Eukaryotic cells restrict protein synthesis under various stress conditions, by inhibiting the eukaryotic translation initiation factor 2B (eIF2B). 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-subunits; its α-, β- and δ-subunits constitute the regulatory subcomplex, while the γ- and ε-subunits form the catalytic subcomplex. 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 complex, which prevents nucleotide exchange on eIF2γ, and thus provide a structural framework for the eIF2B-mediated mechanism of stress-induced translational control.

No statistical methods were used to predetermine sample size. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment. Purification of human eIF2The α-, β-, and γ-subunits of human eIF2, and the eIF2-specific chaperone protein human Cdc123 (33) were co-expressed in FreeStyle 293-F cells, using the four pEBMulti-Neo plasmid vectors (Wako), and eIF2γ was expressed with C-terminal FLAG and His8 tags. The cells were lysed in buffer A [20 mM MES-KOH buffer (pH 6.0), containing 150 mM KCl, 1 mM MgCl2, 10%(v/v) glycerol, and 5 mM 2-mercaptoethanol] supplemented with 20 mM imidazole, 0.5 mM EDTA, 0.1%(v/v) Triton X-100 and protease inhibitors. After 30 min on ice and centrifugation, the supernatant was applied to a HisTrap (GE Healthcare) column equilibrated with buffer A supplemented with 20 mM imidazole, and eluted with a linear gradient of 20-500 mM imidazole. The fraction containing eIF2 was collected and applied to a HiTrap SP (GE Healthcare) column equilibrated with buffer A, and eluted with a linear gradient of 200-640 mM KCl. After three-fold dilution with buffer B [20 mM HEPES-KOH buffer (pH 7.5) containing 100 mM KCl, 0.1 mM MgCl2, 10%(v/v) glycerol, and 1 mM DTT], the sample was applied to a HiTrap Heparin (GE Healthcare) column equilibrated with buffer B, and eluted with a linear gradient of 0.2-1 M KCl. The fraction containing eIF2 was further purified on a Superdex200 (GE Healthcare) column equilibrated with buffer B. eIF2 was phosphorylated by PKR, as described for Komagataella pastoris eIF2 (14). Purification of human eIF2BHuman eIF2Bα and eIF2Bβγδε were purified separately.The fragment encoding human eIF2Bα was cloned into pET-28c (Novagen), in which the thrombin cleavage site was replaced by the HRV 3C protease cleavage site. The T7 Express

Summary The small molecule ISRIB antagonizes the activation of the integrated stress response (ISR) by phosphorylated translation initiation factor 2, eIF2(αP). ISRIB and eIF2(αP) bind distinct sites in their common target, eIF2B, a guanine nucleotide exchange factor for eIF2. We have found that ISRIB-mediated acceleration of eIF2B’s nucleotide exchange activity in vitro is observed preferentially in the presence of eIF2(αP) and is attenuated by mutations that desensitize eIF2B to the inhibitory effect of eIF2(αP). ISRIB’s efficacy as an ISR inhibitor in cells also depends on presence of eIF2(αP). Cryoelectron microscopy (cryo-EM) showed that engagement of both eIF2B regulatory sites by two eIF2(αP) molecules remodels both the ISRIB-binding pocket and the pockets that would engage eIF2α during active nucleotide exchange, thereby discouraging both binding events. In vitro , eIF2(αP) and ISRIB reciprocally opposed each other’s binding to eIF2B. These findings point to antagonistic allostery in ISRIB action on eIF2B, culminating in inhibition of the ISR.

The small molecule ISRIB antagonises the activation of the integrated stress response (ISR) by phosphorylated translation initiation factor 2, eIF2(αP). ISRIB and eIF2(αP) bind distinct sites in their common target, eIF2B, a guanine nucleotide exchange factor (GEF) for eIF2. We have found that ISRIB-mediated acceleration of eIF2B activity in vitro is observed preferentially in the presence of eIF2(αP) and is attenuated by mutations that desensitise eIF2B to the inhibitory effects of eIF2(αP). ISRIB’s efficacy as an ISR inhibitor in cells also depends on presence of eIF2(αP). Cryo-EM showed that engagement of both eIF2B regulatory sites by two eIF2(αP) molecules remodels both the ISRIB-binding pocket and the pockets that would engage eIF2α during active nucleotide exchange, thereby discouraging both binding events. In vitro, eIF2(αP) and ISRIB reciprocally opposed each other’s binding to eIF2B. These findings point to antagonistic allostery in ISRIB action on eIF2B, culminating in inhibition of the ISR.

