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.

我们研究了通过GTP结合蛋白和蛋白质磷酸化调节的真核细胞中蛋白质合成的机制和调节。蛋白质合成的第一步是通过因子EIF2将引发剂Met-tRNA与小核糖体亚基结合。 EIF2是GTP结合蛋白，在翻译启动过程中，GTP被水解为GDP。 EIF2从与GDP复杂的核糖体中释放，并要求鸟嘌呤核苷酸交换因子EIF2B将EIF2-GDP转换为EIF2-GTP。这种交换反应由一个应力反应性蛋白激酶家族调节，该蛋白激酶专门磷酸化在残基51处的丝氨酸上的EIF2的α亚基，从而将EIF2掩盖到EIF2B的抑制剂中。在EIF2Alpha激酶的家族中，GCN2在氨基酸饥饿的条件下被激活，PKR被双链RNA激活，并下调病毒感染细胞中的蛋白质合成，并在ER应力条件下激活了PERK。在与Frank Sicheri合作的情况下，我们确定了与EIF2Alpha复杂的PKR激酶结构域的结构。该结构分析表明，EIF2Alpha与C末端叶结合，与螺旋alphag紧密接触，而催化结构域二聚化是由激酶N末端裂片的背靠背方向介导的（参考文献3）。 PKR Helix Alphag附近的EIF2Alpha Aspartate-83残基的定位将丝氨酸-51残基在激酶的活性位点附近。与结构数据一致，PKR螺旋alphag中的突变特异性损害了eIF2alpha的磷酸化。此外，将PKR映射到催化域二聚体界面并促进激酶结构域二聚体的突变。相反，电荷 - 反转突变破坏了二聚体界面中保守的盐桥的阻滞PKR自磷酸化和EIF2Alpha磷酸化。重要的是，将两个电荷反转突变组合在同一PKR等位基因中，旨在以相反的极性恢复盐桥相互作用，并挽救了PKR活性。最后，PKR中保守的苏氨酸-446自磷酸化位点的突变损害了EIF2Alpha磷酸化和病毒式假子结合。我们提出了一种有序的PKR激活机制，其中催化结构域二聚化触发自磷酸化和特定的底物识别（参考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EIF2Alpha激酶之间是保守的。相应的单个突变旨在破坏GCN2和PERK中假定的盐桥相互作用，以消除了激酶活性。更重要的是，将恢复假定的盐桥相互作用的GCN2和PERK中的双重突变恢复了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EIF2Alpha激酶结构域的活性状态。 翻译起始因子EIF2由三个组装以形成稳定复合物组成的多肽链组成。 EIF2的伽马亚基包含共有的GTP结合（G）结构域，并且该因子必须结合GTP以形成稳定的EIF2？gtp？met-trna三纳学复合物。 GTPase激活蛋白（GAP）EIF5促进EIF2和鸟嘌呤核苷酸交换因子（GEF）EIF2B促进GTP水解，负责在EIF2上将GTP交换为GDP，从而使该因子能够在额外的转换启动过程中在额外的转换启动过程中功能。 GST下拉实验表明，EIF2Alpha，EIF2BETA，EIF5和EIF2B与全长EIF2GAMMA相互作用，而EIF2B和EIF2B则与EIF2Alpha或EIF2Beta相互作用，但与EIF2GAMMA G域结合。重要的是，这些相互作用映射到EIF2Bepsilon的EIF5和C末端结构域的催化临界N端。因此，这些关键的EIF2函数调节剂与EIF2GAMMA的G结构域直接接触，这与它们在EIF2上促进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中催化核糖体亚基。 EIF5B是原核翻译起始因子IF2的直系同源。先前的研究表明，EIF5B由四个结构组装以形成圣杯形分子的结构域组成。 G域和域II和III形成了圣杯的杯子，长α螺旋形成茎，而IV域则是圣杯的基础。此外，我们先前证明了EIF5B的域IV与EIF1A因子的C末端尾巴（原核因子IF1的正源性）结合。 EIF5B-EIF1A相互作用对于有效的核糖体亚基连接至关重要（参考文献1）。我们建议EIF5B-EIF1A相互作用促进EIF5B募集到核糖体，并促进EIF5B GTP水解后释放这些因素的释放。 EIF5B GTP结合结构域的开关1元素中保守的苏氨酸残基的突变消除了GTP水解，但并未损害体外加入的亚基。开关1突变的基因抑制剂未偶联的EIF5B GTPase和翻译刺激活性，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。我们建议在GTP EIF5B存在下结合核糖体并促进亚基连接，进而触发GTP水解，从而导致该因子从核糖体中释放出来。 EIF5B开关2中保守的甘氨酸的突变受损GTP结合，GTP水解，翻译起始和酵母细胞生长。与开关2突变相关的慢增长表型的基因抑制剂映射到开关1和螺旋8（链接域II和III）。基因抑制器恢复了EIF5B的GTP结合和GTPase活性，表明开关2中普遍保守的甘氨酸并不是绝对必要的。 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首选位置。当开关2突变和开关1抑制剂突变图与所有GTP结合蛋白中保守的元素时，我们认为这种相互作用对于所有GTP结合蛋白可能都很重要。