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Evolutionary changes in the Leishmania eIF4F complex involve variations in the eIF4E-eIF4G interactions.

Yoffe Y, Léger M, Zinoviev A, Zuberek J, Darzynkiewicz E, Wagner G, Shapira M - Nucleic Acids Res. (2009)

Bottom Line: LeishIF4G-3 was found to coelute with the parasite eIF4F subunits from an m(7)GTP-Sepharose column and to bind directly to LeishIF4E.A parallel eIF4E-binding peptide was identified in LeishIF4G-3 (20-YPGFSLDE-27).The LeishIF4E-LeishIF4G-3 interaction was also confirmed by nuclear magnetic resonance (NMR) studies.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel.

ABSTRACT
Translation initiation in eukaryotes is mediated by assembly of the eIF4F complex over the m(7)GTP cap structure at the 5'-end of mRNAs. This requires an interaction between eIF4E and eIF4G, two eIF4F subunits. The Leishmania orthologs of eIF4E are structurally diverged from their higher eukaryote counterparts, since they have evolved to bind the unique trypanosomatid cap-4 structure. Here, we characterize a key eIF4G candidate from Leishmania parasites (LeishIF4G-3) that contains a conserved MIF4G domain. LeishIF4G-3 was found to coelute with the parasite eIF4F subunits from an m(7)GTP-Sepharose column and to bind directly to LeishIF4E. In higher eukaryotes the eIF4E-eIF4G interaction is based on a conserved peptide signature [Y(X(4))Lphi], where X is any amino acid and Phi is a hydrophobic residue. A parallel eIF4E-binding peptide was identified in LeishIF4G-3 (20-YPGFSLDE-27). However, the binding motif varies extensively: in addition to Y20 and L25, binding strictly requires the presence of F23, whereas the hydrophobic amino acid (Phi) is dispensable. The LeishIF4E-LeishIF4G-3 interaction was also confirmed by nuclear magnetic resonance (NMR) studies. In view of these diversities, the characterization of the parasite eIF4E-eIF4G interaction may not only serve as a novel target for inhibiting Leishmaniasis but also provide important insight for future drug discovery.

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Binding of a LeishIF4G-3 peptide to LeishIF4E-1. (A) 15N, 1HTROSY-HSQC spectra of 15N-labeled LeishIF4E-1 (250 µM) in the presence of m7GTP (5 mM) for stabilization (LeishIF4E-1 free, in blue). (B) Overlays of 15N, 1HTROSY-HSQC spectra of 15N-LeishIF4E-1 free with increasing amounts of non-labeled LeishIF4G-3 peptide (NYLEPPYPGFSLDEVVRR). The concentrations of the LeishIF4G-3 added were 0 µM (blue, ratio 1:0), 250 µM (red, ratio 1:1) and 1.25 mM (green, ratio 1:5). (C) Enlargement of a region displaying the chemical shift differences between the spectra that is boxed in (B) in dashed lines. Peaks movements are indicated by arrows.
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Figure 8: Binding of a LeishIF4G-3 peptide to LeishIF4E-1. (A) 15N, 1HTROSY-HSQC spectra of 15N-labeled LeishIF4E-1 (250 µM) in the presence of m7GTP (5 mM) for stabilization (LeishIF4E-1 free, in blue). (B) Overlays of 15N, 1HTROSY-HSQC spectra of 15N-LeishIF4E-1 free with increasing amounts of non-labeled LeishIF4G-3 peptide (NYLEPPYPGFSLDEVVRR). The concentrations of the LeishIF4G-3 added were 0 µM (blue, ratio 1:0), 250 µM (red, ratio 1:1) and 1.25 mM (green, ratio 1:5). (C) Enlargement of a region displaying the chemical shift differences between the spectra that is boxed in (B) in dashed lines. Peaks movements are indicated by arrows.

Mentions: The binding to LeishIF4E-1 of a synthetic short peptide of 18 amino acids (14-NYLEPPYPGFSLDEVVRR-31) from the LeishIF4G-3 sequence, encompassing the consensus eIF4E-binding motif, was also investigated by NMR titration. LeishIF4E-1 was used for technical reasons, since the recombinant protein is more soluble than LeishIF4E-4 and stable at 25°C for a prolonged period. Further, LeishIF4E-1 binds m7GTP with an affinity similar to cap-4 (30). m7GTP was added to LeishIF4E-1 prior to recording the NMR spectra in order to stabilize the protein. A [15N,1H]TROSY-HSQC spectrum (36) was recorded using uniformly 15N-labeled LeishIF4E-1 (LeishIF4E-1 free) (Figure 8A). In subsequent experiments, unlabeled LeishIF4G-3 peptide was added in increasing molar ratios. This resulted in chemical shift changes in a select subset of resonances (Figure 8B and C) indicating that LeishIF4G-3 peptide binds to LeishIF4E-1.Figure 8.


