Mechanism and Regulation of Eukaryotic Protein Synthesis
- Thomas E. Dever, PhD, Head, Section on Protein Biosynthesis
- Chune Cao, Biological Laboratory Technician
- Ivaylo P. Ivanov, PhD, Research Fellow
- Joo-Ran Kim, BS, Special Volunteer
- Leda Lotspeich-Cole, BS, Graduate Student
- Jason A. Murray, BS, Graduate Student
- Byung-Sik Shin, PhD, Staff Scientist
- Arya Vindu, PhD, Visiting Fellow
- Sara Young, PhD, Postdoctoral Fellow
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 in yeast and human cells to dissect the structure-function properties of translation factors. Of special interest are 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. We also investigate stress-responsive protein kinases that phosphorylate eIF2alpha, viral regulators of these kinases, and how cellular phosphatases are targeted to dephosphorylate eIF2alpha. We are characterizing eIF2gamma mutations that are associated with a novel X-linked intellectual disability syndrome, and we are investigating the function of the translation factor eIF5A with a focus on its ability to stimulate the peptidyl transferase activity of the ribosome and facilitate the reactivity of poor substrates such as proline. We are also examining the role of the hypusine modification on eIF5A and the role of this factor in gene-specific translational control mechanisms.
Analysis of eIF2gamma mutations that link intellectual disability with impaired translation initiation
The first step of protein synthesis is binding of the initiator Met-tRNA to the small ribosomal subunit by the factor eIF2, which is composed of three subunits. The gamma subunit of eIF2 is a GTPase that resembles the bacterial translation elongation factor EF-Tu. We previously showed that, despite their structural similarity, eIF2 and EF-Tu bind to tRNA in substantially different manners, and we showed that the tRNA–binding domain III of EF-Tu has acquired a new function in eIF2gamma to bind to the ribosome.
While protein synthesis plays a critical role in learning and memory in model systems, human intellectual disability syndromes have not been directly associated with alterations in protein synthesis. Working with collaborators in Israel and Germany, we investigated a human X-linked disorder characterized by intellectual disability, epilepsy, hypogonadism, microcephaly, and obesity. The patients carry a mutation in the EIF2S3 gene, which encodes eIF2gamma. In genetic and biochemical studies, we found that the mutation disrupts eIF2 complex integrity, impairs general translation, alters translational control of mRNAs encoding key regulatory proteins, and reduces the fidelity of translation start codon selection. The findings directly link intellectual disability with impaired translation initiation and provide a mechanistic basis for the human disease resulting from partial loss of eIF2 function (Reference 1). Over the past year, working with additional collaborators, we have been characterizing other mutations in eIF2gamma that cause intellectual disability and we linked these mutations to the MEHMO (Mental retardation, Epileptic seizures, Hypogenitalism, Microcephaly and Obesity) syndrome.
Molecular analysis of eIF2alpha phosphorylation, dephosphorylation, and viral regulation
Phosphorylation of the eIF2alpha subunit is a common mechanism for down-regulating 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 (endoplasmic reticulum) 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 (GCN4 is a transcription factor) required for yeast cells to grow under amino-acid starvation conditions.
We previously 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. Based on our studies, we proposed an ordered mechanism of PKR activation by which catalytic domain dimerization triggers auto-phosphorylation, which in turn is required for specific eIF2alpha substrate recognition. Moreover, our studies on eIF2alpha led us to propose that the Ser51 phosphorylation site is protected in free eIF2alpha, which prevents promiscuous phosphorylation and attendant translational regulation by heterologous kinases. Upon interaction with an eIF2alpha kinase, the Ser51 residue is exposed and phosphorylated by the kinase.
While the protein kinases GCN2, HRI, PKR, and PERK specifically phosphorylate eIF2alpha on Ser51 to regulate global and gene-specific mRNA translation, eIF2alpha is dephosphorylated by the broadly acting serine/threonine protein phosphatase 1 (PP1). In mammalian cells, the regulatory subunits GADD34 and CReP target PP1 to dephosphorylate eIF2alpha. We showed that a novel N-terminal extension on yeast eIF2gamma binds to yeast PP1 (GLC7) and targets GLC7 to dephosphorylate eIF2alpha. Continuing our studies on eIF2alpha dephosphorylation, we reconstituted human GADD34 function in yeast cells. We mapped a novel eIF2a–binding motif to the C-terminus of GADD34 in a region distinct from that where PP1 binds to GADD34. Point mutations altering the 19–residue eIF2alpha–binding motif impaired the ability of GADD34 to interact with eIF2alpha, promote eIF2alpha dephosphorylation, and suppress PKR toxicity in yeast. Interestingly, the eIF2alpha–docking motif is conserved among several viral orthologs of GADD34, and we showed that it is necessary for the proteins produced by African swine fever virus, Canarypox virus, and Herpes simplex virus to promote eIF2alpha dephosphorylation. Taken together, our data demonstrate that GADD34 and its viral orthologs direct specific dephosphorylation of eIF2alpha by interacting with both PP1 and eIF2alpha through independent binding motifs (Reference 2).
