Gordiyenko Yuliya, Schmidt Carla, Jennings Martin D, Matak-Vinkovic Dijana, Pavitt Graham D, Robinson Carol V
1] Department of Chemistry, Physical and Theoretical Chemistry Laboratory, University of Oxford, South Parks Road, Oxford OX1 3QZ, UK [2] MRC Laboratory of Molecular Biology, University of Cambridge, Francis Crick Avenue, Cambridge CB2 0QH, UK [3].
1] Department of Chemistry, Physical and Theoretical Chemistry Laboratory, University of Oxford, South Parks Road, Oxford OX1 3QZ, UK [2].
Nat Commun. 2014 May 23;5:3902. doi: 10.1038/ncomms4902.
eIF2B facilitates and controls protein synthesis in eukaryotes by mediating guanine nucleotide exchange on its partner eIF2. We combined mass spectrometry (MS) with chemical cross-linking, surface accessibility measurements and homology modelling to define subunit stoichiometry and interactions within eIF2B and eIF2. Although it is generally accepted that eIF2B is a pentamer of five non-identical subunits (α-ε), here we show that eIF2B is a decamer. MS and cross-linking of eIF2B complexes allows us to propose a model for the subunit arrangements within eIF2B where the subunit assembly occurs through catalytic γ- and ε-subunits, with regulatory subunits arranged in asymmetric trimers associated with the core. Cross-links between eIF2 and eIF2B allow modelling of interactions that contribute to nucleotide exchange and its control by eIF2 phosphorylation. Finally, we identify that GTP binds to eIF2Bγ, prompting us to propose a multi-step mechanism for nucleotide exchange.
真核起始因子2B（eIF2B）通过介导其伙伴真核起始因子2（eIF2）上的鸟嘌呤核苷酸交换，促进并控制真核生物中的蛋白质合成。我们将质谱（MS）与化学交联、表面可及性测量和同源建模相结合，以确定eIF2B和eIF2内的亚基化学计量和相互作用。尽管人们普遍认为eIF2B是由五个不同亚基（α-ε）组成的五聚体，但我们在此表明eIF2B是一个十聚体。eIF2B复合物的质谱分析和交联使我们能够提出一个eIF2B内亚基排列的模型，其中亚基组装通过催化性的γ-和ε-亚基发生，调节性亚基以不对称三聚体的形式排列并与核心相关联。eIF2和eIF2B之间的交联允许对有助于核苷酸交换及其由eIF2磷酸化控制的相互作用进行建模。最后，我们确定鸟苷三磷酸（GTP）与eIF2Bγ结合，促使我们提出一种核苷酸交换的多步机制。
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