Structural and Thermodynamic Behavior of Eukaryotic Initiation Factor 4E in Supramolecular Formation with 4E-Binding Protein 1 and mRNA Cap Analogue, Studied by Spectroscopic Methods.

Shen, Xu; Tomoo, Koji; Uchiyama, Susumu; Kobayashi, Yuji; Ishida, Toshimasa

doi:10.1248/cpb.49.1299

Cited by 26 publications

(22 citation statements)

References 18 publications

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“…Human eIF4E contains eight tryptophanes, making it a strongly fluorescent protein. This property has been exploited in previous studies (44)(45)(46). We observed that the 4EGI1[E/Z] analogs also induce quenching of the protein intrinsic fluorescence.…”

Section: Mutations and Fluorescence Polarization Experiments A Humanmentioning

confidence: 74%

Structure of the eukaryotic translation initiation factor eIF4E in complex with 4EGI-1 reveals an allosteric mechanism for dissociating eIF4G

Papadopoulos

Jenni

Kabha

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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The interaction of the eukaryotic translation initiation factor eIF4E with the initiation factor eIF4G recruits the 40S ribosomal particle to the 5′ end of mRNAs, facilitates scanning to the AUG start codon, and is crucial for eukaryotic translation of nearly all genes. Efficient recruitment of the 40S particle is particularly important for translation of mRNAs encoding oncoproteins and growth-promoting factors, which often harbor complex 5′ UTRs and require efficient initiation. Thus, inhibiting the eIF4E/eIF4G interaction has emerged as a previously unpursued route for developing anticancer agents. Indeed, we discovered small-molecule inhibitors of this eIF4E/eIF4G interaction (4EGIs) that inhibit translation initiation both in vitro and in vivo and were used successfully in numerous cancer-biology and neurobiology studies. However, their detailed molecular mechanism of action has remained elusive. Here, we show that the eIF4E/eIF4G inhibitor 4EGI-1 acts allosterically by binding to a site on eIF4E distant from the eIF4G binding epitope. Data from NMR mapping and high-resolution crystal structures are congruent with this mechanism, where 4EGI-1 attaches to a hydrophobic pocket of eIF4E between β-sheet 2 (L 60 -T 68 ) and α-helix 1 (E 69 -N 77 ), causing localized conformational changes mainly in the H 78 -L 85 region. It acts by unfolding a short 3 10 -helix (S 82 -L 85 ) while extending α-helix 1 by one turn (H 78 -S 82 ). This unusual helix rearrangement has not been seen in any previous eIF4E structure and reveals elements of an allosteric inhibition mechanism leading to the dislocation of eIF4G from eIF4E. allosteric inhibitor | NMR spectroscopy | inhibitor of protein-protein interaction T he translation initiation factor eIF4E is overexpressed in numerous human cancers and drives cellular transformation, tumorigenesis, and metastatic progression in preclinical and clinical experiments. These oncogenic processes are driven selectively, increasing the translation of a subset of oncogenic mRNAs that has highly structured 5′ UTRs (1, 2) or other regulatory elements (3). eIF4E also seems to play critical roles in learning and memory (4, 5). The protein-protein interaction between eIF4E and eIF4G is a decisive event in eukaryotic protein synthesis, because the interaction of eIF4E with eIF4G is critical for the formation of the trimeric eIF4F complex consisting of eIF4E, the multidomain scaffold protein eIF4G, and RNA helicase eIF4A. This eIF4F complex along with eIF3 mediate the recruitment of the 40S ribosomal particle to the 5′ cap of mRNA. Thus, targeting the eIF4E/eIF4G interaction has emerged as an opportunity for the development of previously unavailable anticancer agents (6). Indeed, small-molecule inhibitors of this interaction (4EGIs, which bind to eIF4E and prevent eIF4G recruitment) were discovered that exhibit this desired activity in vitro and in vivo (7). These agents have now been widely used in numerous cancer-biology (8-15) and neurobiology studies (16-18).

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Section: Mutations and Fluorescence Polarization Experiments A Humanmentioning

confidence: 74%

Structure of the eukaryotic translation initiation factor eIF4E in complex with 4EGI-1 reveals an allosteric mechanism for dissociating eIF4G

Papadopoulos

Jenni

Kabha

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…It is interesting to note that both of these studies evaluate the capacity of in vitro phosphorylation at Thr46 to disrupt pre-existing 4E-BP1:eIF4E complexes and utilize cap column purification to isolate eIF4E-bound 4E-BP1. This experimental detail is particularly relevant, as it has been shown that the RNA cap can exert an allosteric effect stabilizing 4E-BP1:eIF4E binding 14, 36 . Taken together, our data and these previous reports could suggest that Thr46 phosphorylation is sufficient to block the initial binding between eIF4E and 4E-BP1 as observed by far western analyses, but that hyperphosphorylation of 4E-BP1, including at Ser65, is required to disrupt existing 4E-BP1:eIF4E complexes.…”

Section: Discussionmentioning

confidence: 99%

Rapamycin-insensitive mTORC1 activity controls eIF4E:4E-BP1 binding

Livingstone

Bidinosti

2012

F1000Res

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The recent development of mammalian target of rapamycin (mTOR) kinase domain inhibitors and genetic dissection of rapamycin-sensitive and -insensitive mTOR protein complexes (mTORC1 and mTORC2) have revealed that phosphorylation of the mTOR substrate 4E-BP1 on amino acids Thr37 and/or Thr46 represents a rapamycin-insensitive activity of mTORC1. Despite numerous previous reports utilizing serine (Ser)-to-alanine (Ala) and threonine (Thr)-to-Ala phosphorylation site mutants of 4E-BP1 to assess which post-translational modification(s) directly regulate binding to eIF4E, an ambiguous understanding persists. This manuscript demonstrates that the initial, rapamycin-insensitive phosphorylation event at Thr46 is sufficient to prevent eIF4E:4E-BP1 binding. This finding is relevant, particularly as mTOR kinase domain inhibitors continue to be assessed for clinical efficacy, since it clarifies a difference between the action of these second-generation mTOR inhibitors and those of rapamycin analogues.

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“…The excitation wavelength was set at 280 nm and the emission spectra were measured between 300 and 400 nm. RXRαLBD protein was incubated with different concentrations of the test compound at 4°C for 1 h. The dissociation equilibrium constant (K D ) of the test compound binding to RXRαLBD protein was fitted by the previously reported method (Shen et al, 2001).…”

Section: Fluorescence Quenching Assaymentioning

confidence: 99%

Small molecule TBTC as a new selective retinoid X receptor α agonist improves behavioral deficit in Alzheimer's disease model mice

Sun

Fan

Zhu

et al. 2015

European Journal of Pharmacology

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Structural and Thermodynamic Behavior of Eukaryotic Initiation Factor 4E in Supramolecular Formation with 4E-Binding Protein 1 and mRNA Cap Analogue, Studied by Spectroscopic Methods.

Cited by 26 publications

References 18 publications

Structure of the eukaryotic translation initiation factor eIF4E in complex with 4EGI-1 reveals an allosteric mechanism for dissociating eIF4G

Structure of the eukaryotic translation initiation factor eIF4E in complex with 4EGI-1 reveals an allosteric mechanism for dissociating eIF4G

Rapamycin-insensitive mTORC1 activity controls eIF4E:4E-BP1 binding

Small molecule TBTC as a new selective retinoid X receptor α agonist improves behavioral deficit in Alzheimer's disease model mice

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