Crystal Structure of the N-terminal Segment of Human Eukaryotic Translation Initiation Factor 2α

Abstract

Eukaryotic translation initiation factor 2α (eIF2α) is a member of the eIF2 heterotrimeric complex that binds and delivers Met-tRNAiMet to the 40 S ribosomal subunit in a GTP-dependent manner. Phosphorylation/dephosphorylation of eIF2α at Ser-51 is the major regulator of protein synthesis in eukaryotic cells. Here, we report the first structural analysis on eIF2, the three-dimensional structure of a 22-kDa N-terminal portion of human eIF2α by x-ray diffraction at 1.9 Å resolution. This structure contains two major domains. The N terminus is a β-barrel with five antiparallel β-strands in an oligonucleotide binding domain (OB domain) fold. The phosphorylation site (Ser-51) is on the loop connecting β3 and β4 in the OB domain. A helical domain follows the OB domain, and the first helix has extensive interactions, including a disulfide bridge, to fix its orientation with respect to the OB domain. The two domains meet along a negatively charged groove with highly conserved residues, indicating a likely site for protein-protein interaction. Eukaryotic translation initiation factor 2α (eIF2α) is a member of the eIF2 heterotrimeric complex that binds and delivers Met-tRNAiMet to the 40 S ribosomal subunit in a GTP-dependent manner. Phosphorylation/dephosphorylation of eIF2α at Ser-51 is the major regulator of protein synthesis in eukaryotic cells. Here, we report the first structural analysis on eIF2, the three-dimensional structure of a 22-kDa N-terminal portion of human eIF2α by x-ray diffraction at 1.9 Å resolution. This structure contains two major domains. The N terminus is a β-barrel with five antiparallel β-strands in an oligonucleotide binding domain (OB domain) fold. The phosphorylation site (Ser-51) is on the loop connecting β3 and β4 in the OB domain. A helical domain follows the OB domain, and the first helix has extensive interactions, including a disulfide bridge, to fix its orientation with respect to the OB domain. The two domains meet along a negatively charged groove with highly conserved residues, indicating a likely site for protein-protein interaction. Eukaryotic translation initiation factor 2 (eIF2) 1The abbreviations used are: eIF2αeukaryotic translation initiation factor 2αMADmultiple anomalous dispersionSeMetselenomethionineOB foldoligonucleotide-binding fold1The abbreviations used are: eIF2αeukaryotic translation initiation factor 2αMADmultiple anomalous dispersionSeMetselenomethionineOB foldoligonucleotide-binding fold is a GTP-binding protein that plays a central role in initiating translation. It binds charged initiator tRNA (Met-tRNAiMet) in a GTP-dependent manner to form the ternary complex, eIF2·GTP·Met-tRNAiMet. This ternary complex binds to the 40 S ribosomal subunit, and additional initiation factors (including eIF3 and eIF1A) join to form the 43 S preinitiation complex and assist in recognizing the start codon (reviewed in Refs.1.Trachsel H. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996: 113-138Google Scholar, 2.Pain V.M. Eur. J. Biochem. 1996; 236: 747-771Crossref PubMed Scopus (636) Google Scholar, 3.Rhods R.E. J. Biol. Chem. 1993; 268: 3017-3020Abstract Full Text PDF PubMed Google Scholar, 4.Kozak M. Gene. 1999; 234: 187-208Crossref PubMed Scopus (1121) Google Scholar, 5.Merrick W.C. Microbiol. Rev. 1992; 56: 291-315Crossref PubMed Google Scholar).Recognition of the translational site is accompanied by eIF5-mediated hydrolysis of eIF2-bound GTP, which releases an eIF2·GDP binary complex along with several other initiation factors. For another round of initiation, the GDP in the eIF2 binary complex must be exchanged for GTP in a reaction catalyzed by the multimeric protein factor eIF2B (previously called guanine nucleotide exchange factor) (6.Price N. Proud C. Biochimie (Paris). 