Purification and assembly of human eIF2B subcomplexes

Human WT eIFBα2 (pJT075), eIFBαD298A2 (pRL055), WT eIF2Bβγδε (pJT073 and pJT074 coexpression), eIF2Bβγδε δL516A (pRL036 and pJT074 coexpression), eIF2BβγδF443Aε (pRL035 and pJT074 coexpression) and eIF2BβγδE445Aε (pRL049 and pJT074 coexpression) were purified as previously described21.

Purification of viral NSs protein

Viral NSs::6×His was purified as previously described32. Briefly, we used the pMS113 construct to express and purify NSs::6×His. Expi293T cells (Thermo Fisher) were transfected with the NSs construct, as per the manufacturer’s instructions, for the MaxTiter protocol and were collected 5 d after transfection. Cells were pelleted (1,000g, 4 min) and resuspended in lysis buffer (130 mM KCl, 2.5 mM MgCl2, 25 mM HEPES-KOH (pH 7.4), 2 mM EGTA, 1% Triton X-100, 1 mM TCEP and 1× cOmplete protease inhibitor cocktail (Roche)). Cells were then incubated for 30 min at 4 °C and spun at 30,000g for 1 h to pellet cell debris. The lysate was applied to a 5-ml HisTrap HP column (GE Healthcare) equilibrated in Buffer A (20 mM HEPES-KOH (pH 7.5), 200 mM KCl, 5 mM MgCl2 and 15 mM imidazole) and eluted using a gradient of Buffer B (20 mM HEPES-KOH (pH 7.5), 200 mM KCl, 5 mM MgCl2 and 300 mM imidazole). NSs::6×His was concentrated using a 10-kDa molecular weight cutoff spin concentrator (Amicon) and further purified by size-exclusion chromatography over a Superdex 200 Increase 10/300 GL column (GE Healthcare) in elution buffer (20 mM HEPES (pH 7.5), 200 mM KCl, 5 mM MgCl2, 1 mM TCEP and 5% glycerol). The resulting fractions were pooled and flash-frozen in liquid nitrogen.

Purification of heterotrimeric human eIF2

All experiments performed before 1 March 2022 were performed using human eIF2 purified as previously described33. Some experiments were performed using purified eIF2 that was a generous gift from Calico Life Sciences. All experiments performed after 1 March 2022 used eIF2 purified according to a second previously described protocol19.

Phosphorylation of eIF2 trimer (eIF2-P) and eIF2α (eIF2α-P)

To generate phosphorylated eIF2 trimers or eIF2α, 25 µM eIF2 trimer or eIF2α was incubated with 500 nM recombinant PERK kinase domain (purified in-house as previously described18) and 1 mM ATP at 37 °C for 1 h. Phosphorylation of the final product was verified by 12.5% SuperSep PhosTag gels (Wako Chemical Corporation).

Assembly of eIF2B decamer complexes

All eIF2B(αβγδε)2 used throughout was assembled by mixing purified eIF2Bβγδε and eIF2Bα2 at a molar ratio of 2:1.2 eIF2Bβγδε:eIF2Bα2 unless otherwise indicated. Complexes were assembled at 10 µM and incubated at 4 °C for 30 min before dilution to experimental conditions.

Assembly of eIF2B complexes for hydrogen–deuterium exchange–mass spectrometry characterization

For HDX–MS experiments, after preparation of 10 µM eIF2B(αβγδε)2, 10× mixtures containing 5 µM eIF2B(αβγδε)2 and 12 µM NSs or eIF2-P (representing a 1.2-fold molar excess of NSs and eIF2-P relative to available eIF2B binding sites) were prepared in HDX buffer and allowed to assemble overnight at 4 °C. The tetramer sample was prepared by diluting eIF2Bβγδε to 10 µM. The 10× 2BAct samples were prepared by combining 5 µM eIF2B with 10 µM 2BAct dissolved in DMSO. Matched 10× control samples were prepared by combining 5 µM eIF2B with 2% DMSO such that the final experimental DMSO concentration was 0.2%.

