Mechanism and Regulation of Eukaryotic Protein Synthesis

Thomas E. Dever, PhD, Head, Section on Protein Biosynthesis
Byung-Sik Shin, PhD, Staff Scientist
Madhusudan Dey, PhD, Postdoctoral Fellow
Jeanne M. Fringer, PhD, Postdoctoral Fellow
Yvette R. Pittman, PhD, Postdoctoral Fellow
Stefan Rothenburg, PhD, Postdoctoral Fellow
Preeti Saini, PhD, Postdoctoral Fellow
Eun Joo Seo, PhD, Postdoctoral Fellow
Chune Cao, Biological Laboratory Technician
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 have revealed a critical role for eIF2 in start codon selection and have defined the important role of GTP hydrolysis in governing the release of eIF5B and eIF1A from the ribosome following subunit joining. We also study stress-responsive protein kinases that phosphorylate eIF2alpha. Recent studies on the antiviral kinase PKR revealed the importance of dimerization for kinase activation, and we are currently studying viral regulation of PKR by poxvirus inhibitors of the kinase. Finally, in related studies, we have characterized the structure-function properties of IRE1, an endoplasmic reticulum (ER) stress–responsive dual kinase-endonuclease.

Structure-function analysis of eIF2gamma, the GTP- and Met-tRNA–binding subunit of the eIF2 complex

Alone,¹ Cao, Dever; in collaboration with Lorsch

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 to GTP to form a stable eIF2•GTP•Met-tRNA ternary complex. We previously demonstrated that the GTPase-activating protein (GAP) eIF5, which promotes GTP hydrolysis by eIF2, and the guanine-nucleotide exchange factor (GEF) eIF2B, responsible for exchanging GTP for GDP on eIF2, directly bind to the G domain of eIF2gamma (Alone and Dever, 2006).

To gain a deeper understanding of the role of GTP binding and hydrolysis by eIF2, we mutated the conserved Asn135 residue in the eIF2gamma Switch I element to Asp. The N135D mutation impaired Met-tRNA binding to eIF2 and caused a Sui⁻ phenotype, enhancing initiation from a noncanonical UUG codon. Previous studies in the Donahue laboratory correlated a Sui⁻ phenotype with decreased Met-tRNA binding affinity, suggesting that premature release of Met-tRNA from eIF2 led to initiation at the UUG codon. Consistently, an A208V mutation restored Met-tRNA binding affinity and suppressed the slow-growth and Sui⁻ phenotypes of the eIF2gamma-N135D mutant. In contrast, an A382V mutation restored Met-tRNA binding and suppressed the slow-growth, but not the Sui⁻, phenotype. Moreover, an eIF2gamma-A219T mutation impaired Met-tRNA binding but unexpectedly enhanced the fidelity of initiation, suppressing the Sui⁻ phenotype associated with the eIF2gamma-N135D,A382V mutant. This uncoupling of start codon selection and Met-tRNA binding affinity to eIF2 indicates a more direct role for eIF2 in start site recognition. Interestingly, overexpression of eIF1, which is thought to monitor codon-anticodon interaction during translation initiation, likewise suppressed the Sui⁻ phenotype of the eIF2gamma mutants. We propose that structural alterations in eIF2gamma subtly alter the conformation of Met-tRNA on the 40S subunit and thereby affect the fidelity of start codon recognition independently of Met-tRNA binding affinity.

Alone PV, Dever TE. Direct binding of translation initiation factor eIF2gamma-G domain to its GTPase-activating and GDP-GTP exchange factors eIF5and eIF2Bepsilon. J Biol Chem 2006;281:12636-12644.

Structure-function analysis of the universally conserved translational GTPase eIF5B/IF2

Shin, Fringer, Cao, Kim, Dever; in collaboration with Lorsch

In 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 orthologue of the bacterial translation factor IF2, and showed that it catalyzes ribosomal subunit joining. The GTPase binds to GTP and hydrolyzes the nucleotide in the presence of 80S ribosomes. Our current efforts aim to elucidate eIF5B’s structure-function properties and to understand the role played by eIF5B in GTP binding and hydrolysis.