Various stressors such as viral infection lead to the suppression of cap-dependent translation and the activation of the integrated stress response (ISR), since the stress-induced phosphorylated eukaryotic translation initiation factor 2 [eIF2(αP)] tightly binds to eIF2B to prevent it from exchanging guanine nucleotide molecules on its substrate, unphosphorylated eIF2. Sandfly fever Sicilian virus (SFSV) evades this cap-dependent translation suppression through the interaction between its nonstructural protein NSs and host eIF2B. However, its precise mechanism has remained unclear. Here, our cryo-electron microscopy (cryo-EM) analysis reveals that SFSV NSs binds to the α-subunit of eIF2B in a competitive manner with eIF2(αP). Together with SFSV NSs, eIF2B retains nucleotide exchange activity even in the presence of eIF2(αP), in line with the cryo-EM structures of the eIF2B•SFSV NSs•unphosphorylated eIF2 complex. A genome-wide ribosome profiling analysis clarified that SFSV NSs expressed in cultured human cells attenuates the ISR triggered by thapsigargin, an endoplasmic reticulum stress inducer. Furthermore, SFSV NSs introduced in rat hippocampal neurons and human induced-pluripotent stem (iPS) cell-derived motor neurons exhibits neuroprotective effects against the ISR-inducing stress. Since ISR inhibition is beneficial in various neurological disease models, SFSV NSs may be a promising therapeutic ISR inhibitor.

eIF2B (eukaryotic initiation factor 2B) is a key regulator of translation initiation. It catalyzes guanine nucleotide exchange on eIF2, which delivers the methionylated initiator tRNA to the 40S ribosomal subunit. This exchange reaction is inhibited by the stress-induced phosphorylation of the eIF2 alpha subunit, which leads to global repression of cellular protein synthesis. eIF2B is composed of five subunits. The catalytic gamma/epsilon subcomplex is responsible for nucleotide exchange, while the regulatory alpha/beta/delta subcomplex discriminates the phosphorylation status of the eIF2 alpha subunit. We established a bacterial expression system for eIF2B, and determined its crystal structure at 3.2 Å resolution. The crystal structure revealed that eIF2B is a decamer containing two molecules of each subunit. The hexameric regulatory subcomplex is formed by the trimerization of one alpha-alpha homodimer unit and two beta-delta heterodimer units, and two catalytic subcomplexes are individually connected to the regulatory subcomplex through two beta-delta heterodimer units. Photo-cross-linking analyses showed that the N-terminal domain of the eIF2 alpha subunit, which bears the phosphorylation site, is recognized by a composite surface formed by the eIF2B alpha, beta, and delta subunits. Based on these results, we report structural insights into the interaction between eIF2 and eIF2B.

Eukaryotic translation initiation factor 2B (eIF2B), a heterodecameric complex of two sets of the a, b, c, d, and e subunits, is the guanine nucleotide exchange factor (GEF) specific for eIF2, a heterotrimeric G protein consisting of the a, b, and c subunits. The eIF2 protein binds GTP on the c subunits and delivers an initiator methionyl-tRNA (Met-tRNA i Met ) to the ribosome. The GEF activity of eIF2B is inhibited by stress-induced phosphorylation of Ser51 in the a subunit of eIF2, which leads to lower amounts of active eIF2 and a limited quantity of Met-tRNA i Met for the ribosome, resulting in global repression of translation. However, the structural mechanism of the GEF activity inhibition remained enigmatic, and therefore the three-dimensional structure of the entire eIF2B molecule had been awaited. Recently, we determined the crystal structure of Schizosaccharomyces pombe eIF2B. In this Structural Snapshot, we present the structural features of eIF2B and the mechanism underlying the GEF activity inhibition by the phosphorylation of eIF2a, elucidated from structurebased in vitro analyses.

Vanishing white matter disease (VWM) is an autosomal recessive neurological disorder caused by mutation(s) in any subunit of eukaryotic translation initiation factor 2B (eIF2B), an activator of translation initiation factor eIF2. VWM occurs with mutation of the genes encoding eIF2B subunits (EIF2B1, EIF2B2, EIF2B3, EIF2B4, and EIF2B5). However, little is known regarding the underlying pathogenetic mechanisms or how to treat patients with VWM. Here we describe the identification and detailed analysis of a new spontaneous mutant mouse harboring a point mutation in the Eif2b5 gene (p.Ile98Met). Homozygous Eif2b5I98M mutant mice exhibited a small body, abnormal gait, male and female infertility, epileptic seizures, and a shortened lifespan. Biochemical analyses indicated that the mutant eIF2B protein with the Eif2b5I98M mutation decreased guanine nucleotide exchange activity on eIF2, and the level of the endoplasmic reticulum stress marker activating transcription factor 4 was elevated in the 1‐month‐old Eif2b5I98M brain. Histological analyses indicated up‐regulated glial fibrillary acidic protein immunoreactivity in the astrocytes of the Eif2b5I98M forebrain and translocation of Bergmann glia in the Eif2b5I98M cerebellum, as well as increased mRNA expression of an endoplasmic reticulum stress marker, C/EBP homologous protein. Disruption of myelin and clustering of oligodendrocyte progenitor cells were also indicated in the white matter of the Eif2b5I98M spinal cord at 8 months old. Our data show that Eif2b5I98M mutants are a good model for understanding VWM pathogenesis and therapy development. Cover Image for this issue: doi: .