Hydrogen–deuterium exchange

For all HDX experiments, deuterated buffer was prepared by lyophilizing eIF2B assay buffer (20 mM HEPES, 200 mM KCl, 5 mM MgCl2 and 1 mM TCEP, pH 7.9) and resuspending it in deuterium oxide (Sigma-Aldrich, 151882). To initiate the continuous-labeling experiment, samples were diluted tenfold to 1× (final eIF2B tetramer concentration of 1 µM or a final eIF2B decamer concentration of 0.5 µM) into temperature-equilibrated deuterated eIF2B assay buffer. Samples were quenched at the time points outlined below by mixing 30 μl of the partially exchanged protein with 30 μl of 2× quench buffer (6 M urea and 500 mM TCEP, pH 2.4) on ice. Samples were incubated on ice for 1 min to allow for partial unfolding to assist with proteolytic degradation and were then flash-frozen in liquid nitrogen and stored at −80 °C. The HDX time points for these experiments were 10 s, 100 s, 15 min and 3 h.

Protease digestion and liquid chromatography–mass spectrometry

All samples were thawed immediately before injection into a cooled valve system (Trajan LEAP) coupled to an LC (Thermo UltiMate 3000). Sample time points were injected in random order. The temperature of the valve chamber, trap column and analytical column was maintained at 2 °C. The temperature of the protease column was maintained at 10 °C. The quenched sample was subjected to in-line digestion by two immobilized acid proteases in order, aspergillopepsin (Sigma-Aldrich, P2143) and porcine pepsin (Sigma-Aldrich, P6887), at a flow rate of 200 μl min–1 of Buffer A (0.1% formic acid). Protease columns were prepared in-house by coupling protease to beads (Thermo Scientific POROS 20 AL aldehyde activated resin, 1602906) and packed by hand into a column (2 mm (inner diameter) × 2 cm, IDEX C-130B). Following digestion, peptides were desalted for 4 min on a hand-packed trap column (Thermo Scientific POROS R2 reversed-phase resin, 1112906, 1 mm (inner diameter) × 2 cm, IDEX C-128). Peptides were then separated with a C8 analytical column (Thermo Scientific BioBasic-8, 5-μm particle size, 0.5 mm (inner diameter) × 50 mm, 72205-050565) and a gradient of 5–40% Buffer B (100% acetonitrile and 0.1% formic acid) at a flow rate of 40 μl min–1 over 14 min and then a gradient of 40–90% Buffer B over 30 s. The analytical and trap columns were then subjected to a sawtooth wash and equilibrated at 5% Buffer B before the next injection. Protease columns were washed with two injections of 100 μl of 1.6 M guanidinium chloride and 0.1% formic acid before the next injection. Peptides were eluted directly into a Q Exactive Orbitrap mass spectrometer operating in positive mode (resolution of 70,000, automatic gain control target of 3 × 106, maximum injection time of 50 ms and scan range of 300–1,500 m/z). For each eIF2B condition, a tandem MS experiment was performed (full MS settings were the same as described above, and data-dependent MS2 settings included a resolution of 17,500, automatic gain control target of 2 × 105, maximum injection time of 100 ms, loop count of 10, isolation window of 2.0 m/z, normalized collision energy of 28, charge state of 1 and ≥7 excluded and dynamic exclusion of 15 s) on undeuterated samples. LC and MS methods were run using Xcalibur 4.1 (Thermo Scientific).

Peptide identification

Byonic (Protein Metrics) was used to identify peptides in the tandem MS data. Sample digestion parameters were set to nonspecific. Precursor mass tolerance and fragment mass tolerance were set to 6 and 10 ppm, respectively. Peptide lists (sequence, charge state and retention time) were exported from Byonic and imported into HDExaminer 3 (Sierra Analytics). When multiple peptide lists were obtained, all were imported and combined in HDExaminer 3.

HDExaminer 3 analysis

Peptide isotope distributions at each exchange time point were fit in HDExaminer 3. Deuteration levels were determined by subtracting mass centroids of deuterated peptides from undeuterated peptides. All peptides we monitored showed EX2 behavior.

Hydrogen–deuterium exchange data presentation

We define a notable increase or decrease of deuteration as at least three peptides with a change in number of deuterons of >0.5. This was determined after measuring the average noise in our dataset; for biological replicates run on the same day on the instrument, the average standard deviation for the number of deuterons taken up was 0.075. We set a conservative threshold of at least three peptides over 0.5 change in number of deuterons, which represents 6.7 standard deviations.