1994; 76: 748-760Crossref PubMed Scopus (89) Google Scholar). The 40 S·mRNA·Met-tRNAiMet complex joins the 60 S ribosomal subunit to form an 80 S initiation complex that can enter the elongation phase of protein synthesis.In eukaryotes, eIF2 is a heterotrimer composed of α (36 kDa)-, β (38 kDa)-, and γ (52 kDa)-subunits, which appear to remain associated throughout the initiation cycle. Cross-linking and genetic studies have suggested that both β- and γ-subunits are implicated in guanine nucleotide and Met-tRNAiMet binding (7.Erickson F.L. Hannig E.M. EMBO J. 1996; 15: 6311-6320Crossref PubMed Scopus (84) Google Scholar, 8.Gaspar N.J. Kinzy T.G. Scherer B.J. Hümbelin M. Hershey J.W.B. Merrick W.C. J. Biol. Chem. 1994; 269: 3415-3422Abstract Full Text PDF PubMed Google Scholar, 9.Anthony Jr., D.D. Kinzy T.G. Merrick W.C. Arch. Biochem. Biophys. 1990; 281: 157-162Crossref PubMed Scopus (28) Google Scholar). In addition, the β-subunit was shown to interact specifically with eIF5 during GTP hydrolysis and also to bind mRNA (10.Thompson G.M. Pacheco E. Melo E.O. Castilho B.A. Biochem. J. 2000; 347: 703-709Crossref PubMed Scopus (36) Google Scholar, 11.Das S. Maiti T. Das K. Maitra U. J. Biol. Chem. 1997; 272: 31712-31718Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 12.Laurino J.P. Thompson G.M. Pacheco E. Castilho B.A. Mol. Cell. Biol. 1999; 19: 173-181Crossref PubMed Google Scholar). The γ-subunit participates in the recognition of the start site for protein synthesis (13.Dorris D.R. Erickson F.L. Hannig E.M. EMBO J. 1995; 14: 2239-2249Crossref PubMed Scopus (57) Google Scholar).The phosphorylation/dephosphorylation of a conserved serine (Ser-51) in the α-subunit is the major regulator of protein synthesis in eukaryotes (reviewed in Ref. 2.Pain V.M. Eur. J. Biochem. 1996; 236: 747-771Crossref PubMed Scopus (636) Google Scholar). Phosphorylation of Ser-51 shuts off protein synthesis (14.Pathak V.K. Nielsen P.J. Trachsel H. Hershey J.W.B. Cell. 1998; 54: 633-639Abstract Full Text PDF Scopus (87) Google Scholar). Three protein kinases, HRI (heme-regulated inhibitor), PKR (RNA-dependent protein kinase), and GCN2, have been identified that specifically phosphorylate Ser-51 in response to a variety of cellular stresses including viral infection, heat shock, heavy metals, and deprivation of amino acids or serum.The phosphorylated eIF2·GDP complex released during protein synthesis initiation binds eIF2B with much higher affinity than does eIF2·GTP, and binding to eIF2B does not allow the exchange GDP for GTP that is necessary for further rounds of initiation (reviewed in Refs 15.Clemens M.J. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996: 139-172Google Scholar, 16.Hinnebusch A.G. Semin. Cell Biol. 1994; 5: 417-426Crossref PubMed Scopus (121) Google Scholar, 17.Rowlands A.G. Panniers R. Henshaw E.C. J. Biol. Chem. 1988; 263: 5526-5533Abstract Full Text PDF PubMed Google Scholar). A recent paper by Nika and co-authors (18.Nika J. Rippel S. Hannig E.M. J. Biol. Chem. 2001; 276: 1051-1056Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) has shown that unphosphorylated eIF2α also meditates nucleotide exchange. The rate of eIF2B-catalyzed nucleotide exchange increases in the absence of the α-subunit compared with wild type, which suggests that nucleotide exchange requires direct interaction between eIF2α and eIF2B and that phosphorylation strengthens this interaction. There are no structural data on any of the eIF2 monomers to provide a structural context for these important regulatory interactions.Here we report the structure of human eIF2α determined by x-ray crystallography using multiple anomalous dispersion (MAD) on a selenomethionine (SeMet)-labeled baculovirus-expressed eIF2α at 1.9 Å resolution. The crystallization of eIF2α was achieved only by using limited proteolysis techniques, and the final structure comprises residues 3–182, roughly the N-terminal two-thirds of full-length human eIF2α.RESULTS AND DISCUSSIONThe structure of an ∼21.4-kDa N-terminal fragment of human eIF2α was determined using x-ray crystallography at 1.9 Å resolution. The crystal structure contains residues 3–182 with a disordered loop from residues 51 to 68 (Fig. 2). The final model, with approximate