Volume 64, Issue 3, Pages 467-479 (November 2016) The Structures of eIF4E-eIF4G Complexes Reveal an Extended Interface to Regulate Translation Initiation  Stefan Grüner, Daniel Peter, Ramona Weber, Lara Wohlbold, Min-Yi Chung, Oliver Weichenrieder, Eugene Valkov, Cátia Igreja, Elisa Izaurralde  Molecular Cell  Volume 64, Issue 3, Pages 467-479 (November 2016) DOI: 10.1016/j.molcel.2016.09.020 Copyright © 2016 Elsevier Inc. Terms and Conditions

Molecular Cell 2016 64, 467-479DOI: (10.1016/j.molcel.2016.09.020) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 Formation of the Metazoan eIF4E-eIF4G Complex Does Not Require the eIF4E N-Terminal Region (A) Schematic representation of Hs, Dm, and Sc eIF4E proteins. The N-terminal truncations analyzed in this study are indicated by vertical dashed lines. The conserved folded domain is highlighted in dark gray. (B) Schematic organization of the eIF4E-binding regions of Hs, Dm, and Sc eIF4G proteins. The N-terminal region (N), the canonical (C) and non-canonical (NC) 4E-BMs, and the connecting linker (L) are indicated. (C and D) Interaction of N-terminally truncated eIF4E proteins and the eIF4E-binding regions of Hs (C) and Dm (D) eIF4G. The untagged recombinant Hs eIF4E (full-length, Δ23 or Δ35) or purified Dm eIF4E (full-length or Δ68) was pulled down using m7GTP-sepharose beads in the presence of eIF4G fragments. The lanes labeled starting material (SM) show the lysates (2%) and purified proteins (7.5%) used in the pull-down assays. The bound fractions (50% in C and 27% in D) were analyzed by SDS-PAGE and Coomassie blue staining. See also Figure S1A and Table S1. Molecular Cell 2016 64, 467-479DOI: (10.1016/j.molcel.2016.09.020) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 Structures of Hs and Dm eIF4G Proteins Bound to eIF4E (A and B) Surface and cartoon representations of the eIF4E-binding region of Hs eIF4G (green) in complex with cap-bound eIF4E (gray) in two orientations. The bound m7GTP cap analog is shown as sticks. Selected secondary structure elements are labeled. (C and D) Surface and cartoon representations of the structure of Dm eIF4G (orange) bound to eIF4E (gray) in two orientations. Selected secondary structure elements are labeled. (E–H) Schematic representations of eIF4E (E) and of eIF4E bound to Hs (F), Dm (G), and Sc (H) eIF4G. See also Figures S3 and S4; Table S3. Molecular Cell 2016 64, 467-479DOI: (10.1016/j.molcel.2016.09.020) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 Molecular Details of Hs and Dm eIF4E-eIF4G Complexes (A and B) Close-up view of the canonical helix of Hs eIF4G (A) and Dm eIF4G (B) bound to the dorsal surface of the respective eIF4E. In (B), Dm eIF4G residues S601–S616 were omitted for clarity. (C and D) Close-up views of the linker regions of Hs eIF4G (C) and Dm eIF4G (D) contacting the respective eIF4Es. The dashed lines indicate hydrogen bonds or salt bridges. (E and F) Close-up views of the interactions between the non-canonical (NC) loops of Hs eIF4G (E) and Dm eIF4G (F) and the lateral hydrophobic surface of the corresponding eIF4Es. (G) Close-up view of the N-terminal loop of Dm eIF4G assembled on the dorsal surface of Dm eIF4E. Selected residues are shown as sticks. (H) Intramolecular hydrogen bond (dashed lines) network at the N-terminal region of Dm eIF4G. See also Figure S5. Molecular Cell 2016 64, 467-479DOI: (10.1016/j.molcel.2016.09.020) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 Contribution of eIF4G Structural Elements to eIF4E Binding (A) The interaction of Hs His6-eIF4E with the eIF4E-binding region of Hs eIF4G (N+C+L+NC; wild-type or C∗ and NC∗ mutants) was tested using Ni-NTA pull-down assays. The starting material (2% of the lysate or 3% of the purified protein) and bound fractions (25%) were analyzed as described in Figure 1. (B) The interaction of Hs GFP-eIF4G (full-length, wild-type or mutated) with HA-eIF4E in human HEK293T cells was tested by immunoprecipitation