Crystal structure of a minimal eIF4E-Cup complex reveals a general mechanism of eIF4E regulation in translational repression

Abstract

Cup is an eIF4E-binding protein (4E-BP) that plays a central role in translational regulation of localized mRNAs during early Drosophila development. In particular, Cup is required for repressing translation of the maternally contributed oskar, nanos, and gurken mRNAs, all of which are essential for embryonic body axis determination. Here, we present the 2.8 Å resolution crystal structure of a minimal eIF4E-Cup assembly, consisting of the interacting regions of the two proteins. In the structure, two separate segments of Cup contact two orthogonal faces of eIF4E. The eIF4E-binding consensus motif of Cup (YXXXXLΦ) binds the convex side of eIF4E similarly to the consensus of other eIF4E-binding proteins, such as 4E-BPs and eIF4G. The second, noncanonical, eIF4E-binding site of Cup binds laterally and perpendicularly to the eIF4E β-sheet. Mutations of Cup at this binding site were shown to reduce binding to eIF4E and to promote the destabilization of the associated mRNA. Comparison with the binding mode of eIF4G to eIF4E suggests that Cup and eIF4G binding would be mutually exclusive at both binding sites. This shows how a common molecular surface of eIF4E might recognize different proteins acting at different times in the same pathway. The structure provides insight into the mechanism by which Cup disrupts eIF4E-eIF4G interaction and has broader implications for understanding the role of 4E-BPs in translational regulation.

FIGURE 1.

Sequence conservation and interactions in the eIF4E–Cup complex. Structure-based sequence alignment. Above each alignment is a schematic representation of the architecture of the proteins used in this study. Color-filled areas in the scheme identify structural and similarity domains, which include the eIF4E core (purple-blue) and the 4E-T-like region (orange). Indicated are the residue numbers corresponding to the constructs used for the complex reconstitution. (Gray) The portions of the polypeptides ordered in the three-dimensional (3D) structure. The secondary structure elements of Dm eIF4E and Cup are shown below the sequences as rectangles (α-helices) and arrows (β-strands) with the same color code. Dotted lines represent extended/loop regions. Above the sequences, colored circles highlight the residues involved in the interaction with Cup (orange), and eIF4E (purple-blue) as identified using the AquaProt server (Reichmann et al. 2007). Other interactions known from previous structural studies are indicated as colored empty circles above the sequences: interactions with eIF4G (dark green), m⁷GTP (black), and 4E-BP (light green) (Marcotrigiano et al. 1997, 1999; Matsuo et al. 1997; Gross et al. 2003). Residues shown by mutagenesis to affect Cup–eIF4E interactions (Nakamura et al. 2004; Nelson et al. 2004) or that are phosphorylated (Zhai et al. 2008) are surrounded by a red or black circle, respectively. Regions of interaction with Cup binding site I or II are labeled as 4E-BS I and 4E-BS I, respectively. (A) The eIF4E alignment includes orthologs from Drosophila melanogaster (Dm), Drosophila grimshawi (Dgr), Drosophila virilis (Dvi), Drosophila mojavensis (Dmo), Saccharomyces cerevisiae (Sc), and Homo sapiens (Hs) (shown) and also from Xenopus laevis, Danio rerio, Caenorhabditis elegans, Arabidopsis. thaliana, and seven more insect species (data not shown). Conserved sequences shared by all eIF4E proteins are highlighted in gray, whereas residues conserved specifically in insects are highlighted in purple-blue. (B) Cup alignment of the eIF4E interacting region including only the sequences of the orthologous proteins in insects used in A. Conserved residues are marked in orange. The eIF4E binding motif is boxed in black and labeled.

FIGURE 2.

Structure of the eIF4E–Cup complex. (A) Cartoon view of the complex; eIF4E (purple-blue); Cup (orange). Secondary structure elements are labeled. (B) Cartoon view rotated 90° around the y-axis. The eIF4E binding sites of Cup are labeled 4E-BS I and 4E-BS II. Disordered unmodeled stretches are represented as a dashed line for clarity. These and all other protein structure figures were generated using PyMOL (http://www.pymol.org).

FIGURE 3.

Details of the interactions between eIF4E and Cup. Close-up views of the interactions between eIF4E and Cup. (On top) Schematic drawings of the complex with the zoomed regions boxed in black. Interacting residues on eIF4E (purple-blue); interacting side chains on Cup (orange). Secondary structure elements of eIF4E and Cup are labeled. Residues of Cup or eIF4E mutated in this study are highlighted with a red box or with a gray box if part of the 4E binding consensus motif. (A) This view shows the interaction between helices α1 and α2 of eIF4E and the helix α1 of Cup, centered at Trp106_4E (4E-BS I). The molecules are viewed in a similar orientation to that used in Figure 2A. (B) Interactions at Cup binding site II (4E-BS II) showing the prominent interaction of Trp374_Cup. Strands β1 and β3, helix α1, and loop2 of eIF4E and the helix α2 and β turn of Cup are shown. The molecules are viewed in a similar orientation to that used in Figure 2B. (C) Interactions at Cup 4E-BS I. Cartoon view rotated 180° around the y-axis as compared with A. (D) Interactions at Cup 4E-BS II. Cartoon view rotated 150° around the y-axis as compared with B.

FIGURE 4.

Protein recognition at similar molecular surfaces on eIF4E. The complex is shown in a similar view as in Figure 2B. (A) eIF4E is rendered as a surface, with Cup as a cartoon in orange. The surface is colored according to conservation across insects (with a gradient from white to purple indicating increasingly conserved residues). (B) The lateral surface of eIF4E is used for binding both Cup and eIF4G. A cartoon representation of yeast eIF4E (gray) is shown bound to eIF4G (dark green) (pdb id. 1rf8) (Gross et al. 2003). The structures are shown after superimposition in a similar orientation as in Figure 2B. (C) DSF graph of protein stability. (Top) eIF4E–Cup, eIF4E–Cup-Mut I, or eIF4E–Cup-Mut II with or without m⁷GDP and eIF4E with m⁷GDP. The bars show the mean melting temperature difference (ΔT_m), and the error bars represent the standard deviation from three independent measurements. The measurements are normalized to the T_m measurement for unbound eIF4E. (Bottom) eIF4E Mut II–Cup with or without m⁷GDP and eIF4E Mut II with m⁷GDP. The measurements are normalized to the T_m measurement for unbound eIF4E Mut II. (On the left) Schematics showing the complexes used; (red crosses) mutations. (D) ITC measurements of eIF4E, eIF4E–Cup, eIF4E–Cup-Mut I, or eIF4E–Cup-Mut II for m⁷GDP affinity. The bars represent the mean affinity (nanomolar), and the error bars show the standard deviation from three independent measurements (a typical ITC trace and curve are shown in Supplemental Fig. S4). (On top of the graph) Schematics of the complexes used in the measurements. (E) Sequence alignment of consensus eIF4E binding motifs from different 4E-BPs.