dimensions of 57 × 36 × 35 Å, is divided into two major domains: the OB domain and the helical domain.OB DomainThe first 87 N-terminal residues have an oligonucleotide-binding fold (OB fold) (32.Murzin A.G. EMBO J. 1993; 12: 861-867Crossref PubMed Scopus (762) Google Scholar), with a five-stranded antiparallel β-barrel arranged with a Greek key topology capped by a turn of 310 α-helix located between β3 and β4 (Fig. 2).A β-hairpin connects β1 and β2; β2 and β3 are linked by a four-residue loop and β4 and β5 by a three-residue loop. The loop connecting β3 and β4 is longer by 17 residues, and it was not completely modeled due to a lack of interpretable electron density. The visible part, residues 48–50, begins with a turn of 310 helix that is stabilized through a hydrogen bond between the carbonyl group of Leu-46 and nitrogen of Glu-49. Serine 51 comes just after this 310 helix (Fig. 2). Residues 63 and 64 at the end of the loop are also visible with a hydrogen bond between the carbonyl group of Arg-63 and the main chain nitrogen of Arg-66. β1 contains a β-bulge at residue Val-23, and a left-handed Gly-65 (φ and ψ have the same absolute values as the right-handed Gly but with opposite signs) is present at the beginning of β4; both are standard features of the OB fold.The eIF2α N-terminal domain is the latest example of the large oligonucleotide/oligosaccharide-binding fold. The structural classification of the protein data base (SCOP (34.Murzin A.G. Brenner S.E. Hubbard T. Chothia C. J. Mol. Biol. 1995; 247: 536-540Crossref PubMed Scopus (5568) Google Scholar)) counts at present 61 different structures containing an OB fold, divided into seven superfamilies. Using the DALI server software (35.Holm L. Sander C. J. Mol. Biol. 1993; 233: 123-138Crossref PubMed Scopus (3552) Google Scholar), the OB domain of human eIF2α was identified as a member of the nucleic acid-binding superfamily. Examples of members of this family are: S1 RNA-binding domain from the Escherichia coli polynucleotide phosphorylase (33.Bycroft M. Hubbard T.J.P. Proctor M. Freund S.M.V. Murzin A.G. Cell. 1997; 88: 235-242Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar), cold shock proteins A and B (36.Schindelin H. Jiang W. Inouye M. Heinemann U. Proc. Natl. Acad. Sci. U. S. A. 1993; 91: 5119-5123Crossref Scopus (294) Google Scholar, 37.Schindelin H. Marahiel M.A. Heinemann U. Nature. 1993; 364: 164-168Crossref PubMed Scopus (314) Google Scholar), domain II of eukaryotic translation initiation factor 5A (38.Peat T.S. Newman J. Waldo G.S. Berendzen J. Terwilliger T.C. Struct. Fold. Des. 1998; 6: 1207-1214Abstract Full Text Full Text PDF Scopus (101) Google Scholar, 39.Kim K.K. Hung L. Yokota H. Kim R. Kim S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10419-10424Crossref PubMed Scopus (111) Google Scholar), translational initiation factor IF1 (40.Sette M. van Tilborg P. Spurio R. Kaptein R. Paci M. Gualerzi C.O. Boelens R. EMBO J. 1997; 16: 1436-1443Crossref PubMed Scopus (127) Google Scholar), and the N-terminal domain of aspartyl-tRNA synthetase (41.Ruff M. Krishnaswamy S. Boeglin M. Poterszman A. Mitschler A. Podjarny A. Rees B. Thierry J.C. Moras D. Science. 1991; 252: 1682-1689Crossref PubMed Scopus (591) Google Scholar), among others.Although eIF2α shows very little, if any, sequence homology with members of this family, their structures can easily be superimposed, and the root mean square deviation along the coordinates of all Cα atoms is around 2.0 Å. The main differences are restricted to loop regions, especially the length of the loop connecting β3 and β4 containing the phosphorylation site. Among the eIF2α sequences, the β3 to β4 residues are highly conserved (Fig. 3); in human eIF2α, this region contains a turn of a 310 helix (Fig. 2) as observed in the NMR structure of E. coli polynucleotide phosphorylase (33.Bycroft M. Hubbard T.J.P. Proctor M. Freund S.M.V. Murzin A.G. Cell. 1997; 88: 235-242Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar) and the crystal structure of translation initiation factor 5A fromPyrobaculum aerophilum (38.Peat T.S. Newman J. Waldo G.S. Berendzen J. Terwilliger T.C. Struct. Fold. Des. 1998; 6: 1207-1214Abstract Full Text Full Text PDF Scopus (101) Google Scholar). Other family members have different structures in this connecting region. Translational initiation factor IF1 (40.Sette M. van Tilborg P. Spurio R. Kaptein R. Paci M. Gualerzi C.O. Boelens R. EMBO J. 1997; 16: 1436-1443Crossref PubMed Scopus (127) Google Scholar) and the N terminus of aspartyl-tRNA synthetase (41.Ruff M. Krishnaswamy S. Boeglin M. Poterszman A. Mitschler A. Podjarny A. Rees B. Thierry J.C. Moras D. Science. 