Mechanism and Regulation Of Eukaryotic Protein Synthesis

真核蛋白质合成机制及调控

• 批准号: 7333937
• 负责人: THOMAS E DEVER
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7333937
• 关键词: Mechanism; Regulation; Eukaryotic; Protein; Synthesis

We study the mechanism and regulation of protein synthesis in eukaryotic cells focusing on regulation by GTP-binding proteins and protein phosphorylation. The first step of protein synthesis is binding the initiator Met-tRNA to the small ribosomal subunit by the factor eIF2. The eIF2 is a GTP-binding protein and during the course of translation initiation the GTP is hydrolyzed to GDP. The eIF2 is released from the ribosome in complex with GDP and requires the guanine-nucleotide exchange factor eIF2B to convert eIF2-GDP to eIF2-GTP. This exchange reaction is regulated by a family of stress-responsive protein kinases that specifically phosphorylate the alpha subunit of eIF2 on serine at residue 51, and thereby covert eIF2 into an inhibitor of eIF2B. Among the family of eIF2alpha kinases are GCN2, which is activated under conditions of amino acid starvation, PKR, which is activated by double-stranded RNA and downregulates protein synthesis in virally infected cells, and PERK, activated under conditions of ER stress. In collaboration with Frank Sicheri we determined the structure of the PKR kinase domain in complex with eIF2alpha. This structural analysis revealed that eIF2alpha binds to the C-terminal lobe making intimate contact with helix alphaG, while catalytic domain dimerization is mediated by a back-to-back orientation of the kinase N-terminal lobes (reference 3). Positioning of the eIF2alpha aspartate-83 residue near PKR helix alphaG places the serine-51 residue near the active site of the kinase. Consistent with the structural data, mutations in PKR helix alphaG specifically impair phosphorylation of eIF2alpha. Moreover, mutations that activate PKR map to the catalytic domain dimer interface and promote kinase domain dimerization. Conversely, charge-reversal mutations that disrupt a conserved salt-bridge in the dimer interface block PKR autophosphorylation and eIF2alpha phosphorylation. Importantly, combining the two charge-reversal mutations in the same PKR allele, designed to restore the salt-bridge interaction with opposite polarity, rescued PKR activity. Finally, mutation of the conserved threonine-446 autophosphorylation site in PKR impairs eIF2alpha phosphorylation and viral pseudosubstrate binding. We propose an ordered mechanism of PKR activation in which catalytic domain dimerization triggers autophosphorylation and specific substrate recognition (reference 4). Interestingly, the residues forming the salt-bridge interaction in the PKR dimer interface are conserved among the eIF2alpha kinases. The corresponding single mutations designed to disrupt the putative salt-bridge interactions in GCN2 and PERK abolished kinase activity. More importantly, the double mutations in GCN2 and PERK, which would restore the putative salt-bridge interactions, restored the kinases' function both in vivo and in vitro. We conclude that the back-to-back dimer