Bogorad Andrew M, Lin Kai Ying, Marintchev Assen
Boston University School of Medicine, Department of Physiology & Biophysics, Boston, MA 02118, USA.
Nucleic Acids Res. 2017 Nov 16;45(20):11962-11979. doi: 10.1093/nar/gkx845.
Eukaryotic translation initiation factor 2 (eIF2) is a heterotrimeric GTPase, which plays a critical role in protein synthesis regulation. eIF2-GTP binds Met-tRNAi to form the eIF2-GTP•Met-tRNAi ternary complex (TC), which is recruited to the 40S ribosomal subunit. Following GTP hydrolysis, eIF2-GDP is recycled back to TC by its guanine nucleotide exchange factor (GEF), eIF2B. Phosphorylation of the eIF2α subunit in response to various cellular stresses converts eIF2 into a competitive inhibitor of eIF2B, which triggers the integrated stress response (ISR). Dysregulation of eIF2B activity is associated with a number of pathologies, including neurodegenerative diseases, metabolic disorders, and cancer. However, despite decades of research, the underlying molecular mechanisms of eIF2B action and regulation remain unknown. Here we employ a combination of NMR, fluorescence spectroscopy, site-directed mutagenesis, and thermodynamics to elucidate the mechanisms of eIF2B action and its regulation by phosphorylation of the substrate eIF2. We present: (i) a novel mechanism for the inhibition of eIF2B activity, whereby eIF2α phosphorylation destabilizes an autoregulatory intramolecular interaction within eIF2α; and (ii) the first structural model for the complex of eIF2B with its substrate, eIF2-GDP, reaction intermediates, apo-eIF2 and eIF2-GTP, and product, TC, with direct implications for the eIF2B catalytic mechanism.
真核生物翻译起始因子2（eIF2）是一种异源三聚体GTP酶，在蛋白质合成调控中起关键作用。eIF2-GTP与甲硫氨酰-tRNAi结合形成eIF2-GTP•甲硫氨酰-tRNAi三元复合物（TC），该复合物被招募到40S核糖体亚基。GTP水解后，eIF2-GDP通过其鸟嘌呤核苷酸交换因子（GEF）eIF2B循环回到TC。响应各种细胞应激时eIF2α亚基的磷酸化将eIF2转化为eIF2B的竞争性抑制剂，从而触发整合应激反应（ISR）。eIF2B活性失调与多种疾病相关，包括神经退行性疾病、代谢紊乱和癌症。然而，尽管经过数十年的研究，eIF2B作用和调控的潜在分子机制仍然未知。在这里，我们结合核磁共振、荧光光谱、定点诱变和热力学方法来阐明eIF2B的作用机制及其受底物eIF2磷酸化调控的机制。我们提出：（i）一种抑制eIF2B活性的新机制，即eIF2α磷酸化使eIF2α内的一种自动调节分子内相互作用不稳定；（ii）eIF2B与其底物eIF2-GDP、反应中间体、脱辅基eIF2和eIF2-GTP以及产物TC形成复合物的首个结构模型，这对eIF2B催化机制有直接影响。
