Monzingo Arthur F, Dhaliwal Simrit, Dutt-Chaudhuri Anirvan, Lyon Angeline, Sadow Jennifer H, Hoffman David W, Robertus Jon D, Browning Karen S
Department of Chemistry, University of Texas, Austin, Texas 78712, USA.
Plant Physiol. 2007 Apr;143(4):1504-18. doi: 10.1104/pp.106.093146. Epub 2007 Feb 23.
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).
真核生物翻译起始因子4E（eIF4E）识别并结合真核生物信使核糖核酸（mRNA）5'端的7-甲基鸟苷酸；这种蛋白质-RNA相互作用是蛋白质合成起始的关键步骤。利用X射线晶体学和核磁共振（NMR）方法相结合，对小麦（Triticum aestivum）的eIF4E结构进行了研究。结晶蛋白的整体折叠与其他物种的eIF4E相似，有八条β链、三条α螺旋和三个延伸环。令人惊讶的是，尽管配体存在于溶液中，但野生型蛋白在其结合位点并未与7-甲基鸟苷三磷酸（m(7)GTP）一起结晶；帽结合环的构象变化在通常的帽结合位点形成了一个大空腔。eIF4E以二聚体形式结晶，一个单体的一个帽结合环插入另一个单体的空腔中。该蛋白还在仅在植物中保守的两个半胱氨酸（Cys）之间含有一个分子内二硫键。缺乏形成二硫键能力的小麦eIF4E的半胱氨酸到丝氨酸突变体，在其结合口袋中与7-甲基鸟苷二磷酸（m(7)GDP）一起结晶，其结构与其他物种的eIF4E-帽复合物相似。核磁共振光谱用于表明，在晶体中形成二硫键的半胱氨酸在溶液中是还原态的，但在氧化条件下可被诱导形成二硫键。在植物中形成二硫键的半胱氨酸保守存在这一观察结果增加了其氧化态可能在调节蛋白质功能中起作用的可能性。核磁共振提供的证据表明，在氧化的eIF4E中，在无配体晶体二聚体中开放的环在溶液中相对灵活。基于核磁共振的结合试验表明，还原的小麦eIF4E、具有二硫键的氧化形式以及半胱氨酸到丝氨酸突变体蛋白均以相似且不稳定的方式结合m(7)GTP，解离速率在20至100 s(-1)范围内。