Our previous studies on an eIF5B mutant that was unable to hydrolyze GTP revealed that GTP hydrolysis by eIF5B activates a regulatory switch required for eIF5B release from the ribosome following subunit joining (Shin et al., Cell 2002;111:1015). Consistently, mutation of the conserved Gly479 residue in the D-x-x-G G-3 sequence motif, whose movement is thought to be critical for structural transitions of the G domain during GTP binding and hydrolysis, impaired yeast cell growth, eIF5B guanine-nucleotide binding, GTPase, and ribosomal subunit joining activities (Shin et al., 2007). Intragenic suppressor mutations in the G domain (A444V) and in a residue in domain 2 of eIF5B (D740R) that interacts with the G domain restored yeast cell growth and eIF5B nucleotide binding, GTP hydrolysis, and subunit joining activities. We propose that the Gly479 mutation distorted the geometry of the GTP-binding active site, impairing nucleotide binding and the eIF5B domain movements associated with GTP binding. Accordingly, the two suppressor mutations induce the D-x-x-G motif to adopt a conformation favorable for nucleotide binding and hydrolysis and thereby re-establish coupling between GTP binding and eIF5B domain movements (Shin et al., 2007).

In collaboration with Jon Lorsch, we showed that interaction between the C-termini of eIF5A and eIF1A is critical for eIF5B association with 40S ribosomes in vivo and for ribosomal subunit joining and translation-coupled eIF5B GTPase activity in vitro (Fringer et al., 2007; Acker et al., 2006). Moreover, blocking eIF5B GTP hydrolysis led to the accumulation of both eIF1A and eIF5B on the 80S products of subunit joining both in vivo and in vitro. Based on these findings, we propose that eIF1A facilitates the binding of eIF5B to the 40S subunit to promote subunit joining and that subsequent release of eIF1A is dependent on GTP hydrolysis and release of eIF5B from the 80S ribosome (Fringer et al., 2007).

The function of both eIF5B and the translational GTPases that promote elongation and termination of protein synthesis relies on proper interaction with the ribosome. While cryo-EM studies have revealed binding sites for the GTPases on the ribosome, in vivo data supporting these sites have not been reported. As mentioned above, mutations that impair GTP hydrolysis by the eIF5B impair yeast cell growth due to failure to dissociate from the ribosome following subunit joining. After identifying intragenic suppressor mutations of eIF5B GTPase-deficient mutants that restore cell growth by lowering the ribosome binding affinity of eIF5B, we reasoned that it should be possible to obtain mutations in the ribosome that likewise reduce eIF5B binding and suppress the toxic affects associated with expression of GTPase-defective mutants of eIF5B. An eIF5B-H480I mutation abolishes GTPase activity and causes a severe growth defect in yeast. We identified a mutation in helix h5 of the 18S rRNA in the 40S ribosomal subunit and intragenic mutations in domain II of eIF5B that suppress the toxic effects associated with expression of the eIF5B-H480I mutant in yeast. Both the rRNA and intragenic mutations lowered the ribosome binding affinity of eIF5B, indicating that the mutations enable eIF5B release from the ribosome in the absence of GTP hydrolysis. Linking the hydroxyl radical generator BABE at the sites of the domain II suppressors in eIF5B, we mapped the region of the ribosome contacted by domain II of eIF5B. Interestingly, the domain II suppressors contacted the body of the 40S subunit in the vicinity of helix h5. Thus, the rRNA and domain II suppressors affect the same contact surface between eIF5B and the 40S ribosomal subunit. Given that the helix h5 mutation also impairs translation elongation factor function, we propose that the rRNA and eIF5B suppressor mutations provide in vivo evidence supporting a functionally important docking of domain II of the translational GTPases on the body of the small ribosomal subunit.