1991; 252: 1682-1689Crossref PubMed Scopus (591) Google Scholar) contain an α-helix, and cold shock proteins A and B have a long loop lacking a defined secondary structure (36.Schindelin H. Jiang W. Inouye M. Heinemann U. Proc. Natl. Acad. Sci. U. S. A. 1993; 91: 5119-5123Crossref Scopus (294) Google Scholar, 37.Schindelin H. Marahiel M.A. Heinemann U. Nature. 1993; 364: 164-168Crossref PubMed Scopus (314) Google Scholar).Figure 3Sequence alignment of eIF2α proteins. Similar residues are highlighted incyan, and the phosphorylation site is yellow. The conserved positive charges found near the phosphorylation site arehighlighted in pink. The first three sequences are mammalian, and the last four are yeast. Alignment was performed using MULTALIN (45.Corpet F. Nucleic Acids Res. 1988; 16: 10881-10890Crossref PubMed Scopus (4265) Google Scholar) and picture using ALSCRIPT (46.Barton G.J. Protein Eng. 1993; 6: 37-40Crossref PubMed Scopus (1110) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT)The site proposed as the RNA binding site is found in nearly the same position in all members of the nucleic acid-binding superfamily; this is the β-barrel region, where three loops connecting β1 and β2, β3 and β4, and β4 and β5 come together (Fig. 2). In most cases RNA interaction seems to involve solvent-exposed aromatic residues and positively charged residues on the surface of the protein. The only structure available for this group of proteins bound to nucleic acids is the complex of the N-terminal fragment of aspartyl-tRNA synthetase with the anti-codon loop of its cognate tRNA (41.Ruff M. Krishnaswamy S. Boeglin M. Poterszman A. Mitschler A. Podjarny A. Rees B. Thierry J.C. Moras D. Science. 1991; 252: 1682-1689Crossref PubMed Scopus (591) Google Scholar) where there is a π-stacking interaction between one of the bases and a conserved Phe residue. In eIF2α the corresponding region contains two tyrosine residues, Tyr-32 and Tyr-81, clustered with non-polar residues Met-44, Leu-46, Ile-82, and Gly-43 and two residues, Glu-42 and Asp-83, with negatively charged side chains (Fig. 2).The OB domain of human eIF2α does not have any of the clustered positive charges that are observed for the other members of this family. This structural feature is consistent with the observation that, unlike other family members in which biochemical and genetic experiments and limited structural data support the idea of direct interaction with nucleic acids (36.Schindelin H. Jiang W. Inouye M. Heinemann U. Proc. Natl. Acad. Sci. U. S. A. 1993; 91: 5119-5123Crossref Scopus (294) Google Scholar, 37.Schindelin H. Marahiel M.A. Heinemann U. Nature. 1993; 364: 164-168Crossref PubMed Scopus (314) Google Scholar, 40.Sette M. van Tilborg P. Spurio R. Kaptein R. Paci M. Gualerzi C.O. Boelens R. EMBO J. 1997; 16: 1436-1443Crossref PubMed Scopus (127) Google Scholar, 41.Ruff M. Krishnaswamy S. Boeglin M. Poterszman A. Mitschler A. Podjarny A. Rees B. Thierry J.C. Moras D. Science. 