Chune Cao

Mechanistic Link between PKR Dimerization, Autophosphorylation, and eIF2α Substrate Recognition

The antiviral protein kinase PKR inhibits protein synthesis by phosphorylating the translation initiation factor eIF2alpha on Ser51. Binding of double-stranded RNA to the regulatory domains of PKR promotes dimerization, autophosphorylation, and the functional activation of the kinase. Herein, we identify mutations that activate PKR in the absence of its regulatory domains and map the mutations to a recently identified dimerization surface on the kinase catalytic domain. Mutations of other residues on this surface block PKR autophosphorylation and eIF2alpha phosphorylation, while mutating Thr446, an autophosphorylation site within the catalytic-domain activation segment, impairs eIF2alpha phosphorylation and viral pseudosubstrate binding. Mutational analysis of catalytic-domain residues preferentially conserved in the eIF2alpha kinase family identifies helix alphaG as critical for the specific recognition of eIF2alpha. We propose an ordered mechanism of PKR activation in which catalytic-domain dimerization triggers Thr446 autophosphorylation and specific eIF2alpha substrate recognition.

Structure of the dual enzyme ire1 reveals the basis for catalysis and regulation in nonconventional RNA splicing.

Ire1 is an ancient transmembrane sensor of ER stress with dual protein kinase and ribonuclease activities. In response to ER stress, Ire1 catalyzes the splicing of target mRNAs in a spliceosome-independent manner. We have determined the crystal structure of the dual catalytic region of Ire1at 2.4 A resolution, revealing the fusion of a domain, which we term the KEN domain, to the protein kinase domain. Dimerization of the kinase domain composes a large catalytic surface on the KEN domain which carries out ribonuclease function. We further show that signal induced trans-autophosphorylation of the kinase domain permits unfettered binding of nucleotide, which in turn promotes dimerization to compose the ribonuclease active site. Comparison of Ire1 to a topologically disparate ribonuclease reveals the convergent evolution of their catalytic mechanism. These findings provide a basis for understanding the mechanism of action of RNaseL and other pseudokinases, which represent 10% of the human kinome.

Heterologous dimerization domains functionally substitute for the double-stranded RNA binding domains of the kinase PKR

The protein kinase PKR (dsRNA‐dependent protein kinase) phosphorylates the eukaryotic translation initiation factor eIF2α to downregulate protein synthesis in virus‐infected cells. Two double‐stranded RNA binding domains (dsRBDs) in the N‐terminal half of PKR are thought to bind the activator double‐stranded RNA, mediate dimerization of the protein and target PKR to the ribosome. To investigate further the importance of dimerization for PKR activity, fusion proteins were generated linking the PKR kinase domain to heterologous dimerization domains. Whereas the isolated PKR kinase domain (KD) was non‐functional in vivo, expression of a glutathione S‐transferase–KD fusion, or co‐expression of KD fusions containing the heterodimerization domains of the Xlim‐1 and Ldb1 proteins, restored PKR activity in yeast cells. Finally, coumermycin‐mediated dimerization of a GyrB–KD fusion protein increased eIF2α phosphorylation and inhibited reporter gene translation in mammalian cells. These results demonstrate the critical importance of dimerization for PKR activity in vivo, and suggest that a primary function of double‐stranded RNA binding to the dsRBDs of native PKR is to promote dimerization and activation of the kinase domain.

Initiation factor eIF5B catalyzes second GTP-dependent step in eukaryotic translation initiation

Initiation factors IF2 in bacteria and eIF2 in eukaryotes are GTPases that bind Met-tRNA\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}_{i}^{Met}\end{equation*}\end{document} to the small ribosomal subunit. eIF5B, the eukaryotic ortholog of IF2, is a GTPase that promotes ribosomal subunit joining. Here we show that eIF5B GTPase activity is required for protein synthesis. Mutation of the conserved Asp-759 in human eIF5B GTP-binding domain to Asn converts eIF5B to an XTPase and introduces an XTP requirement for subunit joining and translation initiation. Thus, in contrast to bacteria where the single GTPase IF2 is sufficient to catalyze translation initiation, eukaryotic cells require hydrolysis of GTP by both eIF2 and eIF5B to complete translation initiation.

