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J Mol Biol
2015 Jan 30;4272:387-405. doi: 10.1016/j.jmb.2014.11.009.
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Distinct features of cap binding by eIF4E1b proteins.
Kubacka D, Miguel RN, Minshall N, Darzynkiewicz E, Standart N, Zuberek J.
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eIF4E1b, closely related to the canonical translation initiation factor 4E (eIF4E1a), cap-binding protein is highly expressed in mouse, Xenopus and zebrafish oocytes. We have previously characterized eIF4E1b as a component of the CPEB mRNP translation repressor complex along with the eIF4E-binding protein 4E-Transporter, the Xp54/DDX6 RNA helicase and additional RNA-binding proteins. eIF4E1b exhibited only very weak interactions with m(7)GTP-Sepharose and, rather than binding eIF4G, interacted with 4E-T. Here we undertook a detailed examination of both Xenopus and human eIF4E1b interactions with cap analogues using fluorescence titration and homology modeling. The predicted structure of eIF4E1b maintains the α+β fold characteristic of eIF4E proteins and its cap-binding pocket is similarly arranged by critical amino acids: Trp56, Trp102, Glu103, Trp166, Arg112, Arg157 and Lys162 and residues of the C-terminal loop. However, we demonstrate that eIF4E1b is 3-fold less well able to bind the cap than eIF4E1a, both proteins being highly stimulated by methylation at N(7) of guanine. Moreover, eIF4E1b proteins are distinguishable from eIF4E1a by a set of conserved amino acid substitutions, several of which are located near to cap-binding residues. Indeed, eIF4E1b possesses several distinct features, namely, enhancement of cap binding by a benzyl group at N(7) position of guanine, a reduced response to increasing length of the phosphate chain and increased binding to a cap separated by a linker from Sepharose, suggesting differences in the arrangement of the protein's core. In agreement, mutagenesis of the amino acids differentiating eIF4E1b from eIF4E1a reduces cap binding by eIF4E1a 2-fold, demonstrating their role in modulating cap binding.
Fig. 1. Amino acid sequence alignment of vertebrate eIF4E1a and eIF4E1b proteins of H. sapiens, M. musculus, X. laevis and X. tropicalis, D. rerio, B. taurus, R. norvegicus, C. familiaris and G. gallus, performed with CLUSTALW2. Residues in red and blue show negatively and positively charged amino acids within the N-terminus, respectively. The conserved amino acids that distinguish eIF4E1a (gray) and eIF4E1b (black) proteins are highlighted. The amino acids of the eIF4E1a cap-binding pocket and binding sites for eIF4G/4E-BP proteins are marked with circle and triangle symbols, respectively, with green and magenta shading, respectively, showing their conservation. Starred residues indicate the residues whose impact on cap binding we checked experimentally. Secondary structural elements of α-helices (H1–H3) and β-strands (S1–S8) are shown according to the crystal structure of human eIF4E1a in complex with m7GTP or m7GpppA [2].
Fig. 2. Models of the structures of Xenopus eIF4E1b protein in apo and cap-bound form. The models were predicted by MODELLER using human/mouse eIF4E1a as a template (PDB IDs: 2GPQ for apo and 1IPB for cap-bound eIF4E1a). (a) eIF4E1b in complex with the m7GTP. The amino acids forming the cap-binding site are indicated (blue). (b) Structural superimposition of apo (magenta) and m7GTP-bound (blue) Xenopus eIF4E1b showing rearrangements of loops that compose the cap-binding site.
Fig. 3. Analysis of eIF4E1a protein conformational stability. (a) Relative fluorescence intensity over time was determined for human (red) and Xenopus (black) eIF4E1a and eIF4E1b proteins as indicated. Insert shows the analysis of human and Xenopus eIF4E proteins by 15% SDS-PAGE and Coomassie Blue staining. (b) Relative fluorescence intensity over time was determined for Xenopus eIF4E1a and eIF4E1b in the presence of 5% or 10% glycerol in buffer as shown (black) and in the presence only of m7GTP (red).
