MECHANISM AND REGULATION OF EUKARYOTIC PROTEIN SYNTHESIS
, Head, Section on Protein Biosynthesis
Byung-Sik Shin, PhD, Staff Scientist
Pankaj Alone, PhD, Postdoctoral Fellow
Madhusudan Dey, PhD, Postdoctoral Fellow
Jeanne M. Fringer, 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
Structure-function analysis of universally conserved translational GTPase eIF5B/IF2
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 it catalyzes ribosomal subunit joining. In the presence of 80S ribosomes, eIF5B binds to GTP and hydrolyzes it. Our current efforts aim to elucidate the structure-function properties of eIF5B and understand the role of GTP binding and hydrolysis by eIF5B.
All G domains contain two structural elements, referred to as Switch I (SwI) and Switch II (SwII), that undergo marked conformational changes upon GTP binding and hydrolysis. Our previous studies on an eIF5B Switch I mutant 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). To gain further insight into eIF5B's GTP-binding properties and regulatory switch, and by extension the switch and guanine nucleotide–binding behavior of other G proteins, we conducted a mutational and suppressor analysis of the conserved SwII Gly479 residue of yeast eIF5B. Studies of other G proteins have generally suggested that movement of this Gly residue, in the D-X-X-G G-3 sequence motif, is critical for the structural transition of SwII during GTP binding and hydrolysis.
We found that the G479A mutation in eIF5B impaired yeast cell growth and the guanine nucleotide–binding, GTPase, and ribosomal subunit–joining activities of eIF5B (Shin et al., 2007). A screen for mutations that bypassed the critical requirement of SwII Gly in eIF5B identified intragenic suppressors in the SwI element (A444V) and at a residue in domain 2 of eIF5B that interacts with SwII (D740R). The intragenic suppressors restored yeast cell growth and eIF5B nucleotide-binding, GTP hydrolysis, and subunit-joining activities. We propose that the SwII mutation distorts the geometry of the GTP-binding active site, impairing nucleotide binding and the eIF5B domain movements associated with GTP binding. Accordingly, the SwI and domain 2 suppressor mutations induce SwII to adopt a conformation favorable for nucleotide binding and hydrolysis, thereby re-establishing coupling between GTP binding and eIF5B domain movements (Shin et al., 2007).
Previously, we showed that the C-termini of eIF5B and eIF1A directly interact. Working in collaboration with Jon Lorsch, we showed that substitution of the last five residues of eIF1A (DIDDI) with Ala impaired ribosomal subunit joining and translation-coupled eIF5B GTPase activity (Acker et al., 2006). Thus, the eIF5B-eIF1A interaction promotes subunit joining and possibly provides a checkpoint for correct 80S complex formation, with full activation of eIF5B GTPase activity dependent on formation of a properly organized 80S initiation complex.
Even though in vitro assays established that eIF5B catalyzes ribosomal subunit joining, in vivo evidence has been lacking. To address the essential function of eIF5B in vivo, we used a degron approach in which we tagged eIF5B with an unstable ubiquitin fusion protein to deplete eIF5B rapidly in yeast cells. Analysis of translation initiation complexes from the cells revealed accumulation of eIF1A and mRNA on 40S subunits (48S complexes) in addition to a “halfmer” ribosome (an mRNA with bound 80S and 48S complex), providing in vivo evidence that eIF5B promotes ribosomal subunit joining (Fringer et al., 2007). The eIF1A-5A substitution impaired eIF5B binding to eIF1A in cell extracts and to 40S complexes in vivo. Consistent with these observations, overexpression of eIF5B suppressed the growth and translation initiation defects in yeast expressing eIF1A-5A, indicating that eIF1A helps recruit eIF5B to the 40S subunit before subunit joining. Blocking GTP hydrolysis with the eIF5B-T439A SwI mutant led to the accumulation of both eIF1A and eIF5B on the 80S products of subunit joining both in vivo and in vitro. Likewise, eIF5B and eIF1A remained associated with 80S complexes formed in the presence of non-hydrolyzable GDPNP, whereas these factors were released from the 80S complexes in assays containing GTP. 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).
Acker MG, Shin B-S, Dever TE, Lorsch JR. Interaction between eukaryotic initiation factors 1A and 5B is required for efficient ribosomal subunit joining. J Biol Chem 2006;281:8469-75.
Fringer JM, Acker MG, Fekete CA, Lorsch JR, Dever TE. Coupled release of factors eIF5B and eIF1A from 80S ribosomes following subunit joining. Mol Cell Biol 2007;27:2384-97.
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. Wiley-VCH Verlag, 2005;241-322.
Shin BS, Acker MG, Maag D, Kim J-R, Lorsch JR, Dever TE. Intragenic suppressor mutations restore GTPase and translation functions of eIF5B switch II mutant. Mol Cell Biol 2007;27:1677-85.
