Structural basis for eIF2B inhibition in integrated stress response

Abstract

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

Structural differences in translation initiation between pathogenic trypanosomatids and their mammalian hosts

“…Noteworthy, the eIF5-CTD shares a common topology with the CTD of the ε subunit of the nucleotide exchange factor eIF2B (16); they both fold into a W2-type HEAT domain (27) mediating contacts of both factors with the eIF2β-NTT and eIF2γ (31). Based on our structure, the arrangement of the eIF5-CTD HEAT domain binding site on eIF2γ in the context of the 43S PIC is similar to that of the eIF2Bε-CTD HEAT domain in the context of the recently solved eIF2-eIF2B complex (32,33).…”

eIF3b and eIF3i relocate together to the ribosomal subunit interface during translation initiation and modulate start codon selection

“…Finally, there is an unassigned density on top of the β-propeller of eIF3b in contact with eIF2γ, which is more prominently seen in py48S-closed-eIF3 ( Figures 1A, 1D, 2C, 2F, 3A, 3B, 3E and based on its size and proximity to eIF3i or to the C-terminal domain of eIF5 based on the contacts of eIF2γ with the structurally homologous heat domain of eIF2Bε (Kashiwagi et al, 2019;Kenner et al, 2019); although further studies are required to identify it with confidence.…”

Structural insights into ISRIB, a memory‐enhancing inhibitor of the integrated stress response

“…Structures of eIF2B bound to eIF2 [17][18][19][20] revealed that multiple eIF2B subunits mediate binding of the eIF2 heterotrimer. In humans, eIF2a binds across the decamer symmetry interface, bridging the eIF2Bb and eIF2Bd subunits [17,18]. Mutational analyses based on these structures demonstrated that the composite binding surface present in the full eIF2B decamer substantially enhances recruitment of eIF2a.…”