Structural studies of the eIF4E-VPg complex reveal a direct competition for capped RNA: Implications for translation.

Viruses have transformed our understanding of mammalian RNA processing, including facilitating the discovery of the methyl-7-guanosine (m7G) cap on the 5' end of RNAs. The m7G cap is required for RNAs to bind the eukaryotic translation initiation factor eIF4E and associate with the translation machinery across plant and animal kingdoms. The potyvirus-derived viral genome-linked protein (VPg) is covalently bound to the 5' end of viral genomic RNA (gRNA) and associates with host eIF4E for successful infection. Divergent models to explain these observations proposed either an unknown mode of eIF4E engagement or a competition of VPg for the m7G cap-binding site. To dissect these possibilities, we resolved the structure of VPg, revealing a previously unknown 3-dimensional (3D) fold, and characterized the VPg-eIF4E complex using NMR and biophysical techniques. VPg directly bound the cap-binding site of eIF4E and competed for m7G cap analog binding. In human cells, VPg inhibited eIF4E-dependent RNA export, translation, and oncogenic transformation. Moreover, VPg formed trimeric complexes with eIF4E-eIF4G, eIF4E bound VPg-luciferase RNA conjugates, and these VPg-RNA conjugates were templates for translation. Informatic analyses revealed structural similarities between VPg and the human kinesin EG5. Consistently, EG5 directly bound eIF4E in a similar manner to VPg, demonstrating that this form of engagement is relevant beyond potyviruses. In all, we revealed an unprecedented modality for control and engagement of eIF4E and show that VPg-RNA conjugates functionally engage eIF4E. As such, potyvirus VPg provides a unique model system to interrogate eIF4E.

BRAF/MAPK and GSK3 signaling converges to control MITF nuclear export.

The close integration of the MAPK, PI3K, and WNT signaling pathways underpins much of development and is deregulated in cancer. In principle, combinatorial posttranslational modification of key lineage-specific transcription factors would be an effective means to integrate critical signaling events. Understanding how this might be achieved is central to deciphering the impact of microenvironmental cues in development and disease. The microphthalmia-associated transcription factor MITF plays a crucial role in the development of melanocytes, the retinal pigment epithelium, osteoclasts, and mast cells and acts as a lineage survival oncogene in melanoma. MITF coordinates survival, differentiation, cell-cycle progression, cell migration, metabolism, and lysosome biogenesis. However, how the activity of this key transcription factor is controlled remains poorly understood. Here, we show that GSK3, downstream from both the PI3K and Wnt pathways, and BRAF/MAPK signaling converges to control MITF nuclear export. Phosphorylation of the melanocyte MITF-M isoform in response to BRAF/MAPK signaling primes for phosphorylation by GSK3, a kinase inhibited by both PI3K and Wnt signaling. Dual phosphorylation, but not monophosphorylation, then promotes MITF nuclear export by activating a previously unrecognized hydrophobic export signal. Nonmelanocyte MITF isoforms exhibit poor regulation by MAPK signaling, but instead their export is controlled by mTOR. We uncover here an unanticipated mode of MITF regulation that integrates the output of key developmental and cancer-associated signaling pathways to gate MITF flux through the import-export cycle. The results have significant implications for our understanding of melanoma progression and stem cell renewal.

The diversity, plasticity, and adaptability of cap-dependent translation initiation and the associated machinery.

Translation initiation is a critical facet of gene expression with important impacts that underlie cellular responses to stresses and environmental cues. Its dysregulation in many diseases position this process as an important area for the development of new therapeutics. The gateway translation factor eIF4E is typically considered responsible for 'global' or 'canonical' m7G cap-dependent translation. However, eIF4E impacts translation of specific transcripts rather than the entire translatome. There are many alternative cap-dependent translation mechanisms that also contribute to the translation capacity of the cell. We review the diversity of these, juxtaposing more recently identified mechanisms with eIF4E-dependent modalities. We also explore the multiplicity of functions played by translation factors, both within and outside protein synthesis, and discuss how these differentially contribute to their ultimate physiological impacts. For comparison, we discuss some modalities for cap-independent translation. In all, this review highlights the diverse mechanisms that engage and control translation in eukaryotes.

A biochemical framework for eIF4E-dependent mRNA export and nuclear recycling of the export machinery.