1991; 252: 1682-1689Crossref PubMed Scopus (591) Google Scholar), there is little or no evidence that eIF2α binds RNA (18.Nika J. Rippel S. Hannig E.M. J. Biol. Chem. 2001; 276: 1051-1056Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Most cross-linking and genetic experiments suggest that β- and γ-subunits of eIF2 are the subunits involved in initiator tRNA and ribosomal RNA binding (8.Gaspar N.J. Kinzy T.G. Scherer B.J. Hümbelin M. Hershey J.W.B. Merrick W.C. J. Biol. Chem. 1994; 269: 3415-3422Abstract Full Text PDF PubMed Google Scholar, 42.Westermann P. Nygard O. Bielka H. Nucleic Acids Res. 1980; 8: 3065-3071Crossref PubMed Scopus (21) Google Scholar).Helical DomainThe helical domain comprises residues 88–182. In contrast to the highly conserved architecture of the OB domain, the helical domain adopts a previously unreported fold consisting of a 28-residue-long α-helix (α1), a series of small α-helices (α2, α3, α4, and α6), and one 310 helix folded into a very compact domain (Fig. 2). The OB and helical domains are linked by a disulfide bridge between Cys-69 in β4 and Cys-97 in α1 (Fig. 2). Alignment of eIF2α sequences suggests that this disulfide bridge is only possible in mammalian eIF2α (Fig. 3). The last residue in our model, Arg-182, is solvent-exposed, and some uninterpretable electron density can be observed around this region, suggesting that proteolytic attack may not be specific.Groove between the OB and Helical DomainsA negatively charged cavity is formed where the OB and helical domains come together (Fig. 4). The residues defining this groove come from several secondary structural elements: Tyr-8, located at the beginning of the OB domain; Asp-17 at β1; Asp-37 and Tyr-38 in the loop connecting β2 and β3; and Asp-138 on α3. Interestingly, all of the residues found in this region are highly conserved among the species (Fig. 4). The β-subunit of eukaryotic translation initiation factor 2 (eIF2β (12.Laurino J.P. Thompson G.M. Pacheco E. Castilho B.A. Mol. Cell. Biol. 1999; 19: 173-181Crossref PubMed Google Scholar)) contains a polylysine motif. The negatively charged groove in eIF2α might serve as the direct interaction site for the polylysine repeat.Figure 4A view of the highly conserved pocket between α1 of the helical domain (upper right) and the OB domain (lower left).The molecular surface was calculated with SPOCK (47.Christopher J.A. Baldwin T.O. J. Mol. Graph Model. 1998; 16: 285Google Scholar) and colored according to the sequence conservation. Completely conserved residues are indicated in red, those more than 50% conserved are in orange, and those less than 50% conserved are in blue. The completely conserved groove is negatively charged.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Phosphorylation SiteeIF2α regulates protein synthesis through phosphorylation/dephosphorylation of Ser-51. Phosphorylation of eIF2α eIF2 from a into a for the guanine exchange factor The eIF2 binding experiments that eIF2B binds to phosphorylated eIF2 with a higher affinity than to unphosphorylated has been suggested that eIF2α is for the interaction between eIF2 and eIF2B (8.Gaspar N.J. Kinzy T.G. Scherer B.J. Hümbelin M. Hershey J.W.B. Merrick W.C. J. Biol. Chem. 1994; 269: 3415-3422Abstract Full Text PDF PubMed Google Scholar). The affinity interaction between eIF2α and the regulatory domain composed of and of eIF2B is for the binding of eIF2 to the domain of of nucleotide exchange A.G. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus (84) Google Scholar). The phosphorylation site of eIF2α on the long loop connecting β3 and β4 of the OB domain, and it is to the RNA-binding site (Fig. 2). The lack of electron density suggests that this region is very the NMR structure of the translational initiation factor IF1 from E. coli shows for the corresponding loop (40.Sette M. van Tilborg P. Spurio R. Kaptein R. Paci M. Gualerzi C.O. Boelens R. EMBO J. 1997; 16: 1436-1443Crossref PubMed Scopus (127) Google Scholar).The for eIF2B to and to phosphorylation at Ser-51 be for eIF2B to direct with eIF2 in the loop region. feature of the highly conserved loop sequence is the of positively charged residues Ser-51 (Fig. 3). This and with the recent that eIF2α and the regulatory domain eIF2B interact of suggest a in which the positively charged residues in an interaction that is by the present the first structural analysis of any member of the eIF2 heterotrimeric complex and a for further and structural of protein-protein Eukaryotic translation initiation factor 2 (eIF2) 