The structure of eukaryotic translation initiation factor-4E from wheat reveals a novel disulfide bond.

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 beta-strands, three alpha-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(-1).

The crystal structure of the N-terminal region of the alpha subunit of translation initiation factor 2 (eIF2alpha) from Saccharomyces cerevisiae provides a view of the loop containing serine 51, the target of the eIF2alpha-specific kinases.

The alpha subunit of translation initiation factor 2 (eIF2alpha) is the target of specific kinases that can phosphorylate a conserved serine residue as part of a mechanism for regulating protein expression at the translational level in eukaryotes. The structure of the 20 kDa N-terminal region of eIF2alpha from Saccharomyces cerevisiae was determined by X-ray crystallography at 2.5A resolution. In most respects, the structure is similar to that of the recently solved human eIF2alpha; the rather elongated protein contains a five-stranded antiparallel beta-barrel in its N-terminal region, followed by an almost entirely helical domain. The S.cerevisiae eIF2alpha lacks a disulfide bridge that is present in the homologous protein in humans and some of the other higher eukaryotes. Interestingly, a conserved loop consisting of residues 51-65 and containing serine 51, the putative phosphorylation site, is visible in the electron density maps of the S.cerevisiae eIF2alpha; most of this functionally important loop was not observed in the crystal structure of the human protein. This loop is relatively exposed to solvent, and contains two short 3(10) helices in addition to some extended structure. Serine 51 is located at the C-terminal end of one of the 3(10) helices and near several conserved positively charged residues. The side-chain of serine 51 is sufficiently exposed so that its phosphorylation would not necessitate a substantial change in the protein structure. The structures and relative positions of residues that have been implicated in kinase binding and in the interaction with guanine nucleotide exchange factor (eIF2B) are described.