We are studying the mechanism and regulation of protein synthesis, focusing on GTPases and protein kinases that control this fundamental cellular process. We are using molecular genetic and biochemical studies to dissect the structure-function properties of the translation initiation factor eIF2, a GTPase that binds methionyl-tRNA to the ribosome. We are also studying how phosphorylation regulates eIF2 function. Focusing on the mechanism of kinase-substrate recognition, we recently showed that eIF2alpha residues remote from the phosphorylation site are critical for efficient phosphorylation by the endogenous yeast kinase GCN2 as well as by the antiviral kinase PKR. In addition, mutational and structural analyses of PKR revealed a mechanistic link among kinase dimerization, autophosphorylation, and specific substrate recognition. We are also studying viral regulation of PKR by poxvirus inhibitors of the kinase. The translation factor eIF5B is a second GTPase that catalyzes ribosomal subunit joining in the final step of translation initiation. We are using mutational and suppressor analyses combined with biochemical studies to characterize the structure-function properties of this universally conserved factor and to define the role of GTP binding and hydrolysis by the factor.
Structure-function analysis of eIF2gamma, the GTP-binding subunit of the eIF2 complex
Alone, Cao, Dever; in collaboration with Burley
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. In collaboration with Stephen Burley, we obtained the crystal structure of eIF2gamma from the archaeon M. jannaschii. The eIF2gamma structure revealed a three-domain protein with the beta-barrel fold domains II and III packed closely against the N-terminal G domain. The eIF2gamma structure closely resembles the structure of elongation factor EF-Tu (EF1), consistent with the common function of the two proteins to bind aminoacyl-tRNAs to the ribosome. Based on the structure of EF-Tu•Phe-tRNA, we modeled the binding site of Met-tRNA on eIF2gamma and tested the model by mutating conserved residues in the proposed Met-tRNA binding pocket. Consistent with the model, mutation of the conserved Gly-397 to Ala caused a slow-growth phenotype in yeast that could be suppressed by overexpressing initiator Met-tRNA. The result indicates that eIF2gamma and EF-Tu use a common pocket for binding to aminoacyl-tRNA.
To define the architecture of the eIF2 complex and map the interactions of eIF2 with other components of the translational machinery, we mutated conserved surface residues on the eIF2gamma structure and examined the mutant proteins for function in yeast. Mutation of Asp-403 in yeast eIF2gamma to Ala caused a slow-growth phenotype in yeast that was suppressed by overexpressing eIF2alpha. Consistent with the finding, mutation of the corresponding residue in M. jannaschii eIF2gamma impaired the binding of eIF2alpha. Based on these results, we propose that the conserved surface on domain II centered near Asp-403 of eIF2gamma is a binding site for eIF2alpha.
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. Curiously, the two effectors of eIF2 function have been found to interact with eIF2beta but not with the GTP-binding eIF2gamma subunit. To identify proteins that interact with eIF2gamma, we expressed full-length eIF2gamma as well as its individual domains as GST fusion proteins in yeast cells. 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. The latter finding demonstrates that these critical regulators of eIF2 function make direct contacts with the G domain of eIF2gamma, consistent with their roles in promoting GTP hydrolysis and GTP-GDP exchange on eIF2. Future experiments will seek to map these binding sites more precisely on eIF2gamma, eIF5, and eIF2B.
Roll-Mecak A, Alone P, Cao C, Dever TE, Burley SK. X-ray structure of translation initiation factor eIF2gamma: implications for tRNA and eIF2alpha binding. J Biol Chem 2004;279:10634-10642.
Substrate recognition by the eIF2alpha protein kinases PKR and GCN2
Dey, Cao, Dever; in collaboration with Sicheri
Phosphorylation of eIF2alpha is a common mechanism for downregulating protein synthesis under stress conditions. Four kinases phosphorylate eIF2alpha on Ser-51 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 Ser-51, 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 by slowing the growth of yeast cells and paradoxically enhances the translation of the GCN4 mRNA required for yeast cells to grow under conditions of amino acid starvation. To identify the eIF2alpha residues required for kinase recognition and phosphorylation of Ser-51 as well as the residues required for inhibition of eIF2B by phosphorylated eIF2, we mutated eIF2alpha and screened for mutants defective for translational regulation by GCN2. We isolated mutations in the residues flanking Ser-51 and in “KGYID,” a conserved motif located at residues 79 through 83. In the three-dimensional structure of eIF2alpha, the latter mutations are located about 20 Å from Ser-51. Any mutation at residue 49 or residue 83 was found to disrupt translational regulation by GCN2; however, only a subset of the mutations impaired Ser-51 phosphorylation. Substitution of Ala for Asp-83 eliminated phosphorylation of Ser-51 by GCN2 and PKR both in vivo and in vitro. The result establishes the importance of remote residues to kinase-substrate recognition by the eIF2alpha kinases. In contrast, a number of mutations in the residues flanking Ser-51 and in the remote motif blocked translational regulation but not Ser-51 phosphorylation. The latter mutations impaired the binding of eIF2B to phosphorylated eIF2alpha. We conclude that the eIF2alpha kinases and eIF2B recognize the same surfaces and overlapping determinants on eIF2alpha (Dey et al., 2005a).