using anti-GFP antibodies. The inputs (0.75% for GFP-proteins and 0.5% for HA-eIF4E) and immunoprecipitates (15% for GFP-proteins and 25% for HA-eIF4E) were analyzed by western blotting. (C) Competition between Hs eIF4G and Hs 4E-BP1 in vivo. The lysates from human HEK293T cells expressing the indicated GFP-proteins were incubated with m7GTP-sepharose beads. The input fractions (1% for GFP-proteins and 0.75% for eIF4E and eIF4G) and bound fractions (15% for GFP-proteins, 5% for eIF4E, and 20% for eIF4G) were analyzed by western blotting using anti-eIF4E, anti-eIF4G, and anti-GFP antibodies. The GFP-tagged proteins included MBP, a 4E-BP1 fragment (C+L+NC), and a fragment of Hs eIF4G (C+L+NC; wild-type or C∗ or NC∗ mutants). The position of the 4E-BP1 fragment is indicated by a black dot. (D) The interaction of Dm His6-eIF4E (full-length; wild-type or lateral surface mutant [II-AA]) with Dm eIF4G (N+C+L+NC; wild-type or C∗ mutant) was analyzed by Ni-NTA pull-down assay. The starting material (3% for eIF4G and 8% for eIF4E) and bound fractions (13% for eIF4E WT and 40% for eIF4E II-AA) were analyzed by SDS-PAGE. (E) Interaction of HA-tagged full-length Dm eIF4G (wild-type, C∗ or NC∗ mutants) with endogenous eIF4E in S2 cell lysates was analyzed by immunoprecipitation using anti-HA antibodies. The inputs (0.75% for eIF4G and 0.15% for eIF4E) and immunoprecipitates (30%) were analyzed by western blotting using anti-HA and anti-eIF4E antibodies. (F) Cap pull-down assay showing the association of purified, untagged Dm eIF4E (full-length) with the indicated Dm eIF4G fragments. The starting material (SM, 3% purified eIF4E and 2.5% cell lysates) and bound fractions (25%) were visualized on SDS-PAGE followed by Coomassie staining. See also Tables S1 and S4. Molecular Cell 2016 64, 467-479DOI: (10.1016/j.molcel.2016.09.020) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 5 The Linker and Non-Canonical Motif of 4E-BPs Confer a Competitive Advantage over eIF4G (A–H) In vitro competition assay. Dm eIF4E-eIF4G complexes were incubated in the presence of a 3-fold molar excess of the indicated competitor peptides. (A and B) Show the quantification of eIF4G (N+C+L+NC) remaining bound to eIF4E in the presence of the competitor proteins at different time points (n ≥ 3). The half-life of the eIF4E-eIF4G complex (t1/2, mean ± SDs from three independent experiments) in the presence of the competitor protein and the KD values for the competitor peptides are indicated. (C–H) Show Representative SDS-PAGE gels for each competition assay. The competitor and eIF4G peptides are boxed in blue and black, respectively. The lanes labeled starting material (SM) show the purified peptides and complexes used in the competition assay. See also Figure S2 and Tables S1 and S2. Molecular Cell 2016 64, 467-479DOI: (10.1016/j.molcel.2016.09.020) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 6 The Amino Acid Composition in the Linker Regions Modulates the Competitive Behavior of the 4E-BP and eIF4G Peptides (A) Sequence alignment of the eIF4E-interacting regions of human 4E-BP1–3, Drosophila Thor, and eIF4G proteins. Canonical (C) and non-canonical (NC) motifs are boxed in black. The Pro residues present in the linker region of 4E-BP1–3 and Thor, but absent in eIF4G, are in bold and highlighted with a cyan background. The conserved residues and residues with >70% similarity are highlighted with an orange and yellow background, respectively. (B–E) Competition assay. Dm eIF4E-eIF4G complexes were incubated in the presence of the indicated competitor peptides as described in Figure 5. (B and D) Quantification of the amount of eIF4G remaining associated with eIF4E. The KD values determined by ITC are indicated. (C and E) Representative SDS-PAGE gels for each competition assay. See also Figure S2 and Table S2. Molecular Cell 2016 64, 467-479DOI: (10.1016/j.molcel.2016.09.020) Copyright © 2016 Elsevier Inc. Terms and Conditions