Evolutionary changes in the Leishmania eIF4F complex involve variations in the eIF4E-eIF4G interactions.

Yoffe Y, Léger M, Zinoviev A, Zuberek J, Darzynkiewicz E, Wagner G, Shapira M - Nucleic Acids Res. (2009)

Binding of a LeishIF4G-3 peptide to LeishIF4E-1. (A) 15N, 1HTROSY-HSQC spectra of 15N-labeled LeishIF4E-1 (250 µM) in the presence of m7GTP (5 mM) for stabilization (LeishIF4E-1 free, in blue). (B) Overlays of 15N, 1HTROSY-HSQC spectra of 15N-LeishIF4E-1 free with increasing amounts of non-labeled LeishIF4G-3 peptide (NYLEPPYPGFSLDEVVRR). The concentrations of the LeishIF4G-3 added were 0 µM (blue, ratio 1:0), 250 µM (red, ratio 1:1) and 1.25 mM (green, ratio 1:5). (C) Enlargement of a region displaying the chemical shift differences between the spectra that is boxed in (B) in dashed lines. Peaks movements are indicated by arrows.
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Related In: Results  -  Collection

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Figure 8: Binding of a LeishIF4G-3 peptide to LeishIF4E-1. (A) 15N, 1HTROSY-HSQC spectra of 15N-labeled LeishIF4E-1 (250 µM) in the presence of m7GTP (5 mM) for stabilization (LeishIF4E-1 free, in blue). (B) Overlays of 15N, 1HTROSY-HSQC spectra of 15N-LeishIF4E-1 free with increasing amounts of non-labeled LeishIF4G-3 peptide (NYLEPPYPGFSLDEVVRR). The concentrations of the LeishIF4G-3 added were 0 µM (blue, ratio 1:0), 250 µM (red, ratio 1:1) and 1.25 mM (green, ratio 1:5). (C) Enlargement of a region displaying the chemical shift differences between the spectra that is boxed in (B) in dashed lines. Peaks movements are indicated by arrows.
Mentions: The binding to LeishIF4E-1 of a synthetic short peptide of 18 amino acids (14-NYLEPPYPGFSLDEVVRR-31) from the LeishIF4G-3 sequence, encompassing the consensus eIF4E-binding motif, was also investigated by NMR titration. LeishIF4E-1 was used for technical reasons, since the recombinant protein is more soluble than LeishIF4E-4 and stable at 25°C for a prolonged period. Further, LeishIF4E-1 binds m7GTP with an affinity similar to cap-4 (30). m7GTP was added to LeishIF4E-1 prior to recording the NMR spectra in order to stabilize the protein. A [15N,1H]TROSY-HSQC spectrum (36) was recorded using uniformly 15N-labeled LeishIF4E-1 (LeishIF4E-1 free) (Figure 8A). In subsequent experiments, unlabeled LeishIF4G-3 peptide was added in increasing molar ratios. This resulted in chemical shift changes in a select subset of resonances (Figure 8B and C) indicating that LeishIF4G-3 peptide binds to LeishIF4E-1.Figure 8.

Bottom Line: LeishIF4G-3 was found to coelute with the parasite eIF4F subunits from an m(7)GTP-Sepharose column and to bind directly to LeishIF4E.A parallel eIF4E-binding peptide was identified in LeishIF4G-3 (20-YPGFSLDE-27).The LeishIF4E-LeishIF4G-3 interaction was also confirmed by nuclear magnetic resonance (NMR) studies.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel.

ABSTRACT
Translation initiation in eukaryotes is mediated by assembly of the eIF4F complex over the m(7)GTP cap structure at the 5'-end of mRNAs. This requires an interaction between eIF4E and eIF4G, two eIF4F subunits. The Leishmania orthologs of eIF4E are structurally diverged from their higher eukaryote counterparts, since they have evolved to bind the unique trypanosomatid cap-4 structure. Here, we characterize a key eIF4G candidate from Leishmania parasites (LeishIF4G-3) that contains a conserved MIF4G domain. LeishIF4G-3 was found to coelute with the parasite eIF4F subunits from an m(7)GTP-Sepharose column and to bind directly to LeishIF4E. In higher eukaryotes the eIF4E-eIF4G interaction is based on a conserved peptide signature [Y(X(4))Lphi], where X is any amino acid and Phi is a hydrophobic residue. A parallel eIF4E-binding peptide was identified in LeishIF4G-3 (20-YPGFSLDE-27). However, the binding motif varies extensively: in addition to Y20 and L25, binding strictly requires the presence of F23, whereas the hydrophobic amino acid (Phi) is dispensable. The LeishIF4E-LeishIF4G-3 interaction was also confirmed by nuclear magnetic resonance (NMR) studies. In view of these diversities, the characterization of the parasite eIF4E-eIF4G interaction may not only serve as a novel target for inhibiting Leishmaniasis but also provide important insight for future drug discovery.

Show MeSH
Related in: MedlinePlus