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Mechanism and Regulation of Eukaryotic Protein Synthesis
- Thomas E. Dever, PhD, Head, Section on Protein Biosynthesis
- Byung-Sik Shin, PhD, Staff Scientist
- Yvette R. Pittman, PhD, Postdoctoral Fellow
- Margarito Rojas, PhD, Visiting Fellow
- Meghna Thakur, PhD, Visiting Fellow
- Chune Cao, Biological Laboratory Technician
- Erik Gutierrez, BS, Graduate Student
- Jason A. Murray, BS, Graduate Student
- Joo-Ran Kim, BS, Special Volunteer
We study the mechanism and regulation of protein synthesis, focusing on GTPases and protein kinases that control this fundamental cellular process. We use molecular-genetic and biochemical studies to dissect the structure-function properties of the translation initiation factors eIF2, a GTPase that binds methionyl-tRNA to the ribosome, and eIF5B, a second GTPase that catalyzes ribosomal subunit joining in the final step of translation initiation. Our studies revealed a critical role for eIF2 in start codon selection, elucidated how an eIF2gamma mutation that is associated with intellectual disability impairs eIF2 function, and defined a functionally important contact between eIF5B and the ribosome. We also investigate stress-responsive protein kinases that phosphorylate eIF2alpha. Recent studies revealed fast evolution of the antiviral kinase PKR in vertebrates and linked this with altered sensitivity to poxvirus inhibitors of the kinase. Finally, our studies on the factor eIF5A revealed an unanticipated role in the elongation phase of protein synthesis.
Analysis of eIF2 binding to Met-tRNAiMet and the ribosome
The gamma subunit of eIF2 is a GTPase that, based on its sequence and the structure of the archaeal homolog aIF2alpha, resembles the bacterial translation elongation factor EF–Tu. However, in contrast to EF-Tu, which binds to the A-site of the 70S ribosome, eIF2 binds Met-tRNAiMet to the P-site of the 40S subunit. To gain insights into how eIF2 binds to Met-tRNAiMet and then associates with the 40S ribosome, we used directed hydroxyl radical probing to identify eIF2 contacts within the 40S–eIF1–eIF1A–eIF2–GTP–Met-tRNAi–mRNA (48S) complex (1). We generated a cysteine-deficient version of Saccharomyces cerevisiae eIF2 and then introduced single Cys residues at predicted surface-exposed sites (based on the aIF2 structure) on eIF2alpha, eIF2beta, and eIF2gamma. The mutant proteins were purified from yeast and then derivatized with Fe(II)–BABE on the Cys residue. Following addition of hydrogen peroxide, hydroxyl radicals that form in the vicinity of the ferrous iron diffuse and cleave nucleic acid and protein backbones. Based on the structure of the EF-Tu ternary complex, we predicted that linkage of Fe(II)–BABE to domain III of eIF2gamma would result in cleavage of Met-tRNAiMet in the T-stem. However, derivatization of Fe(II)–BABE to domain III resulted in cleavage of the D-stem of Met-tRNAiMet and of 18S rRNA at the top of helix h44, a prominent landmark on the intersubunit surface of the 40S subunit. Based on the results of these and other cleavage experiments, and the fact that Met-tRNAiMet is bound to the P-site of the 40S subunit, we generated a model of the 48S complex in which domain III of eIF2gamma binds near the 18S rRNA helix h44 and in which eIF2gamma–Met-tRNAiMet contacts are restricted to the acceptor stem of the tRNA. In this model of the eIF2 ternary complex, the Met-tRNAiMet is rotated nearly 180° relative to the position of the tRNA in the EF-Tu ternary complex. Consistent with the alternate models of the eIF2 and EF-Tu ternary complexes, we found that the EF–Tu–T394C mutation in domain III severely impaired Phe-tRNA binding, whereas the corresponding eIF2gamma–K507C mutation did not impair Met-tRNAiMet binding to eIF2. Thus, despite their structural similarity, eIF2 and EF-Tu bind to tRNAs in substantially different manners, and we propose that the tRNA-binding domain III of EF-Tu has acquired a new function in eIF2gamma to bind to the ribosome (1).