Fig. 4. The influence of phosphate groups on the stability eIF4E1b–cap analogue complexes relative to eIF4E1a–cap complexes, described as the ratio of Kas(eIF4E1a) to Kas(eIF4E1b). The resultant charges of cap analogues at given pH values were estimated from experimental pKa1 values for dissociation of the N1 proton of N7-methylguanosine and from the experimental pKa2 values for the dissociation of the second proton of the terminal phosphate group [33,34].
Fig. 5. (a) Homology model of eIF4E1b in complex with m7GTP. The residues forming the cap-binding site are indicated (blue). Amino acids conserved in eIF4E1b and distinct to those in eIF4E1a proteins positioned in the neighborhood of the cap-binding site are highlighted in red. Residues mediating cap binding that influence positions of Trp56 and Trp102 are indicated in purple. (b) Far-UV CD spectra of XeIF4E1a and X4E1a6 performed at two protein concentrations: 5 and 10 μM. (c) Influence of mutations in XeIF4E1a on association constants. The mutations that introduce XeIF4E1b residues into XeIF4E1a are listed in brackets under the name of mutants, with the amino acids that directly interact with the cap marked below. (d) The equilibrium association constants, Kas, for complexes of XeIF4E1a, its mutated form X4E1a6 and XeIF4E1b ΔN27 with m7GDP and bn7GDP.
Fig. 6. Cap-Sepharose binding assays. (a) Lysates from mid-stage Xenopus oocytes were analyzed by affinity chromatography with control GTP-Sepharose, m7GTP-Sepharose and m7GpCH2ppA-Sepharose. Aliquots of load (L), flow-through (FT), wash (lanes 1–3), m7GTP elution (lanes 4 and 5) and final SDS-sample buffer (SDS) fractions were analyzed by Western blotting using an-eIF4E1 antibody, which detects eIF4E1a (both an alternatively spliced isoform L, eIF4E1aL, and the canonical isoform, eIF4E1a) and eIF4E1b. (b) Analysis of binding recombinant proteins XeIF4E1a and XeIF4E1b to GTP-Sepharose, m7GTP-Sepharose and m7GpCH2ppA-Sepharose by 15% SDS-PAGE and Coomassie Blue staining. (c) The structures of GTP-Sepharose, m7GTP-Sepharose and m7GpCH2ppA-Sepharose are shown.
Andrei,
A role for eIF4E and eIF4E-transporter in targeting mRNPs to mammalian processing bodies.
2005, Pubmed
Andrei,
A role for eIF4E and eIF4E-transporter in targeting mRNPs to mammalian processing bodies.
2005,
Pubmed Brown,
Crystallographic and mass spectrometric characterisation of eIF4E with N7-alkylated cap derivatives.
2007,
Pubmed Cai,
Quantitative assessment of mRNA cap analogues as inhibitors of in vitro translation.
1999,
Pubmed Charlesworth,
Specificity factors in cytoplasmic polyadenylation.
2013,
Pubmed Chen,
Small-molecule inhibition of oncogenic eukaryotic protein translation in mesothelioma cells.
2014,
Pubmed Cho,
A new paradigm for translational control: inhibition via 5'-3' mRNA tethering by Bicoid and the eIF4E cognate 4EHP.
2005,
Pubmed Dostie,
A novel shuttling protein, 4E-T, mediates the nuclear import of the mRNA 5' cap-binding protein, eIF4E.
2000,
Pubmed Esposito,
Influence of glycerol on the structure and thermal stability of lysozyme: a dynamic light scattering and circular dichroism study.
2009,
Pubmed Evsikov,
Cracking the egg: molecular dynamics and evolutionary aspects of the transition from the fully grown oocyte to embryo.
2006,
Pubmed
,
Xenbase Evsikov,
Evolutionary origin and phylogenetic analysis of the novel oocyte-specific eukaryotic translation initiation factor 4E in Tetrapoda.
2009,
Pubmed
,
Xenbase Ferraiuolo,
A role for the eIF4E-binding protein 4E-T in P-body formation and mRNA decay.