The eIF2alpha kinase PKR is part of the cellular anti-viral defense mechanism. When expressed in yeast, human PKR phosphorylates eIF2alpha, resulting in inhibition of protein synthesis and yeast cell growth. To subvert the anti-viral defense mediated by PKR, viruses produce inhibitors of the kinase. The poxviral protein E3L binds to double-stranded RNA and inhibits PKR by sequestering activators and forming heterodimers with the kinase. We previously showed that a Z-DNA–binding domain near the N-terminus of E3L, but not its Z-DNA–binding activity, is critical for E3L inhibition of PKR. We are currently characterizing mutations in PKR that confer resistance to E3L inhibition. Similarly, we previously discovered that the insect baculovirus PK2 protein is an eIF2alpha kinase inhibitor. PK2 structurally mimics the C-terminal lobe of a protein kinase domain. Genetic screens, yeast two-hybrid assays, and protein-interaction studies revealed that PK2 associates with the N-lobe of PKR. Using yeast-based assays, we showed that PK2 was most effective at inhibiting an insect HRI–like kinase, and our collaborators showed that knockdown of the HRI–like kinase in insects rescued viral defects associated with loss of PK2. We proposed an inhibitory mechanism whereby PK2 engages the N-lobe of an eIF2alpha kinase domain to create a nonfunctional pseudokinase domain complex, possibly through a lobe-swapping mechanism (Reference 3).
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 a model assay for first peptide–bond synthesis. However, the precise cellular role of eIF5A was unknown. Using molecular-genetic and biochemical studies, we previously showed that eIF5A promotes translation elongation and that this activity depends on the hypusine modification. Given that eIF5A is a structural homolog of the bacterial protein EF-P, we proposed that eIF5A/EF-P is a universally conserved translation elongation factor.
Recently, it was shown that EF-P promotes translation of polyproline sequences by bacterial ribosomes. Using in vivo reporter assays, we showed that eIF5A in yeast stimulates the synthesis of proteins containing runs of three or more consecutive proline residues. Consistently, the expression of native yeast proteins containing homopolyproline sequences was impaired in eIF5A mutant strains. To support the in vivo findings, we used reconstituted yeast in vitro translation assays to monitor the impact of eIF5A on protein synthesis. We found that the synthesis of polyproline peptides, but not of polyphenylalanine peptides, was critically dependent on addition of eIF5A. Consistent with the functions of eIF5A in promoting peptide bond synthesis, directed hydroxyl radical probing experiments localized eIF5A binding to near the E site of the ribosome. Thus, we propose that eIF5A, like its bacterial ortholog EF-P, stimulates the peptidyl-transferase activity of the ribosome and facilitates the reactivity of poor substrates such as proline (Reference 4).
Over the last year, and working with the X-ray crystallographer Marat Yusupov, we obtained a 3.25 Å resolution crystal structure of eIF5A bound to the yeast 80S ribosome. The structure reveals interactions between eIF5A and conserved ribosomal proteins and rRNA bases. Moreover, eIF5A occupies the E site of the ribosome, with the hypusine residue projecting toward the acceptor stem of the P-site tRNA. Our studies reveal a previously unseen conformation of an eIF5A–ribosome complex, suggest a function for eIF5A and its hypusine residue in repositioning the peptidyl-tRNA in the P site to alleviate stalling, and they highlight a possible functional link between conformational changes of the ribosome during protein synthesis and eIF5A-ribosome association (Reference 5). In ongoing studies, we are characterizing translational control mechanisms that exploit the fact that polyproline synthesis requires eIF5A.