The identification of kinase recognition determinants 20 Å from the phosphorylation site in eIF2alpha suggests that residues remote from the PKR catalytic site will be important for specific eIF2alpha recognition. 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., 2005). The PKR kinase domain resembles typical eukaryotic protein kinases, with the active site located between a smaller N-terminal lobe and larger C-terminal lobe. In the crystal structure, the PKR kinase domain dimerizes in a back-to-back orientation mediated by conserved residues in the N-terminal lobe. One molecule of eIF2alpha is bound to each PKR kinase domain protomer, with the eIF2alpha “KGYID” motif interacting with helix alphaG in the kinase domain C-terminal lobe and the Ser-51 phosphorylation site positioned near the PKR active site. Given that the PKR residues mediating kinase domain dimerization and eIF2alpha recognition are shared among all four eIF2alpha kinases, we propose that all four kinases dimerize and recognize eIF2alpha in similar manners.
Binding of double-stranded RNA to the regulatory domains in the N-terminal half of PKR is thought to promote dimerization and activation of the kinase. We screened for mutants that would activate PKR in the absence of its regulatory domain. The mutations enhanced PKR kinase domain dimerization and mapped to the dimerization interface on the N-terminal lobe of kinase domain (Dey et al., 2005b). Sequence comparisons of all four eIF2alpha kinases and other unrelated kinases identified 17 residues preferentially conserved among the eIF2alpha kinases. We systematically mutated these conserved residues in PKR and screened for mutants that impaired PKR toxicity in yeast. Mutation of conserved residues on the kinase domain dimerization interface blocked PKR autophosphorylation and eIF2alpha phosphorylation while mutation of the Thr-446 autophosphorylation site in the kinase domain activation segment impaired eIF2alpha phosphorylation and viral pseudosubstrate binding. Finally, mutation of residues in helix alphaG prevented phosphorylation of eIF2alpha but not phosphorylation of the nonspecific substrate histone. Based on our studies, we propose an ordered mechanism of PKR activation in which catalytic domain dimerization triggers Thr-446 autophosphorylation, which in turn is required for specific eIF2alpha substrate recognition (Dey et al., 2005b).
Dar AC, Dever TE, Sicheri F. Higher-order substrate recognition of eIF2alpha by the RNA-dependent protein kinase PKR. Cell 2005;122:887-900.
Dever TE, Hinnebusch AG. GCN2 whets the appetite for amino acids. Mol Cell 2005;18:141-142.
Dey M, Cao C, Dar AC, Tamura T, Ozato K, Sicheri F, Dever TE. Mechanistic link between PKR dimerization, autophosphorylation, and eIF2alpha substrate recognition. Cell 2005b;122:901-913.
Dey M, Trieselmann B, Locke EG, Lu J, Cao C, Dar AC, Krishnamoorthy T, Dong J, Sicheri F, Dever TE. PKR and GCN2 kinases and guanine nucleotide exchange factor eukaryotic translation initiation factor 2B (eIF2B) recognize overlapping surfaces on translation factor eIF2alpha. Mol Cell Biol 2005a;25:3063-3075.
Klann E, Dever TE. Biochemical mechanisms for translational regulation in synaptic plasticity. Nat Rev Neurosci 2004;5:931-942.
Poxvirus regulation of protein kinase PKR
Seo, Cao, Dever; in collaboration with Sicheri
The phosphorylation of eIF2alpha by PKR is one means used by mammalian cells to block viral replication. To subvert this host cell defense mechanism, viruses produce inhibitors of PKR. Several members of the poxvirus family express two different types of PKR inhibitors: 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. In previous studies, we showed that expression of the K3L protein in yeast prevented PKR inhibition of yeast cell growth. Moreover, we showed that the PKR-inhibitory activity of the K3L protein was dependent on residues conserved between the K3L protein and eIF2alpha, including the “KGYID” motif that is critical for recognition of eIF2alpha by the eIF2alpha protein kinases. Recently, we found that the related C3L protein from the smallpox (variola) virus is also able to block PKR toxicity in yeast. Thus, like vaccinia virus, the smallpox virus uses a pseudosubstrate inhibitor to block PKR function.
To gain further insights into pseudosubstrate regulation of PKR, we screened for PKR mutants resistant to K3L inhibition. We isolated 12 PKR mutants that were toxic in yeast cells co-expressing the K3L protein. Interestingly, the 12 PKR mutations mapped to the C-terminal lobe of the kinase domain, consistent with the fact that the eIF2alpha binding site mapped to this region of the kinase. Additional studies in yeast revealed that the mutant kinases were neither simply hyperactive nor resistant to the viral E3L inhibitor. Thus, the PKR mutants appear to be specifically desensitized to inhibition by the viral pseudosubstrate K3L protein. In vitro, the PKR mutants phosphorylate eIF2alpha with the same kinetics as the wild-type kinase. However, whereas recombinant K3L protein inhibited phosphorylation of eIF2alpha by wild-type PKR in vitro, purified PKR mutant kinases were less sensitive to K3L inhibition. The results indicate that the PKR mutations do not affect the Km for eIF2alpha phosphorylation but instead raise the Ki for inhibition by the K3L protein. Consistent with this latter idea, surface plasmon resonance experiments revealed that the PKR mutants bound to the K3L protein with about 20-fold lower affinity than did wild-type PKR. We propose that the PKR mutations either directly or indirectly alter points of contact between PKR and its pseudosubstrate such that the mutations have a greater impact on pseudosubstrate inhibition than on substrate phosphorylation and thus confer resistance to pseudosubstrate inhibition.