The eukaryotic translation initiation factor eIF4E acts in the nuclear export and translation of a subset of mRNAs. Both of these functions contribute to its oncogenic potential. While the biochemical mechanisms that underlie translation are relatively well understood, the molecular basis for eIF4E's role in mRNA export remains largely unexplored. To date, over 3000 transcripts, many encoding oncoproteins, were identified as potential nuclear eIF4E export targets. These target RNAs typically contain a ∼50-nucleotide eIF4E sensitivity element (4ESE) in the 3' UTR and a 7-methylguanosine cap on the 5' end. While eIF4E associates with the cap, an unknown factor recognizes the 4ESE element. We previously identified cofactors that functionally interacted with eIF4E in mammalian cell nuclei including the leucine-rich pentatricopeptide repeat protein LRPPRC and the export receptor CRM1/XPO1. LRPPRC simultaneously interacts with both eIF4E bound to the 5' mRNA cap and the 4ESE element in the 3' UTR. In this way, LRPPRC serves as a specificity factor to recruit 4ESE-containing RNAs within the nucleus. Further, we show that CRM1 directly binds LRPPRC likely acting as the export receptor for the LRPPRC-eIF4E-4ESE RNA complex. We also found that Importin 8, the nuclear importer for cap-free eIF4E, imports RNA-free LRPPRC, potentially providing both coordinated nuclear recycling of the export machinery and an important surveillance mechanism to prevent futile export cycles. Our studies provide the first biochemical framework for the eIF4E-dependent mRNA export pathway.

Importin 8 mediates m7G cap-sensitive nuclear import of the eukaryotic translation initiation factor eIF4E.

Regulation of nuclear-cytoplasmic trafficking of oncoproteins is critical for growth homeostasis. Dysregulated trafficking contributes to malignancy, whereas understanding the process can reveal unique therapeutic opportunities. Here, we focus on eukaryotic translation initiation factor 4E (eIF4E), a prooncogenic protein highly elevated in many cancers, including acute myeloid leukemia (AML). Typically, eIF4E is localized to both the nucleus and cytoplasm, where it acts in export and translation of specific methyl 7-guanosine (m(7)G)-capped mRNAs, respectively. Nuclear accumulation of eIF4E in patients who have AML is correlated with increased eIF4E-dependent export of transcripts encoding oncoproteins. The subcellular localization of eIF4E closely correlates with patients' responses. During clinical responses to the m(7)G-cap competitor ribavirin, eIF4E is mainly cytoplasmic. At relapse, eIF4E reaccumulates in the nucleus, leading to elevated eIF4E-dependent mRNA export. We have identified importin 8 as a factor that directly imports eIF4E into the nucleus. We found that importin 8 is highly elevated in untreated patients with AML, leading to eIF4E nuclear accumulation. Importin 8 only imports cap-free eIF4E. Cap-dependent changes to the structure of eIF4E underpin this selectivity. Indeed, m(7)G cap analogs or ribavirin prevents nuclear entry of eIF4E, which mirrors the trafficking phenotypes observed in patients with AML. Our studies also suggest that nuclear entry is important for the prooncogenic activity of eIF4E, at least in this context. These findings position nuclear trafficking of eIF4E as a critical step in its regulation and position the importin 8-eIF4E complex as a novel therapeutic target.

eIF4E3 acts as a tumor suppressor by utilizing an atypical mode of methyl-7-guanosine cap recognition.

Recognition of the methyl-7-guanosine (m(7)G) cap structure on mRNA is an essential feature of mRNA metabolism and thus gene expression. Eukaryotic translation initiation factor 4E (eIF4E) promotes translation, mRNA export, proliferation, and oncogenic transformation dependent on this cap-binding activity. eIF4E-cap recognition is mediated via complementary charge interactions of the positively charged m(7)G cap between the negative π-electron clouds from two aromatic residues. Here, we demonstrate that a variant subfamily, eIF4E3, specifically binds the m(7)G cap in the absence of an aromatic sandwich, using instead a different spatial arrangement of residues to provide the necessary electrostatic and van der Waals contacts. Contacts are much more extensive between eIF4E3-cap than other family members. Structural analyses of other cap-binding proteins indicate this recognition mode is atypical. We demonstrate that eIF4E3 relies on this cap-binding activity to act as a tumor suppressor, competing with the growth-promoting functions of eIF4E. In fact, reduced eIF4E3 in high eIF4E cancers suggests that eIF4E3 underlies a clinically relevant inhibitory mechanism that is lost in some malignancies. Taken together, there is more structural plasticity in cap recognition than previously thought, and this is physiologically relevant.

