BP1, W424ET, and I444ET have been omitted for clarity.
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7. Figure 5 Effects of Phosphorylation of Thor Linker Residues
(A and B) Half-life of eIF4E-eIF4G complexes. Purified eIF4E-eIF4G complexes containing His6-tagged eIF4E and MBP-eIF4G (residues 578–650) were incubated with a 2-fold molar excess of Thor (residues 32–83, either wild-type [WT] or the S65D, T70D phosphomimetic mutant). MBP served as a negative control. The proteins bound to eIF4E were pulled down using Ni-NTA beads at the indicated time points. Mean values ± SD from five independent experiments (n = 5) are shown. A representative SDS-PAGE gel is shown in (B). The competitor peptides are labeled in blue, and their positions are highlighted by blue dashed boxes. The black dashed boxes indicate the position of MBP-eIF4G. See also Figures S4F and S4G.
(C and D) The half-life of eIF4E-eIF4G complexes in the presence of a 2-fold molar excess of Thor synthetic peptides (residues 50–83, either WT or phosphorylated at S65 and T70) was determined as described in (A) and (B). Note that the phosphorylated peptide does not stain well with Coomassie. See also Figure S5 and Table S2.
(E and F) Translation efficiency of a R-Luc reporter in Dm cells expressing GFP or GFP-tagged Thor (WT or the S65D, T70D phosphomimetic mutant). Mean values ± SD from three independent experiments are shown. The red dashed line indicates the translation efficiency for the maximum repressive activity exhibited by Thor WT, whereas the black dashed line indicates the translation efficiency expected in the absence of repression. Protein expression was analyzed by western blotting (F). The RNA levels were determined by northern blotting (Figure S4H).
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8. Figure 6 Translational Repression by a 4E-BP Mimic Peptide
(A–E) Overview of the structure of eIF4E bound to the 4EGI-1 inhibitor (PDB accession number 4TPW; Papadopoulos et al., 2014) and superposition with the indicated 4E-BP structures. Selected secondary structure elements and residues are labeled in black for eIF4E and in color for the 4E-BPs. See also Figure S4I.
(F) Overview of the structure of eIF4E bound to the triple chimera peptide (crystal form 2). Selected secondary structure elements are labeled in black for eIF4E and in color for the peptide. See also Figures S6 and S7.
(G and H) The half-life of eIF4E-eIF4G complexes in the presence of 1.5-fold molar excess of CUP or of the triple chimera peptide was determined as described in Figures 5A and 5B. See also Figures S7A and S7B.
(I and J) The translation efficiency of a R-Luc reporter in Dm cells expressing GFP or GFP-tagged Thor (WT) or the triple chimera was analyzed as described in Figures 5E and 5F. Mean values ± SD from three independent experiments are shown. The expression of the GFP-tagged proteins was analyzed by western blotting (J). The RNA levels were determined by northern blotting (Figure S7C). See also Figures S7D and S7E.
Molecular Cell  , DOI: ( /j.molcel )
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