Volume 3, Issue 6, Pages 707-716 (June 1999) Cap-Dependent Translation Initiation in Eukaryotes Is Regulated by a Molecular Mimic of eIF4G  Joseph Marcotrigiano, Anne-Claude Gingras, Nahum Sonenberg, Stephen K Burley  Molecular Cell  Volume 3, Issue 6, Pages 707-716 (June 1999) DOI: 10.1016/S1097-2765(01)80003-4

Figure 5 eIF4GII and 4E-BP1 Peptides Binding to the Convex Dorsal Surface of eIF4E GRASP (Gilson et al. 1988) representation of the eIF4E surface, colored coded for electrostatic potential (red < −10 kBT; blue > 10 kBT), calculated in the absence of the peptides (see Figure 5C in Marcotrigiano et al. 1997) using a water probe radius of 1.4 Å. The (A) eIF4GII and (B) 4E-BP1 peptides are depicted with orange and yellow, respectively. (C) Overlay of the eIF4GII (orange) and 4E-BP1 (yellow) peptides with the solvent-accessible convex surface of eIF4E, colored green for residues that are absolutely conserved among all known eIF4E sequences (Marcotrigiano et al. 1997). Molecular Cell 1999 3, 707-716DOI: (10.1016/S1097-2765(01)80003-4)

Figure 1 Characterization of Recombinant 4E-BP1 and the 4E-BP1 and eIF4GII Peptides Schematic representation showing the effects of scanning deletion mutagenesis on translation inhibition in vitro. Chymotrypsin and V8 protease cleavage of 4BEP1 in the presence of eIF4E (residues 28–212) plus 7-methyl-GDP is shown at the bottom of the panel. The solid white area represents the peptide (residues 51–67) crystallized with eIF4E and 7-methyl-GDP. The striped areas correspond to portions of 4E-BP1 that are sensitive to protease digestion. Molecular Cell 1999 3, 707-716DOI: (10.1016/S1097-2765(01)80003-4)

Figure 2 Sequence Alignment of All Known eIF4E Recognition Motifs from eIF4Gs and the eIF4E-Binding Proteins Sequence alignments of the eIF4E recognition motifs of mammalian 4E-BP1 (Pause et al. 1994), 4E-BP2 (Pause et al. 1994), 4E-BP3 (Poulin et al. 1998), mammalian eIF4GI (Imataka et al. 1998), eIF4GII (Gradi et al. 1998a), yeast TIF4631 (p150) (Goyer et al. 1993), yeast TIF4632 (p130) (Goyer et al. 1993), yeast CAF20 (p20) (Altmann et al. 1997), and wheat germ p82 (Allen et al. 1992). Individual and consensus sequences are numbered with respect to the invariant tyrosine (position 0). Interactions with eIF4E are illustrated schematically across the top, with bold type for residues comprising the phylogenetically conserved eIF4E surface feature (green in Figure 5C). (Residues in bold here are underlined in the text.) Symbols: §, oligopeptides used in structure determinations; R/K, partially conserved basic residue (8/9 sequences); b, partially conserved hydrophobic (7/9 sequences); boldface Y, invariant tyrosine; boldface f, partially conserved phenylalanine (7/9 sequences); φ, conserved hydrophobic (9/9 sequences); boldface L, invariant leucine; PO4, possible 4E-BP phosphorylation site; X, variable residue. Molecular Cell 1999 3, 707-716DOI: (10.1016/S1097-2765(01)80003-4)

Figure 3 Structures of Two Murine eIF4E/7-methyl-GDP/Peptide Ternary Complexes Ribbon diagrams of the eIF4E (blue)/7-methyl-GDP (ball-and-stick representation)/peptide ternary complex structures viewed perpendicular to the eIF4E’s β sheet (A and C) and in profile, rotated 90° about the vertical from the previous view (B and D). 7-methyl-GDP is located in the cap-binding slot on the convex face of eIF4E, 35 Å from peptide binding site. Upper: The eIF4GII peptide (orange) forms an L-shaped structure that interacts with the N terminus of eIF4E and two of three long α helices (H1 and H2) on its convex dorsal surface. Lower: The 4E-BP1 peptide (yellow) adopts a very similar conformation on binding to eIF4E. Molecular Cell 1999 3, 707-716DOI: (10.1016/S1097-2765(01)80003-4)

Figure 4 eIF4GII and 4E-BP1 Peptides Interacting with eIF4E All views are colored and orientated similarly to the one shown in Figure 3A and Figure 3C. The positions of the peptide residues are shown in parentheses, with the invariant Tyr defined as position 0. Stick figure representations of the peptide binding sites, showing selected residues involved in ternary complex formation. Hydrogen bonds and water-mediated contacts are depicted as dashed lines. The magenta sphere labeled “B” represents a bridging water molecule. Residues involved in intermolecular contacts are labeled in blue for eIF4E and italics for the (A) eIF4GII and (B) 4E-BP1 peptides. (C) Overlay of the peptide structures based upon superposition of eIF4E only. eIF4GII (orange) and 4E-BP1 (yellow) peptides, showing selected side chains that interact with eIF4E. Molecular Cell 1999 3, 707-716DOI: (10.1016/S1097-2765(01)80003-4)

Figure 6 CD Spectroscopy of Recombinant 4E-BP1 and eIF4GII and 4E-BP1 Peptides Circular dichroism spectra of recombinant 4E-BP1 (°) and eIF4GII (·) and 4E-BP1 (Δ) peptides in solution, plotted as versus mean residue ellipticity (deg cm2 dmol−1) versus wavelength (nm). Molecular Cell 1999 3, 707-716DOI: (10.1016/S1097-2765(01)80003-4)