Abstract
Eukaryotic cells restrict protein synthesis under various stress conditions, by inhibiting the eukaryotic translation initiation factor 2B (eIF2B)1,2. eIF2B is the guanine nucleotide exchange factor for eIF2, a heterotrimeric G protein consisting of α-, β- and γ-subunits. eIF2B exchanges GDP for GTP on the γ-subunit of eIF2 (eIF2γ), and is inhibited by stress-induced phosphorylation of eIF2α. eIF2B is a heterodecameric complex of two copies each of the α-, β-, γ-, δ- and ε-subunits3; its α-, β- and δ-subunits constitute the regulatory subcomplex4, while the γ- and ε-subunits form the catalytic subcomplex5. The three-dimensional structure of the entire eIF2B complex has not been determined. Here we present the crystal structure of Schizosaccharomyces pombe eIF2B with an unprecedented subunit arrangement, in which the α2β2δ2 hexameric regulatory subcomplex binds two γε dimeric catalytic subcomplexes on its opposite sides. A structure-based in vitro analysis by a surface-scanning site-directed photo-cross-linking method identified the eIF2α-binding and eIF2γ-binding interfaces, located far apart on the regulatory and catalytic subcomplexes, respectively. The eIF2γ-binding interface is located close to the conserved ‘NF motif’, which is important for nucleotide exchange. A structural model was constructed for the complex of eIF2B with phosphorylated eIF2α, which binds to eIF2B more strongly than the unphosphorylated form. These results indicate that the eIF2α phosphorylation generates the ‘nonproductive’ eIF2–eIF2B complex5, which prevents nucleotide exchange on eIF2γ, and thus provide a structural framework for the eIF2B-mediated mechanism of stress-induced translational control.
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Acknowledgements
We thank the staff of the beamline BL41XU at SPring-8 for their support. This work was performed with the approval of the Japan Synchrotron Radiation Research Institute (proposals 2012A1335 and 2012B1572). This work was supported by JSPS KAKENHI grants 23687013 and 25121737 (to T.I.), the Targeted Proteins Research Program (TPRP) and the Platform for Drug Discovery, Informatics and Structural Life Science, from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (to S.Y.), and a research program of the UT-RIKEN Cooperation Laboratory of Structural Biology (to S.Y.).
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K.K. and T.I. designed and performed experiments, and wrote the manuscript. M.T., M.N. and T.B.H. purified the samples. T.H. and T.U. established the K. pastoris strain. K.S. provided the materials and technical advice for the surface-scanning photo-cross-linking experiments. S.Y. designed experiments and wrote the manuscript.
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Extended data figures and tables
Extended Data Figure 1 Biochemical properties of recombinant S. pombe eIF2B.
a, Guanine-nucleotide exchange catalysed by S. pombe eIF2B, and its inhibition by eIF2 phosphorylation. K. pastoris eIF2 was labelled with [3H]GDP and incubated with ATP and activated PKR (eIF2(αP); red line), or with ATP only (eIF2; blue line). Reactions were started by the addition of excess unlabelled GDP with eIF2B (solid line) or buffer (dashed line). Individual data points of triplicate analyses are shown by dots and boxes. The dissociation rates of GDP from eIF2 are summarized in the table. b, The SEC profiles of respective proteins (S. pombe eIF2B (green), K. pastoris eIF2 (cyan), eIF2(αP) (pink)) (top), eIF2B with K. pastoris eIF2 (middle) and eIF2B with eIF2(αP) (bottom). The chromatograms of the absorbance at 280 nm and the Coomassie blue stained SDS–PAGE gels are shown. For gel source data, see Supplementary Fig. 1. c, ITC measurements between S. pombe eIF2B (as the decamer) and S. pombe eIF2α (top), eIF2B and P-eIF2α (bottom). We used the eIF2α subunit alone, rather than the trimeric eIF2, in these experiments because of the difficulty in the preparation of highly concentrated eIF2. Representative thermograms are shown. Two runs were concatenated for analysis. The binding stoichiometry (N) and the dissociation constant (Kd) were calculated from triplicate analyses (mean ± s.d.).
