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First published online February 23, 2007; 10.1104/pp.106.093146 Plant Physiology 143:1504-1518 (2007) © 2007 American Society of Plant Biologists OPEN ACCESS ARTICLE
The Structure of Eukaryotic Translation Initiation Factor-4E from Wheat Reveals a Novel Disulfide Bond1,[OA]Department of Chemistry and Biochemistry and the Institute for Cellular and Molecular Biology, University of Texas, Austin, Texas 78712
Eukaryotic translation initiation factor-4E (eIF4E) recognizes and binds the m7 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
1 This work was supported by the National Institutes of Health (grant no. GM 63593 to J.D.R.), by the National Science Foundation (grant no. MCB–0214996 to K.S.B.), by the Welch Foundation (grant nos. F–1225, F–1353, and F–1333 to J.D.R., D.W.H., and K.S.B., respectively), and by the Center for Structural Biology from the College of Natural Sciences. 2 These authors contributed equally to the paper. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Karen S. Browning (kbrowning{at}mail.utexas.edu). [OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.106.093146 * Corresponding author; e-mail kbrowning{at}mail.utexas.edu; fax 512–471–8696. Received November 15, 2006; accepted February 21, 2007; published February 23, 2007. This article has been cited by other articles:
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-strands, three 
-helices, and three extended loops. Surprisingly, the wild-type protein did not crystallize with m7GTP 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 m7GDP 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 m7GTP in a similar and labile manner, with dissociation rates in the range of 20 to 100 s–1. 