1The abbreviations used are: eIF2αeukaryotic translation initiation factor 2αMADmultiple anomalous dispersionSeMetselenomethionineOB foldoligonucleotide-binding fold1The abbreviations used are: eIF2αeukaryotic translation initiation factor 2αMADmultiple anomalous dispersionSeMetselenomethionineOB foldoligonucleotide-binding fold is a GTP-binding protein that plays a central role in initiating translation. It binds charged initiator tRNA (Met-tRNAiMet) in a GTP-dependent manner to form the ternary complex, eIF2·GTP·Met-tRNAiMet. This ternary complex binds to the 40 S ribosomal subunit, and additional initiation factors (including eIF3 and eIF1A) join to form the 43 S preinitiation complex and assist in recognizing the start codon (reviewed in Refs.1.Trachsel H. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996: 113-138Google Scholar, 2.Pain V.M. Eur. J. Biochem. 1996; 236: 747-771Crossref PubMed Scopus (636) Google Scholar, 3.Rhods R.E. J. Biol. Chem. 1993; 268: 3017-3020Abstract Full Text PDF PubMed Google Scholar, 4.Kozak M. Gene. 1999; 234: 187-208Crossref PubMed Scopus (1121) Google Scholar, 5.Merrick W.C. Microbiol. Rev. 1992; 56: 291-315Crossref PubMed Google Scholar). eukaryotic translation initiation factor 2α multiple anomalous dispersion selenomethionine oligonucleotide-binding fold eukaryotic translation initiation factor 2α multiple anomalous dispersion selenomethionine oligonucleotide-binding fold of the translational site is accompanied by eIF5-mediated hydrolysis of eIF2-bound GTP, which releases an eIF2·GDP binary complex along with several other initiation factors. For another round of initiation, the GDP in the eIF2 binary complex must be exchanged for GTP in a reaction catalyzed by the multimeric protein factor eIF2B (previously called guanine nucleotide exchange factor) (6.Price N. Proud C. Biochimie (Paris). 1994; 76: 748-760Crossref PubMed Scopus (89) Google Scholar). The 40 S·mRNA·Met-tRNAiMet complex joins the 60 S ribosomal subunit to form an 80 S initiation complex that can enter the elongation phase of protein In eukaryotes, eIF2 is a heterotrimer composed of α (36 kDa)-, β (38 kDa)-, and γ (52 kDa)-subunits, which appear to remain associated throughout the initiation cycle. Cross-linking and genetic studies have suggested that both β- and γ-subunits are implicated in guanine nucleotide and Met-tRNAiMet binding (7.Erickson F.L. Hannig E.M. EMBO J. 1996; 15: 6311-6320Crossref PubMed Scopus (84) Google Scholar, 8.Gaspar N.J. Kinzy T.G. Scherer B.J. Hümbelin M. Hershey J.W.B. Merrick W.C. J. Biol. Chem. 1994; 269: 3415-3422Abstract Full Text PDF PubMed Google Scholar, 9.Anthony Jr., D.D. Kinzy T.G. Merrick W.C. Arch. Biochem. Biophys. 1990; 281: 157-162Crossref PubMed Scopus (28) Google Scholar). In addition, the β-subunit was shown to interact specifically with eIF5 during GTP hydrolysis and also to bind mRNA (10.Thompson G.M. Pacheco E. Melo E.O. Castilho B.A. Biochem. J. 2000; 347: 703-709Crossref PubMed Scopus (36) Google Scholar, 11.Das S. Maiti T. Das K. Maitra U. J. Biol. Chem. 1997; 272: 31712-31718Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 12.Laurino J.P. Thompson G.M. Pacheco E. Castilho B.A. Mol. Cell. Biol. 1999; 19: 173-181Crossref PubMed Google Scholar). The γ-subunit participates in the recognition of the start site for protein synthesis (13.Dorris D.R. Erickson F.L. Hannig E.M. EMBO J. 1995; 14: 2239-2249Crossref PubMed Scopus (57) Google Scholar). The phosphorylation/dephosphorylation of a conserved serine (Ser-51) in the α-subunit is the major regulator of protein synthesis in eukaryotes (reviewed in Ref. 2.Pain V.M. Eur. J. Biochem. 1996; 236: 747-771Crossref PubMed Scopus (636) Google Scholar). Phosphorylation of Ser-51 shuts off protein synthesis (14.Pathak V.K. Nielsen P.J. Trachsel H. Hershey J.W.B. Cell. 1998; 54: 633-639Abstract Full Text PDF Scopus (87) Google Scholar). Three protein kinases, HRI (heme-regulated inhibitor), PKR (RNA-dependent protein kinase), and GCN2, have been identified that specifically phosphorylate Ser-51 in response to a variety of cellular stresses including viral infection, heat shock, heavy metals, and deprivation of amino acids or The phosphorylated eIF2·GDP complex released during protein synthesis initiation binds eIF2B with much higher affinity than does eIF2·GTP, and binding to eIF2B does not allow the exchange GDP for GTP that is necessary for further rounds of initiation (reviewed in Refs 15.Clemens M.J. