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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.
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