Acker MG, Shin B-S, Dever TE, Lorsch JR. Interaction between eukaryoticinitiation factors 1A and 5B is required for efficient ribosomal subunitjoining. J Biol Chem 2006;281:8469-8475.
Fringer JM, Acker MG, Fekete CA,Lorsch JR, Dever TE. Coupled release of factors eIF5B and eIF1A from 80Sribosomes following subunit joining. Mol Cell Biol 2007;27:2384-2397.
Shin BS,Acker MG , Maag D, Kim J-R, Lorsch JR, Dever TE. Intragenic suppressor mutations restore GTP ase and translation functions of eIF5B switch II mutant. Mol Cell Biol 2007;27:1677-1685.
Shin BS, Dever TE. Molecular geneticstructure-function analysis of translation initiation factor eIF5B. MethodsEnzymol 2007;429:185-201.

Activation and substrate recognition by the eIF2alpha protein kinases PKR and GCN2

Dey, Cao, Rothenburg, Dever; in collaboration with Sicheri

Phosphorylation of eIF2alpha is a common mechanism for downregulating protein synthesis under stress conditions. Four 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 (Dar et al., Cell 2005;122:887). 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. Consistent with this hypothesis, mutations designed to disrupt and then restore an intermolecular salt bridge in the PKR dimer interface had the expected impact on PKR, PERK, and GCN2 kinase activity (Dey et al., Cell 2005;122:901; Dey et al., 2007). 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 the PKR structure represents the active state of the eIF2alpha kinase domain.

In a systematic screen of residues conserved among the eIF2alpha kinase domains, we identified mutations in helix alphaG of PKR that blocked phosphorylation of eIF2alpha but not phosphorylation of the non-specific substrate histone, consistent with the docking of eIF2alpha on helix alphaG in the PKR•eIF2alpha co-crystal structure. 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 (Dey et al., Cell 2005;122:901).

Dever TE, Dar AC, Sicheri F. The eIF2alpha kinases. In: Mathews MB, Sonenberg N, Hershey JWB, eds. Translational Control in Biology and Medicine. Cold Spring Harbor Laboratory Press, 2007;319-344.
Dey M, Cao C, Sicheri F, Dever TE. Conserved intermolecular salt-bridge required for activation ofprotein kinases PKR, GCN2 and PERK. J Biol Chem 2007;282:6653-6660.
Moraes MCS, Jesus TCL, Hashimoto NN, Dey M, Schwartz KJ, Alves VS, Avila CC, Bangs JD,Dever TE, Schenkman S, Castilho BA. Novel membrane-bound eIF2alpha kinase in the flagellar pocket of Trypanosoma brucei. Eukaryot Cell 2007;6:1979-1991.
Rothenburg S, Deigendesch N, Dey M, Dever TE, Tazi L. Double-strandedRNA-activated protein kinase PKR of fishes and amphibians: varying number ofdouble-stranded RNA binding domains and lineage specific duplications. BMCBiology 2008;6:12-31

Poxvirus regulation of protein kinase PKR

Seo, Cao, Rothenburg, Dever; in collaboration with Sicheri

As part of the mammalian cell innate immune response, the double-stranded RNA–activated protein kinase PKR phosphorylates the translation initiation factor eIF2α 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 types of PKR inhibitor: a pseudosubstrate inhibitor and a double-stranded RNA–binding protein called E3L. The vaccinia virus K3L protein resembles the N-terminal third of eIF2alpha, with both proteins containing a beta-barrel fold of the OB-fold family. Whereas high-level expression of human PKR was toxic in yeast, co-expression of the vaccinia virus K3L protein or the related variola (smallpox) virus C3L protein suppressed such growth inhibition. We used this yeast assay to screen for PKR mutants resistant to K3L inhibition and identified 12 mutations mapping to the C-terminal lobe of the PKR kinase domain in the vicinity of the eIF2alpha binding site. The PKR mutations specifically conferred resistance to the K3L protein, but not to the E3L protein, both in yeast and in vitro. In vitro studies revealed that wild-type PKR and the PKR mutants phosphorylated eIF2alpha with the same kinetics; however, the mutant kinase was less sensitive to inhibition by K3L. Consistently, the PKR-D486V mutation led to a nearly 15-fold decrease in K3L binding affinity. Our results support the identification of the eIF2alpha binding site on an extensive face of the C-terminal lobe of the kinase domain and indicate that subtle changes to the PKR kinase domain can drastically affect pseudosubstrate inhibition while leaving substrate phosphorylation intact. We propose that these paradoxical effects of the PKR mutations on pseudosubstrate versus substrate interactions reflect differences between the rigid K3L protein and the plastic nature of eIF2alpha around the Ser51 phosphorylation site.