PKR and GCN2 Kinases and Guanine Nucleotide Exchange Factor Eukaryotic Translation Initiation Factor 2B (eIF2B) Recognize Overlapping Surfaces on eIF2

ABSTRACT Four stress-responsive protein kinases, including GCN2 and PKR, phosphorylate eukaryotic translation initiation factor 2α (eIF2α) on Ser51 to regulate general and gene-specific protein synthesis. Phosphorylated eIF2 is an inhibitor of its guanine nucleotide exchange factor, eIF2B. Mutations that block translational regulation were isolated throughout the N-terminal OB-fold domain in Saccharomyces cerevisiae eIF2α, including those at residues flanking Ser51 and around 20 Å away in the conserved motif K79GYID83. Any mutation at Glu49 or Asp83 blocked translational regulation; however, only a subset of these mutations impaired Ser51 phosphorylation. Substitution of Ala for Asp83 eliminated phosphorylation by GCN2 and PKR both in vivo and in vitro, establishing the critical contributions of remote residues to kinase-substrate recognition. In contrast, mutations that blocked translational regulation but not Ser51 phosphorylation impaired the binding of eIF2B to phosphorylated eIF2α. Thus, two structurally distinct effectors of eIF2 function, eIF2α kinases and eIF2B, have evolved to recognize the same surface and overlapping determinants on eIF2α.

Conserved Intermolecular Salt Bridge Required for Activation of Protein Kinases PKR, GCN2, and PERK

The protein kinases PKR, GCN2, and PERK phosphorylate translation initiation factor eIF2α to regulate general and genespecific protein synthesis under various cellular stress conditions. Recent x-ray crystallographic structures of PKR and GCN2 revealed distinct dimeric configurations of the kinase domains. Whereas PKR kinase domains dimerized in a back-to-back and parallel orientation, the GCN2 kinase domains displayed an antiparallel orientation. The dimerization interfaces on PKR and GCN2 were localized to overlapping surfaces on the N-terminal lobes of the kinase domains but utilized different intermolecular contacts. A key feature of the PKR dimerization interface is a salt bridge interaction between Arg262 from one protomer and Asp266 from the second protomer. Interestingly, these two residues are conserved in all eIF2α kinases, although in the GCN2 structure, the two residues are too remote to interact. To test the importance of this potential salt bridge interaction in PKR, GCN2, and PERK, the residues constituting the salt bridge were mutated either independently or together to residues with the opposite charge. Single mutations of the Asp (or Glu) and Arg residues blocked kinase function both in yeast cells and in vitro. However, for all three kinases, the double mutation designed to restore the salt bridge interaction with opposite polarity resulted in a functional kinase. Thus, the salt bridge interaction and dimer interface observed in the PKR structure is critical for the activity of all three eIF2α kinases. These results are consistent with the notion that the PKR structure represents the active state of the eIF2α kinase domain, whereas the GCN2 structure may represent an inactive state of the kinase.

Translation Initiation Factor 2γ Mutant Alters Start Codon Selection Independent of Met-tRNA Binding

ABSTRACT Selection of the AUG start codon for translation in eukaryotes is governed by codon-anticodon interactions between the initiator Met-tRNAiMet and the mRNA. Translation initiation factor 2 (eIF2) binds Met-tRNAiMet to the 40S ribosomal subunit, and previous studies identified Sui− mutations in eIF2 that enhanced initiation from a noncanonical UUG codon, presumably by impairing Met-tRNAiMet binding. Consistently, an eIF2γ-N135D GTP-binding domain mutation impairs Met-tRNAiMet binding and causes a Sui− phenotype. Intragenic A208V and A382V suppressor mutations restore Met-tRNAiMet binding affinity and cell growth; however, only A208V suppresses the Sui− phenotype associated with the eIF2γ-N135D mutation. An eIF2γ-A219T mutation impairs Met-tRNAiMet binding but unexpectedly enhances the fidelity of initiation, suppressing the Sui− phenotype associated with the eIF2γ-N135D,A382V mutant. Overexpression of eIF1, which is thought to monitor codon-anticodon interactions during translation initiation, likewise suppresses the Sui− phenotype of the eIF2γ mutants. We propose that structural alterations in eIF2γ subtly alter the conformation of Met-tRNAiMet on the 40S subunit and thereby affect the fidelity of start codon recognition independent of Met-tRNAiMet binding affinity.