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996: 139-172Google Scholar, 16.Hinnebusch A.G. Semin. Cell Biol. 1994; 5: 417-426Crossref PubMed Scopus (121) Google Scholar, 17.Rowlands A.G. Panniers R. Henshaw E.C. J. Biol. Chem. 1988; 263: 5526-5533Abstract Full Text PDF PubMed Google Scholar). A recent paper by Nika and co-authors (18.Nika J. Rippel S. Hannig E.M. J. Biol. Chem. 2001; 276: 1051-1056Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) has shown that unphosphorylated eIF2α also meditates nucleotide exchange. The rate of eIF2B-catalyzed nucleotide exchange increases in the absence of the α-subunit compared with wild type, which suggests that nucleotide exchange requires direct interaction between eIF2α and eIF2B and that phosphorylation strengthens this interaction. There are no structural data on any of the eIF2 monomers to provide a structural context for these important regulatory we report the structure of human eIF2α determined by x-ray crystallography using multiple anomalous dispersion (MAD) on a selenomethionine (SeMet)-labeled baculovirus-expressed eIF2α at 1.9 Å resolution. The crystallization of eIF2α was achieved only by using limited proteolysis techniques, and the final structure comprises residues 3–182, roughly the N-terminal two-thirds of full-length human AND DISCUSSIONThe structure of an ∼21.4-kDa N-terminal fragment of human eIF2α was determined using x-ray crystallography at 1.9 Å resolution. The crystal structure contains residues 3–182 with a disordered loop from residues 51 to 68 (Fig. 2). The final model, with approximate dimensions of 57 × 36 × 35 Å, is divided into two major domains: the OB domain and the helical domain.OB DomainThe first 87 N-terminal residues have an oligonucleotide-binding fold (OB fold) (32.Murzin A.G. EMBO J. 1993; 12: 861-867Crossref PubMed Scopus (762) Google Scholar), with a five-stranded antiparallel β-barrel arranged with a Greek key topology capped by a turn of 310 α-helix located between β3 and β4 (Fig. 2).A β-hairpin connects β1 and β2; β2 and β3 are linked by a four-residue loop and β4 and β5 by a three-residue loop. The loop connecting β3 and β4 is longer by 17 residues, and it was not completely modeled due to a lack of interpretable electron density. The visible part, residues 48–50, begins with a turn of 310 helix that is stabilized through a hydrogen bond between the carbonyl group of Leu-46 and nitrogen of Glu-49. Serine 51 comes just after this 310 helix (Fig. 2). Residues 63 and 64 at the end of the loop are also visible with a hydrogen bond between the carbonyl group of Arg-63 and the main chain nitrogen of Arg-66. β1 contains a β-bulge at residue Val-23, and a left-handed Gly-65 (φ and ψ have the same absolute values as the right-handed Gly but with opposite signs) is present at the beginning of β4; both are standard features of the OB fold.The eIF2α N-terminal domain is the latest example of the large oligonucleotide/oligosaccharide-binding fold. The structural classification of the protein data base (SCOP (34.Murzin A.G. Brenner S.E. Hubbard T. Chothia C. J. Mol. Biol. 1995; 247: 536-540Crossref PubMed Scopus (5568) Google Scholar)) counts at present 61 different structures containing an OB fold, divided into seven superfamilies. Using the DALI server software (35.Holm L. Sander C. J. Mol. Biol. 1993; 233: 123-138Crossref PubMed Scopus (3552) Google Scholar), the OB domain of human eIF2α was identified as a member of the nucleic acid-binding superfamily. Examples of members of this family are: S1 RNA-binding domain from the Escherichia coli polynucleotide phosphorylase (33.Bycroft M. Hubbard T.J.P. Proctor M. Freund