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Distinct features of cap binding by eIF4E1b proteins.

Kubacka D, Miguel RN, Minshall N, Darzynkiewicz E, Standart N, Zuberek J - J. Mol. Biol. (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Warsaw 02-089, Poland. Electronic address: dkuba@biogeo.uw.edu.pl.

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(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.
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f0030: (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.

Mentions: Our analysis of vertebrate eIF4E1a and eIF4E1b sequences identified the amino acids that consistently differentiate the two groups of proteins (Fig. 1). Moreover, the three-dimensional model structure of XeIF4E1b with m7GTP showed that some of these residues are proximal to the amino acids that bind the cap (Fig. 5a). These include Ser86(Met), Ser87(Pro), Ser105(Glu), Arg106(Lys), Ala199(Ser), Leu210(Thr) and Ser211(Thr) (XeIF4E1b numbering, in brackets are shown the eIF4E1a amino acids). We hypothesized that these residue changes in eIF4E1b are a significant factor in its lower cap-binding affinity. The presence in eIF4E1b of Ser105 and Arg106 instead of Glu and Lys may directly influence the position of Trp102 and hence modify the stacking interaction with the cap. Additionally, Ala199 in place of Ser may induce changes in the orientation of the Trp102 indole ring by influencing the position of His200 located close to Trp102. Furthermore, cap-binding stacking interaction may be weakened by changes of the Trp56 environment, resulting from alterations in the position of Phe48 likely induced by the presence Ser86 and Ser87 instead of Met and Pro, respectively. Replacement of Thr in positions 210 and 211 by Leu and Ser is also likely to be important, as they are located in the C-terminal loop responsible for binding the phosphate chain and second cap nucleoside.


Distinct features of cap binding by eIF4E1b proteins.

Kubacka D, Miguel RN, Minshall N, Darzynkiewicz E, Standart N, Zuberek J - J. Mol. Biol. (2014)

(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.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4306533&req=5

f0030: (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.
Mentions: Our analysis of vertebrate eIF4E1a and eIF4E1b sequences identified the amino acids that consistently differentiate the two groups of proteins (Fig. 1). Moreover, the three-dimensional model structure of XeIF4E1b with m7GTP showed that some of these residues are proximal to the amino acids that bind the cap (Fig. 5a). These include Ser86(Met), Ser87(Pro), Ser105(Glu), Arg106(Lys), Ala199(Ser), Leu210(Thr) and Ser211(Thr) (XeIF4E1b numbering, in brackets are shown the eIF4E1a amino acids). We hypothesized that these residue changes in eIF4E1b are a significant factor in its lower cap-binding affinity. The presence in eIF4E1b of Ser105 and Arg106 instead of Glu and Lys may directly influence the position of Trp102 and hence modify the stacking interaction with the cap. Additionally, Ala199 in place of Ser may induce changes in the orientation of the Trp102 indole ring by influencing the position of His200 located close to Trp102. Furthermore, cap-binding stacking interaction may be weakened by changes of the Trp56 environment, resulting from alterations in the position of Phe48 likely induced by the presence Ser86 and Ser87 instead of Met and Pro, respectively. Replacement of Thr in positions 210 and 211 by Leu and Ser is also likely to be important, as they are located in the C-terminal loop responsible for binding the phosphate chain and second cap nucleoside.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Warsaw 02-089, Poland. Electronic address: dkuba@biogeo.uw.edu.pl.

Show MeSH
Related in: MedlinePlus