Conformational changes induced in the eukaryotic translation initiation factor eIF4E by a clinically relevant inhibitor, ribavirin triphosphate.

The eukaryotic translation initiation factor eIF4E is highly elevated in human cancers including acute myeloid leukemia (AML). A potential anticancer agent, ribavirin, targets eIF4E activity in AML patients corresponding to clinical responses. To date, ribavirin is the only direct inhibitor of eIF4E to reach clinical trials. We showed that ribavirin acts as a competitive inhibitor of the methyl 7-guanosine (m(7)G) cap, the natural ligand of eIF4E. Here we examine the conformational changes occurring in human eIF4E upon binding the active metabolite of ribavirin, ribavirin triphosphate (RTP). Our NMR data revealed an unexpected concentration dependence on RTP affinity for eIF4E. We observed NMR spectra characteristic of tight binding at low micromolar concentrations (2-5 µM eIF4E) but much weaker affinity at more typical NMR concentrations (50- ). Comparison of chemical shift perturbation and line broadening suggest that the two eIF4E-RTP complexes differ in the precise positioning of RTP within the cap binding pocket, with the high affinity complex showing more extensive changes to the central ß-sheet and dorsal surface of eIF4E, similar to m(7)G cap. The differences between high and low affinity complexes arise due to concentration dependent aggregation of eIF4E and RTP. Given the intracellular concentrations of eIF4E and RTP and the differential binding toward the W56A eIF4E mutant the high affinity complex is the most physiologically relevant. In summary, these findings demonstrate that RTP binds in the cap-binding site but also suggests new features of this pocket that should be considered in drug design efforts and reveal new insights into ligand eIF4E recognition.

Structural characterization of the Z RING-eIF4E complex reveals a distinct mode of control for eIF4E.

The eukaryotic translation initiation factor eIF4E, a potent oncogene, is highly regulated. One class of eIF4E regulators, including eIF4G and the 4E-binding proteins (4E-BPs), interact with eIF4E using a conserved YXXXXLPhi-binding site. The structural basis of this interaction and its regulation are well established. Really Interesting New Gene (RING) domain containing proteins, such as the promyelocytic leukemia protein PML and the arenaviral protein Z, represent a second class of eIF4E regulators that inhibit eIF4E function by decreasing eIF4E's affinity for its m(7)G cap ligand. To elucidate the structural basis of this inhibition, we determined the structure of Z and studied the Z-eIF4E complex using NMR methods. We show that Z interacts with eIF4E via a novel binding site, which has no homology with that of eIF4G or the 4E-BPs, and is different from the RING recognition site used in the ubiquitin system. Z and eIF4G interact with distinct parts of eIF4E and differentially alter the conformation of the m(7)G cap-binding site. Our results provide a molecular basis for how PML and Z RINGs reduce the affinity of eIF4E for the m(7)G cap and thereby act as key inhibitors of eIF4E function. Furthermore, our findings provide unique insights into RING protein interactions.

Identification and Characterization of the Interaction Between the Methyl-7-Guanosine Cap Maturation Enzyme RNMT and the Cap-Binding Protein eIF4E.

The control of RNA metabolism is an important aspect of molecular biology with wide-ranging impacts on cells. Central to processing of coding RNAs is the addition of the methyl-7 guanosine (m7G) "cap" on their 5' end. The eukaryotic translation initiation factor eIF4E directly binds the m7G cap and through this interaction plays key roles in many steps of RNA metabolism including nuclear RNA export and translation. eIF4E also stimulates capping of many transcripts through its ability to drive the production of the enzyme RNMT which methylates the G-cap to form the mature m7G cap. Here, we found that eIF4E also physically associated with RNMT in human cells. Moreover, eIF4E directly interacted with RNMT in vitro. eIF4E is only the second protein reported to directly bind the methyltransferase domain of RNMT, the first being its co-factor RAM. We combined high-resolution NMR methods with biochemical studies to define the binding interfaces for the RNMT-eIF4E complex. Further, we found that eIF4E competes for RAM binding to RNMT and conversely, RNMT competes for binding of well-established eIF4E-binding partners such as the 4E-BPs. RNMT uses novel structural means to engage eIF4E. Finally, we observed that m7G cap-eIF4E-RNMT trimeric complexes form, and thus RNMT-eIF4E complexes may be employed so that eIF4E captures newly capped RNA. In all, we show for the first time that the cap-binding protein eIF4E directly binds to the cap-maturation enzyme RNMT.