Analysis of an eIF2gamma mutation that links intellectual disability to impaired translation initiation
While protein synthesis is known to play a critical role in learning and memory in diverse model systems, human intellectual disability syndromes have not been directly associated with alterations in protein synthesis. Moreover, the consequences of partial loss of eIF2gamma function or eIF2 integrity are unknown in mammals, including humans. Our collaborators in Israel and Germany recently identified a human X-chromosomal neurological disorder characterized by intellectual disability and microcephaly. Mapping studies identified the causative mutation as a single base change resulting in a missense mutation in eIF2gamma (encoded by EIF2S3). Biochemical studies of human cells overexpressing the eIF2gamma mutant and of yeast eIF2gamma with the analogous mutation revealed a defect in binding of the eIF2beta subunit to eIF2gamma. Consistent with this loss of eIF2 integrity, the mutation in yeast eIF2gamma impaired translation start codon selection and eIF2 function in vivo in a manner that was suppressed by overexpression of eIF2beta. The findings directly link intellectual disability to impaired translation initiation and implicate partial loss of eIF2 function as a mechanistic basis for the human disease (2).
Structure-function analysis of the universally conserved translational GTPase eIF5B/IF2
During the final step of translation initiation, the large 60S ribosomal subunit joins with the 40S subunit, already bound to an mRNA, to form an 80S ribosome competent for protein synthesis. We previously discovered the translation initiation factor eIF5B, an ortholog of the bacterial translation factor IF2, and showed that it catalyzes ribosomal subunit joining. The eIF5B is a GTPase that binds to GTP and hydrolyzes the nucleotide in the presence of 80S ribosomes. Moreover, we showed that GTP hydrolysis by eIF5B is a regulatory switch governing the release of eIF5B from the ribosome following subunit joining, and we identified a functionally important contact between domain II of eIF5B and helix h5 of 18S rRNA in the 40S ribosome. More recently, we showed that alpha-helix H12, which forms the stem of the chalice-shaped eIF5B, functions as a ruler connecting the GTPase center of the ribosome to the P site where Met-tRNAiMet is bound and, furthermore, that helix H12 rigidity is required to stabilize Met-tRNAiMet binding.
Molecular analysis of eIF2alpha protein kinase substrate recognition and viral regulation
Phosphorylation of eIF2alpha is a common mechanism for downregulating protein synthesis under stress conditions. Four distinct kinases phosphorylate eIF2alpha on Ser51 under different cellular stress conditions. GCN2 responds to amino acid limitation, HRI to heme deprivation, PERK to ER stress, and PKR to viral infection. Consistent with their common activity to phosphorylate eIF2alpha on Ser51, the kinases show strong sequence similarity in their kinase domains. Phosphorylation of eIF2alpha converts eIF2 from a substrate to an inhibitor of its guanine-nucleotide exchange factor eIF2B. The inhibition of eIF2B impairs general translation, slowing the growth of yeast cells and, paradoxically, enhancing the translation of the GCN4 mRNA required for yeast cells to grow under amino acid starvation conditions. We used structural, molecular, and biochemical studies to define how the eIF2alpha kinases recognize their substrate.
In collaboration with Frank Sicheri, we obtained the X-ray structure of eIF2alpha bound to the catalytic domain of PKR. Back-to-back dimerization enables each PKR protomer to engage a molecule of eIF2alpha in the crystal structure. Given that all four eIF2alpha kinases share the PKR residues mediating kinase domain dimerization and eIF2alpha recognition, we propose that all four kinases similarly dimerize and recognize eIF2alpha. Based on our results, we propose an ordered mechanism of PKR activation in which catalytic domain dimerization triggers autophosphorylation on Thr446, which in turn is required for specific eIF2alpha substrate recognition.
Our previous crystallography and accompanying molecular-genetic analyses revealed that the PKR helix alphaG contacts eIF2alpha on a face remote from the Ser51 phosphorylation site; however, the helix alphaG contact is critical for eIF2alpha phosphorylation. Moreover, when the structure of free eIF2alpha, in which the position of Ser51 is resolved, was docked on the structure of the PKR-eIF2alpha complex, Ser51 was about 20 Å from the kinase active site. Our studies revealed that docking of eIF2alpha onto PKR helix alpha-G disrupts a hydrophobic network that restricts the position of Ser51. The docking thus induces a conformational change, greater than the spontaneous "breathing" of the Ser51 loop, which enables Ser51 to engage the phospho-acceptor binding site of the kinase. Finally, we propose that the protected state of Ser51 in free eIF2alpha prevents promiscuous phosphorylation and the attendant translational regulation by heterologous kinases (3).
As part of the mammalian cell's innate immune response, the double-stranded RNA-activated protein kinase PKR phosphorylates the translation initiation factor eIF2alpha to inhibit protein synthesis and thus block viral replication. To subvert this host cell defense mechanism, viruses produce inhibitors of PKR. Several members of the poxvirus family express two distinct types of PKR inhibitor: a pseudosubstrate inhibitor (such as the vaccinia virus K3L protein that resembles the N-terminal third of eIF2alpha) and a double-stranded RNA-binding protein called E3L. High-level expression of human PKR inhibited the growth of yeast, and co-expression of the vaccinia virus K3L or E3L protein [or the related variola (smallpox) virus C3L or E3L protein, respectively] restored yeast cell growth. We are currently studying the structure-function properties of the E3L protein and characterizing PKR mutations that confer resistance to E3L inhibition.