orientation observed in the PKR crystal structure is critical for the activity of PKR, GCN2 and PERK and that PKR structure represents the active state of the eIF2alpha kinase domain. The translation initiation factor eIF2 is composed of three polypeptide chains that assemble to form a stable complex. The gamma subunit of eIF2 contains a consensus GTP-binding (G) domain, and the factor must bind GTP to form a stable eIF2?GTP?Met-tRNA ternary complex. The GTPase-activating protein (GAP) eIF5 promotes GTP hydrolysis by eIF2 and the guanine-nucleotide exchange factor (GEF) eIF2B is responsible for exchanging GTP for GDP on eIF2 enabling the factor to function in additional rounds of translation initiation. GST pull-down experiments revealed that eIF2alpha, eIF2beta, eIF5 and eIF2B interacted with full-length eIF2gamma, whereas eIF5 and eIF2B, but not eIF2alpha or eIF2beta, bound to the eIF2gamma G domain. Importantly, these interactions were mapped to the catalytically critical N-terminus of eIF5 and C-terminal domain of eIF2Bepsilon. Thus, these critical regulators of eIF2 function make direct contacts with the G domain of eIF2gamma, consistent with their roles to promote GTP hydrolysis and GTP-GDP exchange on eIF2 (reference 2). The GTP-binding protein eIF5B catalyzes ribosomal subunit joining in the final step of translation initiation. The eIF5B is an ortholog of prokaryotic translation initiation factor IF2. Previous studies revealed that eIF5B consists of four domains that structurally assemble to form a chalice-shaped molecule. The G domain plus domains II and III form the cup of the chalice, a long alpha helix forms the stem, and domain IV is the base of the chalice. In addition, we previously showed that the domain IV of eIF5B binds to the C-terminal tail of the factor eIF1A (an ortholog of prokaryotic factor IF1). The eIF5B-eIF1A interaction is critical for efficient ribosomal subunit joining (reference 1). We propose that the eIF5B-eIF1A interaction promotes eIF5B recruitment to the ribosome and also facilitates release of the factors following GTP hydrolysis by eIF5B. Mutation of the conserved threonine residue in the switch 1 element of the eIF5B GTP-binding domain abolished GTP hydrolysis, but did not impair subunit joining in vitro. Intragenic suppressors of the switch 1 mutation uncoupled eIF5B GTPase and translational stimulatory activities indicating a regulatory rather than mechanical role for eIF5B GTP hydrolysis in translation initiation. We propose that in the presence of GTP eIF5B binds the ribosome and promotes subunit joining, which in turn triggers GTP hydrolysis leading to the factor's release from the ribosome. Mutation of the conserved glycine in switch 2 of eIF5B impaired GTP binding, GTP hydrolysis, translation initiation and yeast cell growth. Intragenic suppressors of the slow-growth phenotype associated with the switch 2 mutation mapped to switch 1 and to helix 8 (linking domains II and III). The intragenic suppressors restored both the GTP binding and GTPase activities of eIF5B revealing that the universally conserved glycine in switch 2 is not