1H, 13C and 15N chemical shift assignments of the C-terminal domain of human UDP-Glucuronosyltransferase 2B7 (UGT2B7-C).

The human UDP-glucuronosyltransferase (UGT) family of enzymes catalyze the covalent addition of glucuronic acid to a wide range of compounds, generally rendering them inactive. Although important for clearance of environmental toxins and metabolites, UGT activation can lead to inappropriate glucuronidation of therapeutics underlying drug resistance. Indeed, 50% of medications are glucuronidated. To better understand this mode of resistance, we studied the UGT2B7 enzyme associated with glucuronidation of cancer drugs such as Tamoxifen and Sorafenib. We report 1H, 13C and 15N backbone (> 90%) and side-chain assignments (~ 78% completeness according to CYANA) for the C-terminal domain of UGT2B7 (UGT2B7-C). Given the biomedical importance of this family of enzymes, our assignments will provide a key tool for improving understanding of the biochemical basis for substrate selectivity and other aspects of enzyme activity. This in turn will inform on drug design to overcome UGT-related drug resistance.

Biochemical and Structural Insights into the Eukaryotic Translation Initiation Factor eIF4E.

A major question in cell and cancer biology is concerned with understanding the flow of information from gene to protein. Indeed, many studies indicate that the proteome can be decoupled from the transcriptome. A major source of this decoupling is post-transcriptional regulation. The eukaryotic translation initiation factor eIF4E serves as an excellent example of a protein that can modulate the proteome at the post-transcriptional level. eIF4E is elevated in many cancers thus highlighting the relevance of this mode of control to biology. In this review, we provide a brief overview of various functions of eIF4E in RNA metabolism e.g. in nuclear-cytoplasmic RNA export, translation, RNA stability and/or sequestration. We focus on the modalities of eIF4E regulation at the biochemical and particularly structural level. In this instance, we describe not only the importance for the m7Gcap eIF4E interaction but also of recently discovered non-traditional RNA-eIF4E interactions as well as cap-independent activities of eIF4E. Further, we describe several distinct structural modalities used by the cell and some viruses to regulate or co-opt eIF4E, substantially extending the types of proteins that can regulate eIF4E from the traditional eIF4E-binding proteins (e.g. 4E-BP1 and eIF4G). Finally, we provide an overview of the results of targeting eIF4E activity in the clinic.

Chemical shift assignment of the viral protein genome-linked (VPg) from potato virus Y.

The dysregulation of translation contributes to many pathogenic conditions in humans. Discovering new translational mechanisms is important to understanding the diversity of this process and its potential mechanisms. Such mechanisms can be initially observed in viruses. With this in mind, we studied the viral protein genome-linked VPg factor from the largest genus of plant viruses. Studies in plants show that VPg binds to the eukaryotic translation initiation factor eIF4E for translation of viral RNAs. VPg contains no known eIF4E binding motifs and no sequence homology to any known proteins. Thus, as a first step in understanding the structural basis of this interaction, we carried out NMR assignments of the VPg from the potato virus Y potyvirus protein.

Overcoming Drug Resistance through the Development of Selective Inhibitors of UDP-Glucuronosyltransferase Enzymes.

Drug resistance is a major cause of cancer-related mortality. Glucuronidation of drugs via elevation of UDP-glucuronosyltransferases (UGT1As) correlates with clinical resistance. The nine UGT1A family members have broad substrate specificities attributed to their variable N-terminal domains and share a common C-terminal domain. Development of UGT1As as pharmacological targets has been hampered by toxicity of pan-UGT inhibitors and by difficulty in isolating pure N-terminal domains or full-length proteins. Here, we developed a strategy to target selected UGT1As which exploited the biochemical tractability of the C-domain and its ability to allosterically communicate with the catalytic site. By combining NMR fragment screening with in vitro glucuronidation assays, we identified inhibitors selective for UGT1A4. Significantly, these compounds selectively restored sensitivity in resistant cancer cells only for substrates of the targeted UGT1A. This strategy represents a crucial first step toward developing compounds to overcome unwanted glucuronidation thereby reversing resistance in patients.