BODIPY-GDP exchange assay

In vitro detection of GDP binding to eIF2 was adapted from a published protocol for a fluorescence intensity-based assay describing dissociation of eIF2 and nucleotide34. We first performed a loading assay for fluorescent BODIPY-FL-GDP as previously described21. Purified eIF2 (137.5 nM) was incubated with 100 nM BODIPY-FL-GDP (Thermo Fisher Scientific) in assay buffer (100 mM HEPES-KOH (pH 7.5), 100 mM KCl, 5 mM MgCl2, 1 mM TCEP and 1 mg ml–1 bovine serum albumin) to a volume of 500 µl in a black-walled 1.5-ml tube. This mix was then added to 384-square-well, black-walled, clear-bottom polystyrene assay plates (Corning, 3766) with 18 µl per well. A GEF mix composed of a 10× solution of eIF2B(αβγδε)2 was prepared. To compare nucleotide exchange rates, 2 µl of the 10× GEF mixes was spiked into the 384-well plate wells with a multichannel pipette, such that the resulting final concentration of eIF2B(αβγδε)2 was 5 nM, and the final concentrations of other proteins and drugs are as indicated in the figures. Subsequently, in the same wells, we performed a ‘GDP unloading assay’ as indicated in the figures. After completion of the loading reaction, wells were spiked with 1 mM GDP to start the unloading reaction at t = 0. In the case of inhibition assays with eIF2-P, the eIF2/BODIPY-GDP mix was also incubated with 25 nM eIF2B(αβγδε)2, and a 10× mix of eIF2-P was spiked into the wells so that the final concentration was 50 nM. For all GEF assays involving eIF2-P, an ‘unloading’ assay was used because the eIF2B(αβγδε)2 had been preincubated with eIF2. Fluorescence intensity was recorded every 10 s for 60 min at 25 °C using a Clariostar Plus (BMG LabTech) plate reader (excitation wavelength of 497 nm and bandwidth of 14 nm; emission wavelength of 525 nm and bandwidth of 30 nm). Data collected were fit to a first-order exponential.

FAM-ISRIB binding assay

All fluorescence polarization measurements were performed in 20-μl reactions with 100 nM eIF2B(αβγδε)2 + 2.5 nM FAM-ISRIB (Praxis Bioresearch) in FP buffer (20 mM HEPES-KOH (pH 7.5), 100 mM KCl, 5 mM MgCl2 and 1 mM TCEP) and measured in 384-well, non-stick black plates (Corning, 3820) using a ClarioStar Plus (BMG LabTech) plate reader at room temperature. Before the reaction setup, eIF2B(αβγδε)2 was assembled in FP buffer using eIF2Bβγδε and eIF2Bα2 in a 2:1.5 molar ratio for at least 1 h at 4 °C. FAM-ISRIB was always first diluted to 2.5 μM in 100% NMP before dilution to 50 nM in 2% NMP and then added to the reaction. For titrations with eIF2α and eIF2α-P, dilutions were made in FP buffer. For titrations with ISRIB, ISRIB was first diluted with 100% NMP to 2.5 µM and then to the final concentrations in 4% NMP. The reactions with eIF2B, FAM-ISRIB and the dilutions were incubated at 25 °C for 30 min before measurement of parallel and perpendicular intensities (excitation of 482 nm and emission of 530 nm).

FAM-ISRIB kinetic binding assay

The kinetic characterization of FAM-ISRIB binding during eIF2α phosphorylation was assayed in 18-μl reactions of 100 nM eIF2B(αβγδε)2, 2.5 nM FAM-ISRIB, 100 μM ATP and 5.6 μM eIF2α/eIF2α-P in FP buffer. These solutions were preincubated at 22 °C for 30 min before polarization was measured every 15 s (30 flashes per s). After four cycles, 2 μl of homemade PERK kinase domain at 0.1 mg ml–1 was added for a final concentration of 10 μg ml–1 in the reaction, and measurement was resumed for 1 h.

Structural measurements

Measurement of rotation of the switch-helix between the A-state and I-state was performed by comparing the position of the first Cα away from the amide backbone of PDB 6O81 (eIF2-bound eIF2B) to that of PDB 6O9Z (eIF2α-P-bound eIF2B). For global rotational changes in Fig. 4, the hinge movement between the two eIF2B halves was measured between the lines connecting eIF2Bε H352 and P439.

