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.
- SourceAvailable from: Rashmi Sharma
[Show abstract] [Hide abstract]
- "FACT DNA Drosophila melanogaster  MeCP2 DNA Homo sapiens   MBD2 NurD DNA Homo sapiens  Transcription factors Max DNA Homo sapiens   NKX3.1 DNA Drosophila melanogaster  ApLLP DNA Aplysia kurodai  Neurogenin 1 DNA Homo sapiens  Ultrabithorax DNA, Exd Drosophila melanogaster   HMGB1 DNA Rattus norvegicus  Oct-1 DNA Homo sapiens  Ets-1 DNA Mus musculus   c-Myc Bin1 SH3 domain Homo sapiens  Nrf2 Keap1 Mus musculus  Prothymosin α Keap1 Homo sapiens  Coactivator interactions GCN4 Med15 Saccharomyces cerevisiae   p65 (RelA) CBP TAZ1 Mus Musculus  p53 TAD CBP NCBD Homo sapiens  KID KIX Mus musculus   Interactions with the basal machinery EWS PIC Homo sapiens  SP1 TFIID Homo sapiens  GCN4 PIC Saccharomyces cerevisiae  Gal4 PIC Saccharomyces cerevisiae  PC4 PIC Homo sapiens  Nuclear receptors, transport PPAR-γ DNA Homo sapiens  NLS Importin-α Xenopus laevis   mRNA maturation, translation Cup eIF4E Drosophila melanogaster  UPF2 UPF1 Homo sapiens  RNAP II CTD mRNA maturation factors Saccharomyces cerevisiae  SF1 U2AF 65 Homo sapiens  4E-BP2 eIF4E Homo sapiens  L7/L12 Ribosome Escherichia coli   DNA repair RPA DNA Homo sapiens   UmuD'2 UmuD2 Escherichia coli  UvrD DNA Escherichia coli  "
ABSTRACT: Specific molecular recognition is assumed to require a well-defined set of contacts and devoid of conformational and interaction ambiguities. Growing experimental evidence demonstrates however, that structural multiplicity or dynamic disorder can be retained in protein complexes, termed as fuzziness. Fuzzy regions establish alternative contacts between specific partners usually via transient interactions. Nature often tailors the dynamic properties of these segments via post-translational modifications or alternative splicing to fine-tune affinity. Most experimentally characterized fuzzy complexes are involved in regulation of gene-expression, signal transduction and cell-cycle regulation. Fuzziness is also characteristic to viral protein complexes, cytoskeleton structure, and surprisingly in a few metabolic enzymes. A plausible role of fuzzy complexes in increasing half-life of intrinsically disordered proteins is also discussed. Copyright © 2015. Published by Elsevier B.V.FEBS letters 07/2015; DOI:10.1016/j.febslet.2015.07.022 · 3.34 Impact Factor
[Show abstract] [Hide abstract]
- "In combination with previous studies, our structures show that the canonical motifs of diverse 4E-BPs and eIF4G use a similar mechanism to bind eIF4E and that this binding mode is conserved (Gross et al., 2003; Kinkelin et al., 2012; Mizuno et al., 2008; Paku et al., 2012; Umenaga et al., 2011). In contrast, the binding mode of the linker regions and non-canonical motifs of 4E-BPs differ in the molecular details as expected on the basis of the differences in sequence and length of these regions. "
ABSTRACT: The eIF4E-binding proteins (4E-BPs) represent a diverse class of translation inhibitors that are often deregulated in cancer cells. 4E-BPs inhibit translation by competing with eIF4G for binding to eIF4E through an interface that consists of canonical and non-canonical eIF4E-binding motifs connected by a linker. The lack of high-resolution structures including the linkers, which contain phosphorylation sites, limits our understanding of how phosphorylation inhibits complex formation. Furthermore, the binding mechanism of the non-canonical motifs is poorly understood. Here, we present structures of human eIF4E bound to 4E-BP1 and fly eIF4E bound to Thor, 4E-T, and eIF4G. These structures reveal architectural elements that are unique to 4E-BPs and provide insight into the consequences of phosphorylation. Guided by these structures, we designed and crystallized a 4E-BP mimic that shows increased repressive activity. Our studies pave the way for the rational design of 4E-BP mimics as therapeutic tools to decrease translation during oncogenic transformation. Copyright © 2015 Elsevier Inc. All rights reserved.Molecular Cell 02/2015; 57(6). DOI:10.1016/j.molcel.2015.01.017 · 14.46 Impact Factor
[Show abstract] [Hide abstract]
- "Interestingly, the D. melanogaster protein Cup, related to human 4E–T, has two nearby eIF4E-binding sites, the high-affinity one which conforms to the consensus, being Y342TRSRLM, with a second lower affinity and non-consensus one, ELEGRLR, some 30 residues downstream . The second site binds laterally and perpendicularly in α-helix form to the eIF4E β-sheet , and plays a role in the stabilisation of associated mRNA . "
ABSTRACT: In addition to the canonical eIF4E cap-binding protein, eukaryotes have evolved sequence-related variants with distinct features, some of which have been shown to negatively regulate translation of particular mRNAs, but which remain poorly characterised. Mammalian eIF4E proteins have been divided into three classes, with class I representing the canonical cap-binding protein eIF4E1. eIF4E1 binds eIF4G to initiate translation, and other eIF4E-binding proteins such as 4E-BPs and 4E-T prevent this interaction by binding eIF4E1 with the same consensus sequence YX 4Lϕ. We investigate here the interaction of human eIF4E2 (4EHP), a class II eIF4E protein, which binds the cap weakly, with eIF4E-transporter protein, 4E-T. We first show that ratios of eIF4E1:4E-T range from 50:1 to 15:1 in HeLa and HEK293 cells respectively, while those of eIF4E2:4E-T vary from 6:1 to 3:1. We next provide evidence that eIF4E2 binds 4E-T in the yeast two hybrid assay, as well as in pull-down assays and by recruitment to P-bodies in mammalian cells. We also show that while both eIF4E1 and eIF4E2 bind 4E-T via the canonical YX 4Lϕ sequence, nearby downstream sequences also influence eIF4E:4E-T interactions. Indirect immunofluorescence was used to demonstrate that eIF4E2, normally homogeneously localised in the cytoplasm, does not redistribute to stress granules in arsenite-treated cells, nor to P-bodies in Actinomycin D-treated cells, in contrast to eIF4E1. Moreover, eIF4E2 shuttles through nuclei in a Crm1-dependent manner, but in an 4E-T-independent manner, also unlike eIF4E1. Altogether we conclude that while both cap-binding proteins interact with 4E-T, and can be recruited by 4E-T to P-bodies, eIF4E2 functions are likely to be distinct from those of eIF4E1, both in the cytoplasm and nucleus, further extending our understanding of mammalian class I and II cap-binding proteins.PLoS ONE 11/2013; 8(8):e72761. DOI:10.1371/journal.pone.0072761 · 3.23 Impact Factor