Molecular mechanism of the dual activity of 4EGI-1: Dissociating eIF4G from eIF4E but stabilizing the binding of unphosphorylated 4E-BP1

Abstract

The eIF4E-binding protein (4E-BP) is a phosphorylation-dependent regulator of protein synthesis. The nonphosphorylated or minimally phosphorylated form binds translation initiation factor 4E (eIF4E), preventing binding of eIF4G and the recruitment of the small ribosomal subunit. Signaling events stimulate serial phosphorylation of 4E-BP, primarily by mammalian target of rapamycin complex 1 (mTORC1) at residues T 37 /T 46 , followed by T 70 and S 65 . Hyperphosphorylated 4E-BP dissociates from eIF4E, allowing eIF4E to interact with eIF4G and translation initiation to resume. Because overexpression of eIF4E is linked to cellular transformation, 4E-BP is a tumor suppressor, and up-regulation of its activity is a goal of interest for cancer therapy. A recently discovered small molecule, eIF4E/eIF4G interaction inhibitor 1 (4EGI-1), disrupts the eIF4E/eIF4G interaction and promotes binding of 4E-BP1 to eIF4E. Structures of 14-to 16-residue 4E-BP fragments bound to eIF4E contain the eIF4E consensus binding motif, 54 YXXXXLΦ 60 (motif 1) but lack known phosphorylation sites. We report here a 2.1-Å crystal structure of mouse eIF4E in complex with m 7 GTP and with a fragment of human 4E-BP1, extended C-terminally from the consensus-binding motif (4E-BP1 50-84 ). The extension, which includes a proline-turn-helix segment (motif 2) followed by a loop of irregular structure, reveals the location of two phosphorylation sites (S 65 and T 70 ). Our major finding is that the C-terminal extension (motif 3) is critical to 4E-BP1-mediated cell cycle arrest and that it partially overlaps with the binding site of 4EGI-1. The binding of 4E-BP1 and 4EGI-1 to eIF4E is therefore not mutually exclusive, and both ligands contribute to shift the equilibrium toward the inhibition of translation initiation.translation initiation | phosphorylation | protein-protein interaction inhibitor | structure

Mitotic protein kinase CDK1 phosphorylation of mRNA translation regulator 4E-BP1 Ser83 may contribute to cell transformation

“…Although we did not find S83 phosphorylation affecting general cap-binding or capdependent translation, we cannot exclude the possibility that an mRNA subpopulation is regulated by S83-phosphorylated 4E-BP1, such as terminal oligopyrimidine tract (TOP)-containing mRNAs that are reported to be translationally active during mitosis (43). A recent study shows that the interaction between the C-terminal loop with eIF4E is required for eIF4F complex formation and translation repression in vivo using 4E-BP1 truncation mutants, which, in light of our results, prompts the examination of other loop residues (36). Expression of full-length 4E-BP1 constructs with mutations at canonical and noncanonical eIF4E binding motifs would provide robust evidence for this phenomenon in cells.…”

“…The consensus binding motif Y(X) 4 LΦ (where X is any amino acid residue and Φ is a hydrophobic residue) is shared by other 4E-BPs and translation initiation factor eIF4G. Recently, a C-terminal loop region of 4E-BP1, 78 PGVTS 83 , was reported to also play an auxiliary role in binding eIF4E at a hydrophobic pocket separate from the canonical 54 YDRKFLM 60 binding pocket (33)(34)(35)(36). If S83 phosphorylation destabilizes this auxiliary domain, it may affect 4E-BP1:eIF4E interaction and capdependent initiation and translation.…”

The 4E-BP growth pathway regulates the effect of ambient temperature on Drosophila metabolism and lifespan

“…The small molecule 4EGI-1 promotes 4E-BP activity, stabilizing the binding of 4E-BP to eIF4E and inducing the dissociation of the translation initiation complex (39,40). The effect of 4EGI-1 treatment on survival was identical to that of rotenone; 4EGI-1 had no effect at 25°C but attenuated lifespan extension by cold (Fig.…”

GIGYF1/2 proteins use auxiliary sequences to selectively bind to 4EHP and repress target mRNA expression

“…The 4E-BP1 and GYF1/2 canonical motifs adopt a helical conformation on the dorsal surface of 4EHP, whereas the noncanonical sequences bind to the lateral surface of 4EHP using a binding mode similar to that described for 4E-BP1 in complex with eIF4E ( Fig Sekiyama et al 2015). The binding mode and conformation for the GYF2 fragment comprising the canonical motif and the noncanonical sequences were not influenced by the auxiliary region, as the two GYF2 structures in complex with 4EHP are very similar across common elements irrespective of whether the auxiliary region was present (Supplemental Fig.…”

“…eIF4G and the 4E-BPs share a conserved, canonical (C) 4E-binding motif with the sequence YX 4 LΦ (where Y, X, L, and Φ represent Tyr, any amino acid, Leu, and a hydrophobic residue, respectively), which binds to the dorsal surface of eIF4E opposite to the cap-binding pocket (Matsuo et al 1997;Marcotrigiano et al 1999;Gross et al 2003). Both eIF4G and the 4E-BPs also contain variable noncanonical (NC) 4E-binding motifs that bind to an eIF4E hydrophobic lateral surface, increasing the affinity of the interaction (Kinkelin et al 2012;Paku et al 2012;Lukhele et al 2013;Igreja et al 2014;Peter et al 2015a,b;Sekiyama et al 2015;Grüner et al 2016). Because eIF4G and 4E-BPs bind to the same surfaces on eIF4E, their binding is mutually exclusive, resulting in translation activation and inhibition, respectively.…”