Backbone assignment of the apo-form of the human C-terminal domain of UDP-glucuronosyltransferase 1A (UGT1A).

A major component of phase II drug metabolism is the covalent addition of glucuronic acid to metabolites and xenobiotics. This activity is carried out by UDP-glucuronosyltransferases (UGT) which bind the UDP-glucuronic acid donor and catalyze the covalent addition of glucuronic acid sugar moieties onto a wide variety of substrates. UGTs play important roles in drug detoxification and were recently shown to act in an inducible form of multi-drug resistance in cancer patients. Despite their biological importance, structural understanding of these enzymes is limited. The C-terminal domain is identical for all UGT1A family members and required for binding to UDP-glucuronic acid as well as involved in contacts with substrates. Here, we report the backbone assignments for the C-terminal domain of UGT1A. These assignments are a critical tool for the development of a deeper biochemical understanding of substrate specificity and enzymatic activity.

A TFEB nuclear export signal integrates amino acid supply and glucose availability.

How cells coordinate the response to fluctuating carbon and nitrogen availability required to maintain effective homeostasis is a key issue. Amino acid limitation that inactivates mTORC1 promotes de-phosphorylation and nuclear translocation of Transcription Factor EB (TFEB), a key transcriptional regulator of lysosome biogenesis and autophagy that is deregulated in cancer and neurodegeneration. Beyond its cytoplasmic sequestration, how TFEB phosphorylation regulates its nuclear-cytoplasmic shuttling, and whether TFEB can coordinate amino acid supply with glucose availability is poorly understood. Here we show that TFEB phosphorylation on S142 primes for GSK3ß phosphorylation on S138, and that phosphorylation of both sites but not either alone activates a previously unrecognized nuclear export signal (NES). Importantly, GSK3ß is inactivated by AKT in response to mTORC2 signaling triggered by glucose limitation. Remarkably therefore, the TFEB NES integrates carbon (glucose) and nitrogen (amino acid) availability by controlling TFEB flux through a nuclear import-export cycle.

NMR structure determination of a synthetic analogue of bacillomycin Lc reveals the strategic role of L-Asn1 in the natural iturinic antibiotics.

Iturins are a group of antifungal produced by Bacillus subtilis. All are cyclic lipopeptides with seven alpha-amino acids of configuration LDDLLDL and one beta-amino fatty acid. The bacillomycin L is a member of this family and its NMR structure was previously resolved using the sequence Asp-Tyr-Asn-Ser-Gln-Ser-Thr. In this work, we carefully examined the NMR spectra of this compound and detected an error in the sequence. In fact, Asp1 and Gln5 need to be changed into Asn1 and Glu5, which therefore makes it identical to bacillomycin Lc. As a consequence, it now appears that all iturinic peptides with antibiotic activity share the common beta-amino fatty acid 8-L-Asn1-D-Tyr2-D-Asn3 sequence. To better understand the conformational influence of the acidic residue L-Asp1, present, for example in the inactive iturin C, the NMR structure of the synthetic analogue SCP [cyclo (L-Asp1-D-Tyr2-D-Asn3-L-Ser4-L-Gln5-D-Ser6-L-Thr7-beta-Ala8)] was determined and compared with bacillomycin Lc recalculated with the corrected sequence. In both cases, the conformers obtained were separated into two families of similar energy which essentially differ in the number and type of turns. A detailed analysis of both cyclopeptide structures is presented here. In addition, CD and FTIR spectra were performed and confirmed the conformational differences observed by NMR between both cyclopeptides.

NMR structure of the enzyme GatB of the galactitol-specific phosphoenolpyruvate-dependent phosphotransferase system and its interaction with GatA.

The phosphoenolpyruvate-dependent carbohydrate transport system (PTS) couples uptake with phosphorylation of a variety of carbohydrates in prokaryotes. In this multienzyme complex, the enzyme II (EII), a carbohydrate-specific permease, is constituted of two cytoplasmic domains, IIA and IIB, and a transmembrane channel IIC domain. Among the five families of EIIs identified in Escherichia coli, the galactitol-specific transporter (II(gat)) belongs to the glucitol family and is structurally the least well-characterized. Here, we used nuclear magnetic resonance (NMR) spectroscopy to solve the three-dimensional structure of the IIB subunit (GatB). GatB consists of a central four-stranded parallel beta-sheet flanked by alpha-helices on both sides; the active site cysteine of GatB is located at the beginning of an unstructured loop between beta1 and alpha1 that folds into a P-loop-like structure. This structural arrangement shows similarities with other IIB subunits but also with mammalian low molecular weight protein tyrosine phosphatases (LMW PTPase) and arsenate reductase (ArsC). An NMR titration was performed to identify the GatA-interacting residues.