Extended Data Figure 2 Architecture of the eIF2B subcomplexes.
a, b, Two different views of the crystal structure of S. pombe eIF2B (wall-eyed stereo view), coloured as in Fig. 1a and from the same view as in Fig. 1b and Fig. 1c, respectively. The assembly of the subcomplexes is primarily mediated by the β–ε and δ–γ interactions. In this regard, the results of the two prior mutational analyses12,13 are consistent with the present structure, except that one suggested that the β-helical region of the γ-subunit is involved in the inter-subunit interactions. c–k, Ribbon models of the structures of S. pombe eIF2B subcomplexes (c–e, g, h, j) and proteins with structural similarity (f, i, k). c, d, The α2 homodimer (c) and the βδ heterodimer (d) in the eIF2B decamer. The conformations of each subunit are similar to those in the human α2 homodimer43 and the Chaetomium thermophilum βδ heterotetramer3, even though the relative orientations of the subunits in the dimers are slightly different from those represented in these partial structures. e, f, The regulatory subcomplex in the eIF2B decamer (e) and ribose-1,5-bisphosphate (R15Pi) homohexamer44 (PDB 3A11) (f). The architecture of the regulatory subcomplex is an assembly of three similarly shaped dimer moieties: one homodimer of the α-subunit and two heterodimers of the β- and δ-subunits. The arrangement of the regulatory subunits resembles that in the C. thermophilum βδ heterotetramer3, and shares some similarity to that in the homohexameric structure of R15Pi44. g, The γ-subunit of eIF2B. h, The ε-subunit of eIF2B. i, Potato tuber ADP-glucose pyrophosphorylase (AGP) (PDB 1YP3)45. The dimerization interfaces between the catalytic subunits are coloured in deeper shades (g, h). j, k, The subunit heterodimerization mode in the catalytic subcomplex of eIF2B (j) and the subunit homodimerization mode in the potato tuber AGP (k). The dimerization manner of the γ- and ε-subunits is novel: each of their structures resembles the subunit structure of the AGP homotetramer45, but they dimerize through their N-terminal domains, in a different manner than the AGP homotetramer45.
Extended Data Figure 3 Mapping of the residues corresponding to missense VWM mutations on the subunit interfaces and the distal face of eIF2B.
The eIF2B residues corresponding to VWM-causing missense mutations in human (Supplementary Table 1) are mapped on the S. pombe eIF2B structure, with the same subunit colouring as in Fig. 1. The S. pombe eIF2B residues corresponding to VWM-causing missense mutations are shown in parentheses. a, VWM-related residues are mapped as spheres on the overall structure (ribbon model). The environments of the residues are colour-coded (green, solvent-exposed; yellow, subunit interface; brown, structural core) on the spheres. b, VWM-related residues are mapped on the surface model of the inter-subcomplex interfaces on the catalytic subcomplex side (left), with the interfaces for the α-, β- and δ-subunits coloured blue, cyan and green, respectively, and on the βδ dimer side (right), with the interfaces for the γ- and ε-subunits coloured orange and pink, respectively. VWM-related residues around the βδ dimerization interface are shown as spheres in the inset. The mutations in the regulatory subunits are clustered around the dimerization interface between the β- and δ-subunits, as mentioned in ref. 3. Our structure further revealed that the binding site for the ε-subunit is formed by the correct interaction between the β- and δ-subunits, thus explaining the abundance of mutations around this interface. c, VWM-related residues in the α2 homodimer are located around the homodimerization interface, and shown as spheres in the inset. These VWM-related mutations around the subunit interfaces (b, c) may cause appreciable degrees of subunit dissociation from the eIF2B decamer, leading to incomplete complexes, destabilization of eIF2B resulting in aggregation/degradation, and/or changes in the conformation and activity of the intact eIF2B decamer. d, VWM-related residues on the distal face of the catalytic subcomplex are mapped on the surface model. The NF motif is shown in red. Several VWM-related residues are located near the NF motif, including Arg111(2Bε), corresponding to the human Arg136His(2Bε) mutation, for which the mouse model is available46. e, The ε-subunit further contains several exposed missense VWM mutations, especially in the β-helical domain. f, VWM-related residues in the central cavity. Only one residue, corresponding to the human Lys110Glu(2Bα) mutation, is exposed to the solvent.