Analytical ultracentrifugation

Analytical ultracentrifugation sedimentation velocity experiments were performed as previously described using the ProteomeLab XL-I system (Beckman Coulter)21. Briefly, samples were loaded into cells in a buffer consisting of 20 mM HEPES-KOH (pH 7.5), 150 mM KCl, 1 mM TCEP and 5 mM MgCl2. A buffer-only reference control was also loaded. Samples were then centrifuged in an AN-50 Ti rotor at 40,000 r.p.m. at 20 °C, and 280-nm absorbance was monitored. Subsequent data analysis was conducted with Sedfit using a non-model-based continuous c(S) distribution.

Sample preparation for cryo-electron microscopy

Decameric eIF2Bδ δL516A was prepared by incubating 20 μM eIF2BδL516A βγδε with 11 µM eIF2Bα2 in a final solution containing 20 mM HEPES-KOH, 200 mM KCl, 5 mM MgCl2 and 1 mM TCEP. eIF2Bδ δL516A decamers and eIF2B tetramers were diluted to 750 nM in 20 mM HEPES-KOH, 200 mM KCl, 5 mM MgCl2 and 1 mM TCEP before grid application. For grid freezing, a 3-μl aliquot of the sample was applied to a Quantifoil R1.2/1/3 400-mesh gold grid, followed by a 30-s waiting period. A 0.5-μl aliquot of 0.1–0.2% Nonidet P-40 substitute was added immediately before blotting. The entire blotting procedure was performed using Vitrobot (FEI) at 10 °C and 100% humidity.

Electron microscopy data collection

Cryo-EM data were collected on a Titan Krios transmission electron microscope operating at 300 keV. Micrographs were acquired using a Gatan K3 direct electron detector. The total dose was 67 e– Å–2, and 117 frames were recorded during a 5.9-s exposure. Data were collected at 105,000× nominal magnification (0.835 Å per pixel at the specimen level), with a nominal defocus range of −0.6 to −2.0 μm.

Image processing

The micrograph frames were aligned using MotionCor2 (ref. 35). The contrast transfer function (CTF) parameters were estimated with GCTF36. For the decameric eIF2Bδ δL516A, particles were picked in Cryosparc v3.3.2 using apo eIF2B (Electron Microscopy Data Bank (EMDB) EMD-23209) as a template14,37. Particles were extracted using an 128-pixel box size and were classified in 2D. Classes that showed clear protein features were selected and extracted for heterogeneous refinement using models of an apo decamer, a tetramer and an impurity class, followed by homogenous refinement. Particles belonging to the decamer class were then reclassified using heterogeneous refinement to sort the best resolution class. Particles from the resulting best class were then reextracted with a pixel size of 0.835 Å and subjected to nonuniform refinement, yielding a reconstruction of 3.0 Å. These particles were subjected to CTF refinement to correct for the per-particle CTF and beam tilt. A final round of nonuniform refinement yielded the final structure of 2.9 Å.

For the tetramer structure, particles were picked by Gautomatch and extracted at a pixel size of 3.34 Å per pixel. Particles were imported into Relion 3.0 for autorefinement to generate a consensus structure. These particles were then subjected to multiple rounds of 2D classification, where particles that represent proteins were selected and reextracted. The resulting set of particles were subjected to autorefinement, followed by reextraction at 1.67 Å per pixel and another round of autorefinement, yielding a reconstruction at 4.5 Å. These particles were then subjected to 3D classification (k = 4), and the best class was selected for further refinement, which generated a 4.2-Å reconstruction. Particles belonging to this set (~72,000) were extracted at 0.835 Å per pixel and subjected to autorefinement, yielding a 3.9-Å structure. Particles belonging to this class were imported into Cryosparc v3.3.2, where they were subjected to nonuniform refinement and CTF refinement to yield the final structure at a resolution of 3.1 Å.