Kazemi S, Papadopoulou S, Li S, Su Q, Wang S, Yoshimura A, Matlashewski G, Dever TE, Koromilas AE. Control of eukaryotic translation initiation factor 2alpha (eIF2alpha) phosphorylation by the human papillomavirus type 18 E6 oncoprotein: implications for eIF2alpha-dependent gene expression and cell death. Mol Cell Biol 2004;24:3415-3429.
Structure-function analysis of 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 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 eIF5B catalyzed ribosomal subunit joining. The eIF5B binds to GTP and hydrolyzes it in the presence of 80S ribosomes. The x-ray structure of eIF5B revealed a chalice-shaped protein in which GTP binding to the G domain residing in the cup of the chalice triggers lever-type domain rearrangements through a long alpha-helix forming the stem of the chalice. The result is significant movement of domain IV, which is the base of the chalice. Our current efforts aim at elucidating the structure-function properties of eIF5B and understanding the translation initiation factor’s role in GTP binding and hydrolysis.
All G domains contain two structural elements referred to as Switch 1 (Sw1) and Switch 2 (Sw2), which undergo marked conformational changes upon GTP binding and hydrolysis. Conserved residues in Sw1 and Sw2 directly interact with the guanine nucleotide and play important roles in GTP binding and hydrolysis. Mutation of a conserved Thr in Sw1 of yeast eIF5B severely impaired yeast cell growth and eliminated eIF5B GTPase and translational stimulatory properties. However, the mutation did not impair GTP binding or ribosomal subunit joining. An intragenic suppressor of the eIF5B Sw1 mutant restored yeast cell growth and eIF5B translational stimulatory activities but not the eIF5B GTPase activity; thus, the eIF5B suppressor mutant uncoupled eIF5B GTPase and translational stimulatory activities. Additional studies revealed that the suppressor mutation lowered eIF5B’s ribosome-binding affinity, leading us to propose that a GTP-regulated switch governs eIF5B’s ribosome-binding affinity. In the presence of GTP, eIF5B binds to the ribosome and, following GTP hydrolysis, eIF5B is released (Shin et al., Cell 2002;111:1015). In further studies, we mutated a conserved Gly residue in Sw2 that is thought to be important for the structural flexibility of Sw2. Substitution of Ala for the conserved Gly in eIF5B impaired yeast cell growth and the guanine-nucleotide–binding, GTPase, and ribosomal subunit–joining activities of the factor. An intragenic suppressor mutation mapping to Sw1 restored nucleotide-binding–, GTPase, and subunit-joining activities. We propose that the Gly mutation in Sw2 distorts the geometry of the eIF5B active site, impairing nucleotide binding and hydrolysis and the associated eIF5B domain movements. Interestingly, the suppressor mutation resides in a portion of Sw1 that closely approaches Sw2. The suppressor mutation may perturb the structure of Sw1 and cause it to clash with Sw2. We propose that the consequential movement of Sw2 restores a favorable active site geometry, enabling the factor to bind to and hydrolyze GTP and to couple eIF5B domain movements with guanine-nucleotide binding.
We propose the following model for the role of eIF5B in translation initiation: following scanning of the 40S ribosomal complex to an AUG codon on an mRNA, eIF2 hydrolyzes GTP, causing the release of eIF2 and perhaps other factors; binding of eIF5B•GTP stabilizes Met-tRNA binding to the 40S complex and promotes joining of the 60S ribosomal subunit; formation of the 80S ribosome triggers GTP hydrolysis by eIF5B, thereby releasing the factor and enabling the 80S ribosome to begin translation elongation.
Hinnebusch AG, Dever TE, Sonenberg N. Mechanism and regulation of protein synthesis initiation in eukaryotes. In: Nierhaus KH, Wilson DN, eds. Protein Synthesis and Ribosome Structure. Weinheim, Germany: Wiley-VCH, 2005;241-322.
Sonenberg N, Dever TE. Eukaryotic translation initiation factors and regulators. Curr Opin Struct Biol 2003;13:56-63.
COLLABORATORS
Stephen K. Burley, PhD, Howard Hughes Medical Institute, The Rockefeller University, New York, NY, and Structural GenomiX, Inc., San Diego, CA
Jon R. Lorsch, PhD, The Johns Hopkins University, Baltimore, MD
Frank Sicheri, PhD, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, and University of Toronto, Toronto, Canada
For further information, contact devert@mail.nih.gov.