In related studies, we are identifying and characterizing PKR mutants resistant to inhibition by the E3L protein. These PKR mutations map to both the kinase domain dimerization interface and the N-terminal regulatory [double-stranded RNA–(dsRNA) binding] domain of the protein. Consistent with the different natures of the two poxvirus inhibitors of PKR, the PKR mutations that confer resistance to E3L do not confer resistance to K3L. The identification of E3L-resistant mutations in the PKR dimerization interface is consistent with the notion that E3L inhibits PKR by forming inactive heterodimers with the kinase. Future in vitro studies will directly examine the impact of the mutations on PKR dimerization and E3L inhibition.

Kazemi S, Papadopoulou S, Li S, Su Q, Wang S, Yoshimura A, Matlashewski G, Dever TE, Koromilas AE. Control of eukaryotic translation initiation factor2alpha (eIF2alpha) phosphorylation by the human papillomavirus type 18 E6oncoprotein: implications for eIF2alpha-dependent gene expression and celldeath. Mol Cell Biol 2004;24:3415-3429

Structural and molecular analysis of the ER stress–responsive kinase-endonuclease IRE1

Dey, Cao, Dever; in collaboration with Sicheri

Accumulation of misfolded proteins in the ER under stress conditions activates a stress response pathway referred to as the unfolded protein response (UPR). The primary sensor of ER stress in the UPR is the protein IRE1. IRE1 is a transmembrane protein with an N-terminal domain situated in the ER lumen and a cytoplasmic domain consisting of a protein kinase domain and C-terminal kinase-associated nuclease (KEN) domain. Dimerization of IRE1 under ER stress conditions activates kinase autophosphorylation and nuclease activity. The KEN domain then splices, in a spliceosomeindependent manner, mRNAs encoding transcriptional regulators of the UPR—HAC1 mRNA in yeast and Xbp1 mRNA in mammals. In collaboration with Frank Sicheri, we obtained the crystal structure of the cytoplasmic catalytic domain of IRE1, which revealed the structure of both the kinase and KEN domain (Lee et al., 2008). Back-to-back dimerization of the kinase domain in the IRE1 crystal structure juxtaposes the KEN domains and activates the ribonuclease. We identified four autophosphorylation sites in the IRE1 kinase domain, and mutational and biochemical studies revealed that autophosphorylation facilitates ATP binding and the accompanying dimerization of the kinase domain. Yeast cells expressing IRE1 mutants with mutations in the dimer-contact residues were unable to grow in medium containing tunicamycin, an inhibitor of protein glycosylation and inducer of ER stress. Likewise, the dimerization mutants were unable to splice the Xbp1 mRNA in vitro. Comparison of the structure of the IRE1 KEN domain with the structure of the structurally distinct tRNA endonuclease identified putative nuclease active site-residues. Mutation of these residues blocked Xbp1 mRNA splicing in vitro and yeast cell growth under ER stress conditions. Taken together, the data reveal an unexpected convergent evolution of the tRNA endonuclease and IRE1 KEN-domain catalytic mechanism (Lee et al., 2008). We propose that dimerization of IRE1 lumenal domains in response to ER stress promotes kinase domain trans-autophosphorylation, which in turn facilitates nucleotide binding and back-to-back dimerization of the kinase domains. Dimerization of the KEN domains in the resultant structure enables recognition and splicing of the HAC1/Xbp1 mRNA.

Lee KPK, Dey M, Neculai D, Cao C, Dever TE, Sicheri F. Crystal structure of the dual catalytic region of Ire1 reveals the basis for catalysis and regulation in non-conventional RNA splicing. Cell 2008;132:89-100.

¹Pankaj Alone, PhD, former Postdoctoral Fellow

Collaborators

Jon R. Lorsch, PhD, The Johns Hopkins University, Baltimore, MD
FrankSicheri, PhD, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, andUniversity of Toronto, Toronto, Canada

For further information, contact devert@mail.nih.gov or visit http://spb.nichd.nih.gov.