absolutely essential. Interestingly, the intragenic suppressors in switch 1 and helix 8 are located close to contact sites with switch 2, and the suppressor mutations are predicted to allosterically affect the position of switch 2. We propose that mutation of the conserved glycine in switch 2 alters the structure of the eIF5B active site, and that the two intragenic suppressor mutations restore a favorable geometry to the eIF5B active site by re-positioning switch 2 into a preferred location. As the switch 2 mutation and the switch 1 suppressor mutation map to elements conserved in all GTP-binding proteins, we believe that this interaction may be of importance for all GTP-binding proteins.

我们研究真核细胞中蛋白质合成的机制和调控，重点关注 GTP 结合蛋白和蛋白质磷酸化的调控。蛋白质合成的第一步是通过因子 eIF2 将起始子 Met-tRNA 与核糖体小亚基结合。 eIF2 是一种 GTP 结合蛋白，在翻译起始过程中，GTP 被水解为 GDP。 eIF2 从核糖体中与 GDP 复合释放，需要鸟嘌呤核苷酸交换因子 eIF2B 将 eIF2-GDP 转化为 eIF2-GTP。这种交换反应由应激反应性蛋白激酶家族调节，这些蛋白激酶特异性磷酸化 eIF2 丝氨酸残基 51 上的 α 亚基，从而将 eIF2 转化为 eIF2B 抑制剂。 eIF2α 激酶家族包括 GCN2（在氨基酸饥饿条件下激活）、PKR（由双链 RNA 激活并下调病毒感染细胞中的蛋白质合成）和 PERK（在内质网应激条件下激活）。我们与 Frank Sicheri 合作，确定了与 eIF2alpha 复合的 PKR 激酶结构域的结构。该结构分析表明，eIF2alpha 与 C 端叶结合，与螺旋 alphaG 紧密接触，而催化域二聚化是由激酶 N 端叶的背对背方向介导的（参考文献 3）。将 eIF2α 天冬氨酸 83 残基定位在 PKR 螺旋 αG 附近，将丝氨酸 51 残基置于激酶的活性位点附近。与结构数据一致，PKR 螺旋 αG 的突变特异性损害 eIF2α 的磷酸化。此外，激活 PKR 的突变映射到催化结构域二聚体界面并促进激酶结构域二聚化。相反，破坏二聚体界面中保守盐桥的电荷反转突变会阻断 PKR 自磷酸化和 eIF2α 磷酸化。重要的是，将两个电荷反转突变结合在同一个 PKR 等位基因中，旨在恢复具有相反极性的盐桥相互作用，从而挽救了 PKR 活性。最后，PKR 中保守的苏氨酸 446 自磷酸化位点的突变会损害 eIF2α 磷酸化和病毒假底物结合。我们提出了一种 PKR 激活的有序机制，其中催化域二聚化触发自磷酸化和特定底物识别（参考文献 4）。有趣的是，在 PKR 二聚体界面中形成盐桥相互作用的残基在 eIF2α 激酶中是保守的。相应的单突变旨在破坏 GCN2 和 PERK 中假定的盐桥相互作用，从而消除了激酶活性。更重要的是，GCN2 和 PERK 的双突变将恢复假定的盐桥相互作用，从而恢复激酶在体内和体外的功能。我们得出的结论是，在 PKR 晶体结构中观察到的背对背二聚体方向对于 PKR、GCN2 和 PERK 的活性至关重要，并且 PKR 结构代表 eIF2α 激酶结构域的活性状态。 翻译起始因子 eIF2 由三个多肽链组成，它们组装形成稳定的复合物。 eIF2的γ亚基含有共有的GTP结合（G）结构域，并且该因子必须结合GTP才能形成稳定的eIF2→GTP→Met-tRNA三元复合物。 GTP 酶激活蛋白 (GAP) eIF5 通过 eIF2 促进 GTP 水解，而鸟嘌呤核苷酸交换因子 (GEF) eIF2B 负责在 eIF2 上将 GTP 交换为 GDP，从而使该因子能够在额外的翻译起始轮中发挥作用。 GST 下拉实验表明，eIF2alpha、eIF2beta、eIF5 和 eIF2B 与全长 eIF2gamma 相互作用，而 eIF5 和 eIF2B（而非 eIF2alpha 或 eIF2beta）与 eIF2gamma G 结构域结合。重要的是，这些相互作用被映射到 eIF5 的催化关键 N 端和 eIF2Bepsilon 的 C 端结构域。因此，这些 eIF2 功能的关键调节因子与 eIF2gamma 的 G 结构域直接接触，这与它们促进 eIF2 上 GTP 水解和 GTP-GDP 交换的作用一致（参考文献 2）。 GTP 结合蛋白 eIF5B 在翻译起始的最后一步催化核糖体亚基连接。 eIF5B 是原核翻译起始因子 IF2 的直系同源物。先前的研究表明，eIF5B 由四个结构域组成，这些结构域在结构上组装形成圣杯状分子。 G 结构域加上结构域 II 和 III 形成圣杯的杯部，长的 α 螺旋形成茎，结构域 IV 是圣杯的底部。此外，我们之前表明，eIF5B 的结构域 IV 与因子 eIF1A（原核因子 IF1 的直系同源物）的 C 末端尾部结合。 eIF5B-eIF1A 相互作用对于核糖体亚基的有效连接至关重要（参考文献 1）。我们认为 eIF5B-eIF1A 相互作用促进 eIF5B 募集到核糖体，并且还促进 eIF5B 水解 GTP 后因子的释放。 eIF5B GTP 结合结构域开关 1 元件中保守的苏氨酸残基的突变消除了 GTP 水解，但不损害体外亚基连接。 switch 1 突变的基因内抑制子使 eIF5B GTP 酶和翻译刺激活性解偶联，表明 eIF5B GTP 水解在翻译起始中具有调节作用，而不是机械作用。我们提出，在 GTP 存在的情况下，eIF5B 会结合核糖体并促进亚基连接，进而触发 GTP 水解，导致该因子从核糖体中释放。 eIF5B 开关 2 中保守甘氨酸的突变会损害 GTP 结合、GTP 水解、翻译起始和酵母细胞生长。与开关 2 突变相关的缓慢生长表型的基因内抑制因子映射到开关 1 和螺旋 8（连接结构域 II 和 III）。基因内抑制因子恢复了 eIF5B 的 GTP 结合和 GTP 酶活性，表明开关 2 中普遍保守的甘氨酸并不是绝对必需的。有趣的是，开关1和螺旋8中的基因内抑制子位于靠近与开关2的接触位点，并且抑制子突变预计会以变构方式影响开关2的位置。我们提出开关2中保守甘氨酸的突变改变了eIF5B活性位点的结构，并且两个基因内抑制子突变通过将开关2重新定位为a，恢复了eIF5B活性位点的有利几何结构。 首选位置。由于开关 2 突变和开关 1 抑制突变映射到所有 GTP 结合蛋白中保守的元件，我们认为这种相互作用可能对所有 GTP 结合蛋白都很重要。