Atomic model building, refinement and visualization

For both the decamer and the tetramer structures, the previously published apo eIF2B model (PDB 7L70) was used as a starting model14. Each subunit was docked into the EM density individually and subjected to rigid body refinement in Phenix38. The models were then manually adjusted in Coot and refined in phenix.real_space_refine using global minimization, secondary structure restraints, Ramachandran restraints and local grid search39. Iterative cycles of manual rebuilding in Coot and phenix.real_space_refine were then performed. The final model statistics were tabulated using Molprobity40. Molecular graphics and analyses were performed with the University of California San Francisco (UCSF) Chimera package. UCSF Chimera is developed by the Resource for Biocomputing, Visualization and Informatics and is supported by NIGMS P41-GM103311. The atomic model is deposited at PDB under the accession codes 8TQZ (eIF2Bδ δL516A) and 8TQO (tetramer). The EM map is deposited at EMDB under the accession codes EMD-41566 (eIF2Bδ δL516A) and EMD-41510 (tetramer).

Generation of endogenously edited cells

Editing of the Eif2b1 and Eif2b4 genes to introduce D298A and E446A mutations, respectively, into mouse AN3-12 pseudohaploid embryonic stem cells obtained from the Austrian Haplobank (https://haplobank.org) was performed using nucleofection of CRISPR–Cas9 ribonucleoproteins, as previously described (https://www.protocols.io/view/cas9-sgrna-ribonucleoprotein-nucleofection-using-l-261ge1xyv479/v10) using single guide RNAs and single-stranded homology-directed repair templates listed in Supplementary Table 4. Nucleofection was performed using a 4D-Nucleofector with X-unit attachment (Lonza) and with pulse program CG-104. Two days after nucleofection, genomic DNA was extracted using a PureLink Genomic DNA mini kit from a portion of cells, relevant genes were PCR amplified, and editing efficiency was determined using the Synthego ICE tool (https://ice.synthego.com/#/). When editing efficiency was confirmed to be at least 10%, cells were diluted to an expected density of 0.0625 cells per well, plated onto 96-well plates and allowed to grow up from single colonies, with media changes every 3–5 d depending on the media acidification rate. Genomic DNA was extracted from colonies derived from single clones, and successful editing was determined by PCR amplification of the gene of interest and analysis using the Synthego ICE tool. All cell lines were negative for Mycoplasma contamination.

Western blotting

Cells were seeded at 1 × 106 cells per well of a six-well plate and grown at 37 °C and 5% CO2 for 24 h. Cells were treated using the indicated Tg concentrations (Invitrogen) for 1 h, ensuring that the final DMSO concentration was 0.1% across all conditions. Plates were put on ice, and cells were washed once with ice-cold PBS and lysed in 200 μl of ice-cold lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% (vol/vol) Triton X-100, 10% (vol/vol) glycerol, 1× cOmplete protease inhibitor cocktail (Roche) and 1× PhosSTOP (Roche)). Cells were scraped, collected in an Eppendorf tube and rotated for 30 min at 4 °C. Debris was pelleted at 12,000g for 10 min at 4 °C, and the supernatant was removed to a new tube on ice. Protein concentration was normalized to 15 µg of total protein per lane using Bio-Rad Protein Assay Dye. A 5× Laemmli loading buffer (250 mM Tris-HCl (pH 6.8), 30% glycerol, 0.25% bromophenol blue, 10% SDS and 5% β-mercaptoethanol) was added to each sample to 1×, and samples were denatured at 95 °C for 5 min and spun down. Wells of AnyKd Mini-Protean TGX precast protein gels (Bio-Rad) were loaded with equal amounts of total protein in between Precision Plus Dual Color protein ladder (Bio-Rad). After electrophoresis, proteins were transferred onto a nitrocellulose membrane and blocked for 2 h at room temperature in PBS with 0.1% Tween + 3% milk (blocking buffer) while rocking. Primary antibody staining was performed with gentle agitation at 4 °C overnight using the conditions outlined in Supplementary Table 3. After washing four times in appropriate blocking buffer, secondary antibody staining was performed for 1 h at room temperature using anti-rabbit horseradish peroxidase or anti-mouse horseradish peroxidase (Promega, 1:10,000) in blocking buffer. Membranes were washed three times in blocking buffer and then one time in PBS with 0.1% Tween without milk. Membranes were incubated with SuperSignal West Dura or Femto (Thermo Fisher Scientific) for 5 min. Membranes were imaged on a LI-COR Odyssey gel imager for 0.5–10 min depending on band intensity.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.