2005,
Pubmed Furuichi,
Viral and cellular mRNA capping: past and prospects.
2000,
Pubmed Gekko,
Mechanism of protein stabilization by glycerol: preferential hydration in glycerol-water mixtures.
1981,
Pubmed Ghosh,
Nontoxic chemical interdiction of the epithelial-to-mesenchymal transition by targeting cap-dependent translation.
2009,
Pubmed Gillian-Daniel,
Modifications of the 5' cap of mRNAs during Xenopus oocyte maturation: independence from changes in poly(A) length and impact on translation.
1998,
Pubmed
,
Xenbase Gkogkas,
Translational control and autism-like behaviors.
2013,
Pubmed Gross,
Ribosome loading onto the mRNA cap is driven by conformational coupling between eIF4G and eIF4E.
2003,
Pubmed Grudzien,
Novel cap analogs for in vitro synthesis of mRNAs with high translational efficiency.
2004,
Pubmed Gu,
Insights into the structure, mechanism, and regulation of scavenger mRNA decapping activity.
2004,
Pubmed Haghighat,
eIF4G dramatically enhances the binding of eIF4E to the mRNA 5'-cap structure.
1997,
Pubmed Hooft,
Errors in protein structures.
1996,
Pubmed Igreja,
4E-BPs require non-canonical 4E-binding motifs and a lateral surface of eIF4E to repress translation.
2014,
Pubmed Jackson,
The mechanism of eukaryotic translation initiation and principles of its regulation.
2010,
Pubmed Jemielity,
Novel "anti-reverse" cap analogs with superior translational properties.
2003,
Pubmed Jia,
Cap-dependent translation initiation factor eIF4E: an emerging anticancer drug target.
2012,
Pubmed Joshi,
Characterization of mammalian eIF4E-family members.
2004,
Pubmed Joshi,
Phylogenetic analysis of eIF4E-family members.
2005,
Pubmed Kamenska,
eIF4E-binding proteins: new factors, new locations, new roles.
2014,
Pubmed Kamenska,
Human 4E-T represses translation of bound mRNAs and enhances microRNA-mediated silencing.
2014,
Pubmed Kinkelin,
Crystal structure of a minimal eIF4E-Cup complex reveals a general mechanism of eIF4E regulation in translational repression.
2012,
Pubmed Kiraga-Motoszko,
Thermodynamics of molecular recognition of mRNA 5' cap by yeast eukaryotic initiation factor 4E.
2011,
Pubmed Kong,
Translational control in cellular and developmental processes.
2012,
Pubmed Kubacka,
Investigating the consequences of eIF4E2 (4EHP) interaction with 4E-transporter on its cellular distribution in HeLa cells.
2013,
Pubmed
,
Xenbase Kuge,
Cytoplasmic 3' poly(A) addition induces 5' cap ribose methylation: implications for translational control of maternal mRNA.
1995,
Pubmed
,
Xenbase Li,
Treatment of breast and lung cancer cells with a N-7 benzyl guanosine monophosphate tryptamine phosphoramidate pronucleotide (4Ei-1) results in chemosensitization to gemcitabine and induced eIF4E proteasomal degradation.
2013,
Pubmed Liou,
trans-spliced Caenorhabditis elegans mRNAs retain trimethylguanosine caps.
1990,
Pubmed Liu,
Structural basis for nematode eIF4E binding an m(2,2,7)G-Cap and its implications for translation initiation.
2011,
Pubmed Lüthy,
Assessment of protein models with three-dimensional profiles.
1992,
Pubmed Mamane,
eIF4E--from translation to transformation.
2004,
Pubmed Marcotrigiano,
Cocrystal structure of the messenger RNA 5' cap-binding protein (eIF4E) bound to 7-methyl-GDP.
1997,
Pubmed Minshall,
CPEB interacts with an ovary-specific eIF4E and 4E-T in early Xenopus oocytes.
2007,
Pubmed
,
Xenbase Mishra,
Efficient refolding of aggregation-prone citrate synthase by polyol osmolytes: how well are protein folding and stability aspects coupled?