Structure-function analysis of eIF2gamma, the GTP- and Met-tRNA–binding subunit of the eIF2 complex
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. 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, previous studies revealed that these two effectors of eIF2 (GAP and GEF) interact with eIF2beta, but not with the GTP-binding eIF2gamma subunit. Thus, it is reasonable to propose that eIF5 and eIF2B modulate eIF2gamma nucleotide hydrolysis and exchange activities indirectly (allosterically) through eIF2beta. Alternatively, the interactions with eIF2beta may serve as a docking site to facilitate the functional interaction of these proteins with eIF2gamma. Expressing GST fusion proteins containing full-length, or individual domains, of eIF2gamma in yeast cells, we used GST pull-down assays to examine the interaction of eIF5 and the catalytic eIF2Bepsilon subunit with eIF2gamma in crude cell extracts (Alone and Dever, 2006). As expected, full-length GST-eIF2gamma bound to eIF2alpha, eIF2beta, eIF5, and eIF2Bepsilon. Mutation of the eIF2beta K-boxes, which eliminated the binding of eIF2beta to eIF5 and eIF2Bepsilon, did not interfere with the binding of eIF5 and eIF2Bepsilon to the full-length GST-eIF2gamma fusion protein. Our results suggest that eIF5 and eIF2Bepsilon interact with eIF2gamma in a manner independent of eIF2beta. Consistent with this hypothesis, eIF5 and eIF2beta bound to a GST-eIF2gamma–G domain fusion protein that failed to pull down either eIF2alpha or eIF2beta; purified GST-eIF5 was able to pull down purified eIF2gamma or the isolated eIF2gamma-G domain. Finally, we mapped the eIF2gamma-interacting region to the N-terminal half of eIF5, which we showed possesses GAP activity, and to the C-terminal catalytic portion eIF2Bepsilon. Thus, we provided the first evidence that eIF5 and eIF2Bepsilon directly contact the eIF2gamma G-domain to modulate the hydrolysis and exchange of GTP on eIF2 (Alone and Dever, 2006).
To gain further 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 non-canonical 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. However, we identified intragenic suppressors of the eIF2gamma-N135D mutation that functionally separated Met-tRNA binding affinity and start codon selection. Whereas an A208V mutation restored Met-tRNA binding affinity and suppressed the slow-growth and Sui− phenotypes associated with the eIF2gamma-N135D mutant, an A382V mutation restored Met-tRNA binding and suppressed the slow-growth, but not the Sui−, phenotype. Moreover, an A219T mutation impaired Met-tRNA binding but did not confer a Sui− phenotype. 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 translation factor eIF1, which is thought to monitor codon-anticodon interaction during translation initiation, suppressed the Sui− phenotype of the eIF2gamma mutants. We propose that structural alterations in the eIF2gamma GTP-binding domain alter the conformation of Met-tRNA on the 40S subunit and thereby affect the fidelity of start codon recognition.
Alone PV, Dever TE. Direct binding of translation initiation factor eIF2gamma-G domain to its GTPase-activating and GDP-GTP exchange factors eIF5 and eIF2Bepsilon. J Biol Chem 2006;281:12636-44.
Activation and substrate recognition by the eIF2alpha protein kinases PKR and GCN2
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. This inhibition of eIF2B impairs general translation, slowing the growth of yeast cells, and paradoxically enhances 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., 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 conserved eIF2alpha “KGYID” motif interacting with helix alphaG in the kinase domain C-terminal lobe and the Ser51 phosphorylation site positioned near the PKR active site. Given that all four eIF2alpha kinases share the PKR residues mediating kinase domain dimerization and eIF2alpha recognition, we propose that all four kinases dimerize and recognize eIF2alpha in similar manners.
A hallmark of the PKR dimer interface is the presence of reciprocal salt-bridge interactions between R262 of one PKR protomer and D266 of the other protomer. Charge-reversal mutations of R262 to D or D266 to R, designed to disrupt the salt-bridge interaction, abolished PKR function both in yeast cells and in vitro. However, combining the two mutations into the same PKR allele, which would restore the salt-bridge interaction with opposite polarity, rescued PKR function both in vivo and in vitro (Dey et al., 2005a). Likewise, the corresponding single mutations designed to disrupt putative salt-bridge interactions in GCN2 and PERK abolished kinase activity. More important, 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 (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.
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 identified mutations in the PKR dimer interface that enhanced PKR kinase domain dimerization and activated the kinase in the absence of its regulatory domain (Dey et al., 2005a). In a systematic screen of residues conserved among the eIF2alpha kinase domains, we identified mutations in helix alphaG 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 studies, 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., 2005a).
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, 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;319-44.
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 2005a;122:901-13.
Dey M, Cao C, Sicheri F, Dever TE. Conserved intermolecular salt-bridge required for activation of protein kinases PKR, GCN2 and PERK. J Biol Chem 2007;282:6653-60.
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 2005b;25:3063-75.
Poxvirus regulation of protein kinase PKR
The phosphorylation of eIF2alpha by PKR is one means used by mammalian cells to block viral replication (Kazemi et al., Mol Cell Biol 2004;24:3415). 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 inhibitors 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. More 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 that are resistant to K3L inhibition. We isolated 12 PKR mutants that were toxic in yeast cells co-expressing the K3L protein. Interestingly, the 12 mutations mapped to the C-terminal lobe of the kinase domain, consistent with the fact that the eIF2alpha binding site mapped to the same region of the kinase. Additional in vitro studies with the PKR-D486V mutant revealed reduced K3L binding. Moreover, the PKR mutant phosphorylated eIF2alpha with the same kinetics as the wild-type kinase; however, the mutant kinase was less sensitive to inhibition by K3L in vitro. Interestingly, the PKR mutant was not resistant to the viral E3L inhibitor. Thus, the D486V mutation appears to specifically desensitize PKR to inhibition by the viral pseudosubstrate K3L protein. 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.
In related studies, we are identifying and characterizing PKR mutants that are resistant to inhibition by the E3L protein. The mutations map to both the kinase domain and the double-stranded RNA–binding domain of the protein. Consistent with the alternate nature of the two poxvirus inhibitors of PKR, the PKR mutations that confer resistance to E3L do not confer resistance to K3L. Further characterization of the PKR mutants may provide novel insights into the double-stranded RNA–binding properties of PKR, the mechanism of dsRNA activation of the kinase, and the mechanism by which E3L blocks PKR function.
COLLABORATORS
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.