Protein kinase PKR mutants resistant to the poxvirus pseudosubstrate K3L protein

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. Poxviruses including vaccinia and smallpox viruses express PKR inhibitors such as the vaccinia virus K3L protein that resembles the N-terminal substrate-targeting domain of eIF2α. Whereas high-level expression of human PKR was toxic in yeast, this growth inhibition was suppressed by coexpression of the K3L protein. We used this yeast assay to screen for PKR mutants that are resistant to K3L inhibition, and we identified 12 mutations mapping to the C-terminal lobe of the PKR kinase domain. The PKR mutations specifically conferred resistance to the K3L protein both in yeast and in vitro. Consistently, the PKR-D486V mutation led to nearly a 15-fold decrease in K3L binding affinity yet did not impair eIF2α phosphorylation. Our results support the identification of the eIF2α-binding site on an extensive face of the C-terminal lobe of the kinase domain, and they indicate that subtle changes to the PKR kinase domain can drastically impact pseudosubstrate inhibition while leaving substrate phosphorylation intact. We propose that these paradoxical effects of the PKR mutations on pseudosubstrate vs. substrate interactions reflect differences between the rigid K3L protein and the plastic nature of eIF2α around the Ser-51 phosphorylation site.

Baculovirus protein PK2 subverts eIF2α kinase function by mimicry of its kinase domain C-lobe

Significance Many pathogens use molecular mimicry to subvert key cellular processes of the host. RNA-dependent protein kinase (PKR), a member of the eukaryotic translation initiation factor 2α (eIF2α) kinase family, is an important component of innate immunity in vertebrates and has often been subjected to inhibition by viral mimicry. In this study we show that the paradigm of host–virus mimicry extends to invertebrates where there is no discernable PKR homologue. We characterize an eIF2α kinase-mimic protein called “PK2,” encoded by baculoviruses, that inhibits a heme-regulated inhibitor kinase (HRI)-like eIF2α kinase, possibly through a lobe-swap mechanism. The inhibition of the HRI-like kinase confers a growth advantage to the baculovirus during infection of its insect host. These experiments suggest the independent emergence of eIF2α kinase antiviral defense mechanisms in vertebrates and invertebrates. Phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) by eIF2α family kinases is a conserved mechanism to limit protein synthesis under specific stress conditions. The baculovirus-encoded protein PK2 inhibits eIF2α family kinases in vivo, thereby increasing viral fitness. However, the precise mechanism by which PK2 inhibits eIF2α kinase function remains an enigma. Here, we probed the mechanism by which PK2 inhibits the model eIF2α kinase human RNA-dependent protein kinase (PKR) as well as native insect eIF2α kinases. Although PK2 structurally mimics the C-lobe of a protein kinase domain and possesses the required docking infrastructure to bind eIF2α, we show that PK2 directly binds the kinase domain of PKR (PKRKD) but not eIF2α. The PKRKD–PK2 interaction requires a 22-residue N-terminal extension preceding the globular PK2 body that we term the “eIF2α kinase C-lobe mimic” (EKCM) domain. The functional insufficiency of the N-terminal extension of PK2 implicates a role for the adjacent EKCM domain in binding and inhibiting PKR. Using a genetic screen in yeast, we isolated PK2-activating mutations that cluster to a surface of the EKCM domain that in bona fide protein kinases forms the catalytic cleft through sandwiching interactions with a kinase N-lobe. Interaction assays revealed that PK2 associates with the N- but not the C-lobe of PKRKD. We propose an inhibitory model whereby PK2 engages the N-lobe of an eIF2α kinase domain to create a nonfunctional pseudokinase domain complex, possibly through a lobe-swapping mechanism. Finally, we show that PK2 enhances baculovirus fitness in insect hosts by targeting the endogenous insect heme-regulated inhibitor (HRI)–like eIF2α kinase.

Polyamine Control of Translation Elongation Regulates Start Site Selection on Antizyme Inhibitor mRNA via Ribosome Queuing

Translation initiation is typically restricted to AUG codons, and scanning eukaryotic ribosomes inefficiently recognize near-cognate codons. We show that queuing of scanning ribosomes behind a paused elongating ribosome promotes initiation at upstream weak start sites. Ribosomal profiling reveals polyamine-dependent pausing of elongating ribosomes on a conserved Pro-Pro-Trp (PPW) motif in an inhibitory non-AUG-initiated upstream conserved coding region (uCC) of the antizyme inhibitor 1 (AZIN1) mRNA, encoding a regulator of cellular polyamine synthesis. Mutation of the PPW motif impairs initiation at the uCCs upstream near-cognate AUU start site and derepresses AZIN1 synthesis, whereas substitution of alternate elongation pause sequences restores uCC translation. Impairing ribosome loading reduces uCC translation and paradoxically derepresses AZIN1 synthesis. Finally, we identify the translation factor eIF5A as a sensor and effector for polyamine control of uCC translation. We propose that stalling of elongating ribosomes triggers queuing of scanning ribosomes and promotes initiation by positioning a ribosome near the start codon.