Mechanism and Regulation Of Eukaryotic Protein Synthesis

我们研究了真核细胞中蛋白质合成的机制和调控，重点是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eIF2因子将启动子Met-tRNA与核糖体小亚基结合。EIF2是一种GTP结合蛋白，在翻译启动过程中，GTP被水解为GDP。EIF2从核糖体中释放出来，与GDP形成复合体，需要鸟嘌呤核苷酸交换因子eIF2B将eIF2-GDP转化为eIF2-GTP。这种交换反应受一系列应激反应蛋白激酶的调节，这些蛋白激酶特异性地磷酸化51位丝氨酸上eIF2的阿尔法亚单位，从而将eIF2转化为eIF2B的抑制物。在eIF2α激酶家族中，有GCN2，它在氨基酸饥饿的条件下被激活，以及PKR，它被双链RNA激活，并下调病毒感染细胞的蛋白质合成。截断分析表明，在体外和体内，eIF2α残基1-180是GCN2和PKR有效磷酸化丝氨酸-51的最低底物。定点突变和随机突变分析发现酵母eIF2α的N-末端OB-折叠域(残基1-90)中存在大量的突变，这些突变损害了翻译调控(参考文献2)。谷氨酸-49或远端天冬氨酸-83的任何突变都会阻止翻译调节，然而只有一小部分突变会损害丝氨酸-51的磷酸化。丙氨酸替代天冬氨酸-83在体内和体外都消除了丝氨酸-51的磷酸化，证实了远离磷酸化位点的残基对于激酶底物识别的重要性。其他阻止翻译调控的突变，但不是丝氨酸-51磷酸化，削弱了eIF2B与磷酸化的eIF2α的结合。因此，eIF2α激酶和eIF2B识别eIF2α上相同的表面和重叠的决定因素。在与Frank Sinheri的合作中，我们确定了与eIF2α形成的复合体中的PKR激酶域的结构。这一结构分析表明，eIF2α与C-末端叶结合，与螺旋αG密切接触，而催化结构域的二聚化是由激酶N-末端叶的背靠背取向介导的(参考文献4)。将eIF2α天冬氨酸-83残基定位在PKR螺旋αG附近，将丝氨酸-51残基放置在该激酶的活性部位附近。与结构数据一致，PKR螺旋αG的突变特异性地削弱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，促进激活域的二聚化。相反，破坏二聚体界面保守盐桥的突变会阻止PKR自动磷酸化和eIF2α磷酸化。最后，PKR中保守的苏氨酸-446自动磷酸化位点的突变会损害eIF2α的磷酸化和病毒假底物的结合。我们提出了一种PKR激活的有序机制，其中催化结构域二聚触发了自动磷酸化和特定底物识别(参考文献5)。 GTP结合蛋白eIF5B催化核糖体亚基加入翻译起始的最后一步。EIF5B是原核生物翻译起始因子IF2的同源基因。先前的研究表明，eIF5B由四个结构域组成，它们在结构上组装形成圣杯形状的分子。G结构域加上结构域II和III形成圣杯，一个长的阿尔法螺旋形成茎，结构域IV是圣杯的底部。此外，我们先前证明eIF5B的IV结构域与因子eIF1A(原核因子IF1的同源)的C-末端结合。我们认为eIF5B-eIF1A的相互作用对于eIF5B与核糖体的结合以及eIF5B亚基连接和GTP水解后这两种因子从核糖体中的释放也是重要的。EIF5B的G结构域包含典型的GTP结合蛋白的特征标记，包括保守的Switch 1和Switch 2基序。Switch 1中保守的苏氨酸残基的突变阻止了GTP的水解，但不影响亚基在体外的连接。Switch 1突变的基因内抑制物解偶联eIF5B GTP酶和翻译刺激活性，表明eIF5B GTP在翻译启动中的调节作用而不是机械作用。我们认为，在GTP存在的情况下，eIF5B与核糖体结合并促进亚基连接，这反过来又触发GTP水解，导致因子从核糖体中释放。EIF5B开关2中保守的甘氨酸突变会影响GTP结合、GTP水解、翻译启动和酵母细胞的生长。与Switch 2突变相关的缓慢生长表型的基因内抑制基因被映射到Switch 1和Helix 8(连接结构域II和III)。开关1中的基因内抑制子恢复了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与开关2接触的位置附近，并且抑制突变被预测为变构影响开关2的位置。我们认为开关2中保守的甘氨酸突变改变了eIF5B活性部位的结构，两个基因内抑制突变通过将开关2重新定位到首选位置而恢复了eIF5B活性部位的有利几何构型。由于Switch 2突变和Switch 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结合蛋白都是重要的。

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, and PKR, which is activated by double-stranded RNA and downregulates protein synthesis in virally infected cells. Truncation analyses revealed that eIF2alpha residues 1-180 form the minimal substrate for efficient phosphorylation of serine-51 by GCN2 and PKR both in vitro and in vivo. Site-directed and random mutational analyses identified a large number of mutations throughout the N-terminal OB-fold domain (residues 1-90) of yeast eIF2alpha that impaired translational regulation (reference 2). Any mutation at glutamate-49 or the remote aspartate-83 blocked translational regulation, however only a subset of the mutations impaired serine-51 phosphorylation. Substitution of alanine for aspartate-83 eliminated serine-51 phosphorylation both in vivo and in vitro establishing the importance of residues remote from the phosphorylation site for kinase substrate recognition. Other mutations that blocked translational regulation, but not serine-51 phosphorylation, impaired the binding of eIF2B to phosphorylated eIF2alpha. Thus, the eIF2alpha kinases and eIF2B recognize the same surface and overlapping determinants on eIF2alpha. 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 4). 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, mutations that disrupt a conserved salt-bridge in the dimer interface block PKR autophosphorylation and eIF2alpha phosphorylation. 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 5). 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). We propose that the eIF5B-eIF1A interaction is important for binding eIF5B to the ribosome and possibly also for release of both factors from the ribosome following subunit joining and GTP hydrolysis by eIF5B. The G domain of eIF5B contains the hallmarks associated with typical GTP-binding proteins including the conserved switch 1 and switch 2 motifs. Mutation of the conserved threonine residue in switch 1 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 suppressor in switch 1 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.