2005,
Pubmed Modrak-Wojcik,
Eukaryotic translation initiation is controlled by cooperativity effects within ternary complexes of 4E-BP1, eIF4E, and the mRNA 5' cap.
2013,
Pubmed Morita,
A novel 4EHP-GIGYF2 translational repressor complex is essential for mammalian development.
2012,
Pubmed Nakamura,
Drosophila cup is an eIF4E binding protein that associates with Bruno and regulates oskar mRNA translation in oogenesis.
2004,
Pubmed Nelson,
Drosophila Cup is an eIF4E-binding protein that functions in Smaug-mediated translational repression.
2004,
Pubmed Niedzwiecka,
Biophysical studies of eIF4E cap-binding protein: recognition of mRNA 5' cap structure and synthetic fragments of eIF4G and 4E-BP1 proteins.
2002,
Pubmed Niedzwiecka,
Biophysical approach to studies of cap-eIF4E interaction by synthetic cap analogs.
2007,
Pubmed Nishimura,
On the base-stacking in the 5'-terminal cap structure of mRNA: a fluorescence study.
1980,
Pubmed Okumura,
ISG15 modification of the eIF4E cognate 4EHP enhances cap structure-binding activity of 4EHP.
2007,
Pubmed Osborne,
eIF4E3 acts as a tumor suppressor by utilizing an atypical mode of methyl-7-guanosine cap recognition.
2013,
Pubmed Paku,
A conserved motif within the flexible C-terminus of the translational regulator 4E-BP is required for tight binding to the mRNA cap-binding protein eIF4E.
2012,
Pubmed Rhoads,
eIF4E: new family members, new binding partners, new roles.
2009,
Pubmed Robalino,
Two zebrafish eIF4E family members are differentially expressed and functionally divergent.
2004,
Pubmed
,
Xenbase Rosettani,
Structures of the human eIF4E homologous protein, h4EHP, in its m7GTP-bound and unliganded forms.
2007,
Pubmed Sali,
Comparative protein modelling by satisfaction of spatial restraints.
1993,
Pubmed Sonenberg,
Regulation of translation initiation in eukaryotes: mechanisms and biological targets.
2009,
Pubmed Standart,
Translational control in early development: CPEB, P-bodies and germinal granules.
2008,
Pubmed
,
Xenbase Szczepaniak,
Affinity resins containing enzymatically resistant mRNA cap analogs--a new tool for the analysis of cap-binding proteins.
2012,
Pubmed Tomoo,
Structural features of human initiation factor 4E, studied by X-ray crystal analyses and molecular dynamics simulations.
2003,
Pubmed Topisirovic,
Cap and cap-binding proteins in the control of gene expression.
2011,
Pubmed Umenaga,
Identification and function of the second eIF4E-binding region in N-terminal domain of eIF4G: comparison with eIF4E-binding protein.
2011,
Pubmed Villaescusa,
Cytoplasmic Prep1 interacts with 4EHP inhibiting Hoxb4 translation.
2009,
Pubmed Volpon,
Cap-free structure of eIF4E suggests a basis for conformational regulation by its ligands.
2006,
Pubmed Watts,
Weighing up the possibilities: Controlling translation by ubiquitylation and sumoylation.
2014,
Pubmed Weill,
Translational control by changes in poly(A) tail length: recycling mRNAs.
2012,
Pubmed Wieczorek,
Fluorescence and absorption spectroscopic properties of RNA 5'-cap analogues derived from 7-methyl-, N2,7-dimethyl- and N2,N2,7-trimethyl-guanosines.
1995,
Pubmed Yoffe,
Cap-binding activity of an eIF4E homolog from Leishmania.
2004,
Pubmed Ziemniak,
Potential therapeutic applications of RNA cap analogs.
2013,
Pubmed Zuberek,
Weak binding affinity of human 4EHP for mRNA cap analogs.
2007,
Pubmed Zuberek,
Influence of electric charge variation at residues 209 and 159 on the interaction of eIF4E with the mRNA 5' terminus.
2004,
Pubmed