Analysis of the role of eEF2 and its diphthamide modification in translation elongation and CrPV IRES translation
Together with collaborator Venki Ramakrishnan, we are studying the translation elongation factor eEF2. The eEF2, like its bacterial ortholog EF-G, promotes translocation of tRNAs and mRNA on the ribosome following peptide bond formation. In all eukaryotes and archaea, a conserved histidine residue at the tip of eEF2 is post-translationally modified to diphthamide through the action of seven non-essential proteins. The function of diphthamide and rationale for its evolutionary conservation are not well understood, and to date the only known function of diphthamide is to serve as a substrate for inactivation by diphtheria toxin. To gain insights into the role of eEF2 and diphthamide, we reconstituted the function of the cricket paralysis virus (CrPV) internal ribosome entry site (IRES) in a yeast in vitro translation assay system. The CrPV IRES is unique in bypassing the requirement for any translation initiation factors. Thus, the IRES binds directly to the A-site of the ribosome. Following eEF2-directed pseudo-translocation of the IRES to the P site, an aminoacyl–tRNA binds to the A-site followed by a second pseudo-translocation to move the IRES to the E site and the A-site tRNA to the P site. Following binding of the next aminoacyl–tRNA to the A site and ribosome-catalyzed peptide bond formation, normal translation elongation ensues, requiring the factors eEF1A and eEF2 and the yeast-specific factor eEF3. Using the canonical initiation pathway to direct the synthesis of the peptide Met-Phe-Lys revealed no distinction between unmodified eEF2 and eEF2 with the diphthamide modification. In contrast, synthesis of the same peptide directed by the CrPV IRES was sensitive to loss of diphthamide. As the pseudo-translocation steps are the main distinguishing feature of the CrPV IRES system, we propose that the precise phasing of pseudo-translocation is dependent on the diphthamide modification on eEF2.
Consistent with this interpretation, using electron cryomicroscopy, our collaborators revealed that eEF2 stabilizes the ribosome–IRES complex in a rotated state with key residues in domain IV of eEF2 interacting with the CrPV–IRES and stabilizing it in a conformation reminiscent of a hybrid tRNA state. Interestingly, diphthamide appears to interact directly with the tRNA–mimicking pseudo-knot I of the CrPV IRES, perhaps to facilitate its precise translocation in the ribosome and to break decoding interactions between conserved rRNA bases and the pseudo-knot. Thus, our studies provide the first evidence that diphthamide plays a role in protein synthesis, and we propose that diphthamide functions to disrupt the decoding interactions of rRNA in the A-site and to maintain codon-anticodon interactions as the A-site tRNA is translocated to the P site (Reference 6).
Publications
- 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;48:641-646.
- Rojas M, Vasconcelos G, Dever TE. An eIF2alpha-binding motif in protein phosphatase 1 subunit GADD34 and its viral orthologs is required to promote dephosphorylation of eIF2alpha. Proc Natl Acad Sci USA 2015;112:E3466-3475.
- Li JJ, Cao C, Fixsen SM, Young JM, Ono C, Bando H, Elde NC, Katsuma S, Dever TE, Sicheri F. Baculovirus protein PK2 subverts eIF2a kinase function by mimicry of its kinase domain C-lobe. Proc Natl Acad Sci USA 2015;112:E4364-4373.
- Gutierrez E, Shin BS, Woolstenhulme CJ, Kim JR, Saini P, Buskirk AR, Dever TE. eIF5A promotes translation of polyproline motifs. Mol Cell 2013;51:35-45.
- Melnikov S, Mailliot J, Shin BS, Rigger L, Yusupova G, Micura R, Dever TE, Yusupov M. Crystal structure of hypusine-containing translation factor eIF5A bound to a rotated eukaryotic ribosome. J Mol Biol 2016;428:3570-3576.
- Murray J, Savva CG, Shin BS, Dever TE, Ramakrishnan V, Fernández IS. Structural characterization of ribosome recruitment and translocation by type IV IRES. Elife 2016;5:e13567.
Collaborators
- Lina Basel-Vanigaite, MD, Tel Aviv University, Tel Aviv, Israel
- Guntram Borck, MD, PhD, Universität Ulm, Ulm, Germany
- Alan Hinnebusch, PhD, Section on Nutrient Control of Gene Expression, NICHD, Bethesda, MD
- Venkatraman Ramakrishnan, PhD, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
- Frank Sicheri, PhD, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, and University of Toronto, Toronto, Canada
- Marat Yusupov, PhD, L'Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Strasbourg, France
Contact
For more information, email thomas.dever@nih.gov or visit http://deverlab.nichd.nih.gov.