MNKs act as a regulatory switch for eIF4E1 and eIF4E3 driven mRNA translation in DLBCL.

The phosphorylation of eIF4E1 at serine 209 by MNK1 or MNK2 has been shown to initiate oncogenic mRNA translation, a process that favours cancer development and maintenance. Here, we interrogate the MNK-eIF4E axis in diffuse large B-cell lymphoma (DLBCL) and show a distinct distribution of MNK1 and MNK2 in germinal centre B-cell (GCB) and activated B-cell (ABC) DLBCL. Despite displaying a differential distribution in GCB and ABC, both MNKs functionally complement each other to sustain cell survival. MNK inhibition ablates eIF4E1 phosphorylation and concurrently enhances eIF4E3 expression. Loss of MNK protein itself downregulates total eIF4E1 protein level by reducing eIF4E1 mRNA polysomal loading without affecting total mRNA level or stability. Enhanced eIF4E3 expression marginally suppresses eIF4E1-driven translation but exhibits a unique translatome that unveils a novel role for eIF4E3 in translation initiation. We propose that MNKs can modulate oncogenic translation by regulating eIF4E1-eIF4E3 levels and activity in DLBCL.

The oncogene eIF4E reprograms the nuclear pore complex to promote mRNA export and oncogenic transformation.

The eukaryotic translation initiation factor eIF4E is a potent oncogene that promotes the nuclear export and translation of specific transcripts. Here, we have discovered that eIF4E alters the cytoplasmic face of the nuclear pore complex (NPC), which leads to enhanced mRNA export of eIF4E target mRNAs. Specifically, eIF4E substantially reduces the major component of the cytoplasmic fibrils of the NPC, RanBP2, relocalizes an associated nucleoporin, Nup214, and elevates RanBP1 and the RNA export factors, Gle1 and DDX19. Genetic or pharmacological inhibition of eIF4E impedes these effects. RanBP2 overexpression specifically inhibits the eIF4E mRNA export pathway and impairs oncogenic transformation by eIF4E. The RanBP2 cytoplasmic fibrils most likely slow the release and/or recycling of critical export factors to the nucleus. eIF4E overcomes this inhibitory mechanism by indirectly reducing levels of RanBP2. More generally, these results suggest that reprogramming the NPC is a means by which oncogenes can harness the proliferative capacity of the cell.

Structural insights into the allosteric effects of 4EBP1 on the eukaryotic translation initiation factor eIF4E.

The eukaryotic translation initiation factor eIF4E plays key roles in cap-dependent translation and mRNA export. These functions rely on binding the 7-methyl-guanosine moiety (5'cap) on the 5'-end of all mRNAs. eIF4E is regulated by proteins such as eIF4G and eIF4E binding proteins (4EBPs) that bind the dorsal surface of eIF4E, distal to the cap binding site, and modulate cap binding activity. Both proteins increase the affinity of eIF4E for 5'cap. Our understanding of the allosteric effects and structural underpinnings of 4EBP1 or eIF4G binding can be advanced by obtaining structural data on cap-free eIF4E bound to one of these proteins. Here, we report the crystal structure of apo-eIF4E and cap-free eIF4E in complex with a 4EBP1 peptide. We also monitored 4EBP1 binding to cap-free eIF4E in solution using NMR. Together, these studies suggest that 4EBP1 transforms eIF4E into a cap-receptive state. NMR methods were also used to compare the allosteric routes activated by 4EBP1, eIF4G, and the arenavirus Z protein, a negative regulator of cap binding. We observed chemical shift perturbation at the dorsal binding site leading to alterations in the core of the protein, which were ultimately communicated to the unoccupied cap binding site of eIF4E. There were notable similarities between the routes taken by 4EBP1 and eIF4G and differences from the negative regulator Z. Thus, binding of 4EBP1 or eIF4G allosterically drives alterations throughout the protein that increase the affinity of eIF4E for the 5'cap.