Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan.
RIKEN Systems and Structural Biology Center, Tsurumi-ku, Yokohama 230-0045, Japan.
Nature. 2016 Mar 3;531(7592):122-5. doi: 10.1038/nature16991. Epub 2016 Feb 22.
Eukaryotic cells restrict protein synthesis under various stress conditions, by inhibiting the eukaryotic translation initiation factor 2B (eIF2B). eIF2B is the guanine nucleotide exchange factor for eIF2, a heterotrimeric G protein consisting of α-, β- and γ-subunits. eIF2B exchanges GDP for GTP on the γ-subunit of eIF2 (eIF2γ), and is inhibited by stress-induced phosphorylation of eIF2α. eIF2B is a heterodecameric complex of two copies each of the α-, β-, γ-, δ- and ε-subunits; its α-, β- and δ-subunits constitute the regulatory subcomplex, while the γ- and ε-subunits form the catalytic subcomplex. The three-dimensional structure of the entire eIF2B complex has not been determined. Here we present the crystal structure of Schizosaccharomyces pombe eIF2B with an unprecedented subunit arrangement, in which the α2β2δ2 hexameric regulatory subcomplex binds two γε dimeric catalytic subcomplexes on its opposite sides. A structure-based in vitro analysis by a surface-scanning site-directed photo-cross-linking method identified the eIF2α-binding and eIF2γ-binding interfaces, located far apart on the regulatory and catalytic subcomplexes, respectively. The eIF2γ-binding interface is located close to the conserved 'NF motif', which is important for nucleotide exchange. A structural model was constructed for the complex of eIF2B with phosphorylated eIF2α, which binds to eIF2B more strongly than the unphosphorylated form. These results indicate that the eIF2α phosphorylation generates the 'nonproductive' eIF2-eIF2B complex, which prevents nucleotide exchange on eIF2γ, and thus provide a structural framework for the eIF2B-mediated mechanism of stress-induced translational control.
真核细胞在各种应激条件下通过抑制真核翻译起始因子 2B(eIF2B)来限制蛋白质合成。eIF2B 是 eIF2 的鸟嘌呤核苷酸交换因子,eIF2 由α、β和γ亚基组成的异三聚 G 蛋白。eIF2B 将 GDP 替换为 eIF2 的γ亚基(eIF2γ)上的 GTP,并且受应激诱导的 eIF2α 磷酸化抑制。eIF2B 是由两个α、β、γ、δ和ε亚基各两个拷贝组成的异十二聚体复合物;其α、β和δ亚基构成调节亚基,而γ和ε亚基形成催化亚基。整个 eIF2B 复合物的三维结构尚未确定。在这里,我们呈现了史氏酿酒酵母 eIF2B 的晶体结构,具有前所未有的亚基排列方式,其中α2β2δ2 六聚体调节亚基在其相对侧结合两个γε 二聚体催化亚基。通过表面扫描定点光交联方法进行的基于结构的体外分析确定了 eIF2α 结合和 eIF2γ 结合界面,分别位于调节亚基和催化亚基上相隔很远的位置。eIF2γ 结合界面位于保守的“NF 基序”附近,该基序对于核苷酸交换很重要。构建了 eIF2B 与磷酸化 eIF2α 的复合物的结构模型,该模型与未磷酸化形式相比更强烈地结合 eIF2B。这些结果表明,eIF2α 的磷酸化产生了“非生产性”的 eIF2-eIF2B 复合物,从而阻止了 eIF2γ 上的核苷酸交换,为 eIF2B 介导的应激诱导翻译控制机制提供了结构框架。