The Structure of Eukaryotic Translation Initiation Factor-4E from Wheat Reveals a Novel Disulfide Bond

Abstract

Eukaryotic translation initiation factor-4E (eIF4E) recognizes and binds the m 7 guanosine nucleotide at the 5# end of eukaryotic messenger RNAs; this protein-RNA interaction is an essential step in the initiation of protein synthesis. The structure of eIF4E from wheat (Triticum aestivum) was investigated using a combination of x-ray crystallography and nuclear magnetic resonance (NMR) methods. The overall fold of the crystallized protein was similar to eIF4E from other species, with eight b-strands, three a-helices, and three extended loops. Surprisingly, the wild-type protein did not crystallize with m 7 GTP in its binding site, despite the ligand being present in solution; conformational changes in the cap-binding loops created a large cavity at the usual cap-binding site. The eIF4E crystallized in a dimeric form with one of the cap-binding loops of one monomer inserted into the cavity of the other. The protein also contained an intramolecular disulfide bridge between two cysteines (Cys) that are conserved only in plants. A Cys-to-serine mutant of wheat eIF4E, which lacked the ability to form the disulfide, crystallized with m 7 GDP in its binding pocket, with a structure similar to that of the eIF4E-cap complex of other species. NMR spectroscopy was used to show that the Cys that form the disulfide in the crystal are reduced in solution but can be induced to form the disulfide under oxidizing conditions. The observation that the disulfide-forming Cys are conserved in plants raises the possibility that their oxidation state may have a role in regulating protein function. NMR provided evidence that in oxidized eIF4E, the loop that is open in the ligand-free crystal dimer is relatively flexible in solution. An NMR-based binding assay showed that the reduced wheat eIF4E, the oxidized form with the disulfide, and the Cys-to-serine mutant protein each bind m 7 GTP in a similar and labile manner, with dissociation rates in the range of 20 to 100 s 21 .

Two Arabidopsis Loci Encode Novel Eukaryotic Initiation Factor 4E Isoforms That Are Functionally Distinct from the Conserved Plant Eukaryotic Initiation Factor 4E

“…S1). Residues that have been identified as being involved in cap binding from crystal structures of wheat (Triticum aestivum; Monzingo et al, 2007) and pea (Pisum sativum) eIF4E (Ashby et al, 2011) or by mutational analysis (Yeam et al, 2007;GermanRetana et al, 2008) are well conserved in eIF4E1b-type proteins. One exception is the conserved positively charged residue at K78, which is predicted in the wheat eIF4E crystal structure to stabilize the negatively charged phosphate backbone of the cap structure.…”

“…Residues marked * are predicted to be involved in cap binding in plant eIF4E crystal structures (Monzingo et al, 2007;Ashby et al, 2011), and the residue marked^is predicted from the wheat eIF4E structure to form a salt bridge with the negatively charged phosphate backbone of the cap. B, Diverging residues in Arabidopsis eIF4E1b/eIF4E1c were modeled with PyMOL (DeLano, 2002) on the wheat eIF4E structure (Monzingo et al, 2007) as indicated. (Zimmermann et al, 2004) supports expression in shoots and reproductive tissue.…”

Structural Insights into Parasite eIF4E Binding Specificity for m7G and m2,2,7G mRNA Caps

“…Protein Preparation for Crystallization-Because of the flexibility of the eIF4E N terminus, most eIF4E crystals have been produced and the structures have been solved using N-terminal truncated proteins (25,27,31). From eIF4E sequence alignments and limited trypsin digestions, we chose to truncate the schistosome protein by 22 residues at the N terminus to generate schistosome eIF4E- .…”

“…Structural studies on mammalian, plant, and yeast eIF4E using NMR (25)(26)(27)(28)(29) and crystallography (5, 27, 30 -34) have provided insight into the recognition of the m 7 GpppN cap by eIF4E. The eIF4E core resembles a "cupped hand" within which the cap-binding pocket residues are located.…”

Phosphorylation of Plant Translation Initiation Factors by CK2 Enhances the in Vitro Interaction of Multifactor Complex Components

“…NMR analysis was performed by Dr. David Hoffman, University of Texas Department of Chemistry and Biochemistry. Sample preparation, spectra measurement, and evaluation for one-dimensional NMR spectroscopy were performed as described previously (23).…”