Extended Data Figure 4 Photo-cross-linking between pBpa-labelled eIF2B and eIF2.
a, The S. pombe eIF2B variants bearing a single site-specific pBpa substitution in the catalytic subunits were mixed with K. pastoris eIF2, and irradiated with ultraviolet (365 nm) for 5 min on ice. Since eIF2γ harbours the Flag-tag at the C terminus, the products cross-linked with eIF2γ were detected by western blotting with an anti-Flag antibody. Site-specific slow-migrating bands that appeared after ultraviolet irradiation were judged as cross-linked bands. The relevant bands are indicated with teal dots. b, The eIF2γ-cross-linked sites are shown in teal, except for the selected ones explained below, and the cross-link-negative sites are shown in grey, on the surface model of the catalytic subcomplex, coloured in the same manner as in Fig. 1. The NF motif is shown in red. c, Time-course analysis of cross-linking with eIF2γ. Four selected sites on the distal face (Gln117(2Bε) (light green in b), Leu257(2Bε) (blue), Glu204(2Bε) (purple) and Ser258(2Bγ) (violet)) were examined by time courses of the cross-linking with eIF2 or eIF2(αP). The ratio of the band intensity of the eIF2B γ- or ε-subunit cross-linked with eIF2(αP) to that with unphosphorylated eIF2 at each time point is shown below the lane. ND means that no band of the eIF2B subunit cross-linked with eIF2(αP) was detected at the time point. For gel source data, see Supplementary Fig. 1.
Extended Data Figure 5 Photo-cross-linking between pBpa-labelled eIF2B and eIF2α.
a, The S. pombe eIF2B variants bearing a single site-specific pBpa substitution in the regulatory subunit were mixed with S. pombe P-eIF2α, and irradiated with ultraviolet (365 nm) for 5 min. Cross-linking was detected as described in Extended Data Fig. 4. The relevant bands are indicated with orange dots. b, The eIF2α-cross-linked sites are shown in orange, and the cross-link-negative sites are shown in grey, on the surface model of the overall structure of eIF2B, with the same subunit colouring as in Fig. 1. c, The eIF2B variants were similarly mixed with unphosphorylated eIF2α and irradiated with ultraviolet. The bands that were also observed in a are indicated with orange dots, and the unphosphorylated eIF2α-specific bands are indicated with magenta dots. d, Arg84(2Bβ) and Gln91(2Bβ), which exhibited unphosphorylated eIF2α-specific cross-links, are shown in magenta. The view is the same as in Fig. 2a. For gel source data, see Supplementary Fig. 1.
Extended Data Figure 6 Solvent-exposed Gcn– mutations located in the central cavity and buried Gcn– mutations clustered around the trimerization interface.
a, The residues corresponding to Gcn– mutations19,20 are mapped in blue on the surface model of the S. pombe eIF2B structure, with the same subunit colouring as in Fig. 1. b, c, The residues corresponding to exposed Gcn– mutations are mainly located in the central cavity (b), and their locations coincide with the P-eIF2α cross-link sites shown in orange (c, Extended Data Fig. 5b). d, e, The residues corresponding to Gcn– mutations on the α2 homodimer (d) and the βδ heterodimer (e). The residues of S. pombe eIF2B corresponding to S. cerevisiae Gcn– mutations are indicated in parentheses. The interfaces for the trimerization of the regulatory dimers are coloured grey. Most of the Gcn–-related residues are mapped only on one face of the subunits, as predicted43,47. The present eIF2B structure revealed that these ‘mutation-rich’ faces are used for the assembly of the regulatory subunits to form the subcomplex, and the Gcn–-related residues are clustered on the interface for the trimeric assembly.