As described above, we previously demonstrated that, when expressed in yeast, human PKR could phosphorylate the alpha subunit of eIF2 on Ser51, resulting in inhibition of protein synthesis and yeast cell growth. We also identified the mechanism of activation of PKR that requires back-to-back dimerization of two PKR kinase domains. Further demonstrating the importance of kinase domain dimerization, we showed that appending heterologous dimerization domains to the PKR kinase domain activated the kinase in yeast. Kinases resembling the eIF2alpha kinases have been identified in the genome sequences of a variety of eukaryotes, including pathogens such as Plasmodium falciparum, the protozoan that causes malaria. Plasmodium expresses three kinases related by sequence to the eIF2alpha kinases. Working in collaboration with scientists in Victor Nussenzweig's group, we demonstrated that the Plasmodium kinase PfPK4 is an eIF2alpha kinase. We first fused the PfPK4 kinase domain (KD) to the constitutive dimer GST, forming GST–PfPK4–KD. GST–PfPK4–KD phosphorylated yeast eIF2alpha on Ser51 in vivo, leading to inhibition of yeast cell growth. This toxicity was suppressed in cells expressing a non-phosphorylatable form of eIF2alpha in which Ser51 was replaced by Ala. In addition, our collaborators showed that PfPK4 and eIF2alpha phosphorylation are essential for the blood-stage growth of Plasmodium. Thus, PfPK4 is an attractive candidate for drugs to alleviate disease and inhibit malaria transmission (4).
Molecular analysis of the hypusine-containing protein eIF5A
The translation factor eIF5A, the sole protein containing the unusual amino acid hypusine [Ne-(4-amino-2-hydroxybutyl)lysine], was originally identified based on its ability to stimulate the yield (endpoint) of methionyl-puromycin synthesis, a model assay for first peptide bond synthesis. However, the precise cellular role of eIF5A is unknown. Using molecular-genetic and biochemical studies, we recently showed that eIF5A promotes translation elongation and that this activity is dependent on the hypusine modification. Given that eIF5A is a structural homologue of the bacterial protein EF-P, we propose that eIF5A/EF-P is a universally conserved translation elongation factor (5).
- Shin BS, Kim JR, Walker SE, Dong J, Lorsch JR, Dever TE. Initiation factor eIF2gamma promotes eIF2–GTP–Met-tRNAiMet ternary complex binding to the 40S ribosome. Nat Struct Mol Biol 2011;18:1227-1234.
- Borck G, Shin BS, Stiller B, Mimouni-Bloch A, Thiele H, Kim JR, Thakur M, Skinner C, Aschenbach L, Smirin-Yosef P, Har-Zahav A, Nürnberg G, Altmüller J, Frommolt P, Hofmann K, Konen O, Nürnberg P, Munnich A, Schwartz CE, Gothelf D, Colleaux L, Dever TE, Kubisch C, Basel-Vanagaite L. eIF2gamma mutation that disrupts eIF2 complex integrity links intellectual disability to impaired translation initiation. Mol Cell 2012;48:in press.
- Dey M, Velyvis A, Li JJ, Chiu E, Chiovitti D, Kay LE, Sicheri F, Dever TE. Requirement for kinase-induced conformational change in eukaryotic initiation factor 2alpha (eIF2alpha) restricts phosphorylation of Ser51. Proc Natl Acad Sci USA 2011;108:4316-4321.
- Zhang M, Mishra S, Sakthivel R, Rojas M, Ranjan R, Sullivan WJ Jr, Fontoura BM, Ménard R, Dever TE, Nussenzweig V. PK4, a eukaryotic initiation factor 2alpha (eIF2alpha) kinase, is essential for the development of the erythrocytic cycle of Plasmodium. Proc Natl Acad Sci USA 2012;109:3956-3961.
- Saini P, Eyler DE, Green R, Dever TE. Hypusine-containing protein eIF5A promotes translation elongation. Nature 2009;459:118-121.
- Guntram Borck, MD, PhD, Universität Ulm, Ulm, Germany
- Rachel Green, PhD, The Johns Hopkins University, Baltimore, MD
- Jon R. Lorsch, PhD, The Johns Hopkins University, Baltimore, MD
- Victor Nussenzweig, MD, PhD, New York University, New York, NY
- Frank Sicheri, PhD, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, and University of Toronto, Toronto, Canada