Extended Data Figure 7 Cross-linking of pBpa-labelled eIF2α with the central cavity formed by the eIF2B regulatory subunits.
a, b, The S. pombe eIF2α variants bearing a single site-specific pBpa residue were mixed with epitope-tagged S. pombe eIF2B, and irradiated with ultraviolet (365 nm) for 5 min on ice. Cross-linking was detected as described in Extended Data Fig. 4. Cross-links with eIF2Bα were detected with an anti-myc antibody (a), and those with eIF2Bβ were detected with an anti-HA antibody (b). The relevant bands are indicated with blue dots in a, and cyan dots in b. These cross-linked sites are mapped on the human eIF2α structure21 in Fig. 2b. For gel source data, see Supplementary Fig. 1. c, The model of the eIF2B–eIF2α complex, built on the basis of the cross-linking experiments, is shown in Fig. 2c. The phosphorylated residue Ser51(2α) is highlighted with the magenta circle. d, The electrostatic surface potential of eIF2B, from the same viewpoint as in c. Red and blue colours represent negative and positive potentials, respectively, of ±10kT/e. The cavity has no positively charged patch; therefore, the mechanism underlying the enhanced affinity for P-eIF2α is still unclear. One possible mechanism is a cation-mediated recognition of the phosphoserine residue. The phosphorylated Ser51(2α) may coordinate a cation together with the negatively charged residues at the bottom of the central cavity, although we did not observe any electron density for such cations in the cavity. Another possibility is a phosphorylation-induced conformational change of the Ser51-flanking loop. The phosphorylation of Ser51(2α) may induce the rearrangement of adjacent arginine residues, and enable a stronger interaction with the negatively charged residues at the bottom of the central cavity.
Extended Data Figure 8 Analyses of eIF2B Gcn– mutations.
a, The locations of the Gcn–-related residues mutated for the SEC and ITC analyses (Glu57(2Bα) and Asp248(2Bδ)) are indicated on the S. pombe eIF2B structure, with the same view and colouring as in Extended Data Fig. 6b. b, The SEC analyses of the interaction between K. pastoris eIF2(αP) and S. pombe eIF2B, bearing Gcn–-related mutations. The chromatograms of the absorbance at 280 nm and the SDS–PAGE gels of each run are shown. The green bar represents the elution point of free eIF2B (Extended Data Fig. 1b). For gel source data, see Supplementary Fig. 1. c, ITC measurements between S. pombe eIF2α or P-eIF2α and eIF2B, bearing Gcn–-related mutations. Representative thermograms for the ITC experiments are shown. d, e, The nucleotide exchange activities of S. pombe eIF2B, bearing Gcn–-related mutations, on K. pastoris eIF2(αP) (d) and eIF2 (e) were examined as described in Extended Data Fig. 1a (αGlu57Lys mutant, blue line; δAsp248Lys mutant, green line; wild type, grey line). Individual data points of triplicate analyses are shown by dots.
Extended Data Figure 9 The locations of ISRIB-resistant mutations on the eIF2B structure.
Residues corresponding to the ISRIB-resistant mutations26 are mapped onto the eIF2B structure in red. The residue corresponding to the Arg171Gln(2Bδ) mutation (Lys112(2Bδ)) is in the disordered region, at the N terminus of the δ-subunit. The disordered N-terminal segment of the δ-subunit is indicated by the dotted green line.
Supplementary information
Supplementary Figure 1
This file contains full scanned gels in uncropped form. (PDF 425 kb)
Supplementary Table 1
This file contains the list of missense VWM mutations and corresponding residues. (XLSX 18 kb)
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Kashiwagi, K., Takahashi, M., Nishimoto, M. et al. Crystal structure of eukaryotic translation initiation factor 2B. Nature 531, 122–125 (2016). https://doi.org/10.1038/nature16991
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DOI: https://doi.org/10.1038/nature16991
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