Binding of ISRIB reveals a regulatory site in the nucleotide exchange factor eIF2B

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Science  30 Mar 2018:
Vol. 359, Issue 6383, pp. 1533-1536
DOI: 10.1126/science.aar5129

ISRIB mechanism of action

In rodents, a druglike small molecule named ISRIB enhances cognition and reverses cognitive deficits after traumatic brain injury. ISRIB activates a protein complex called eIF2B that is required for the synthesis of new proteins. Tsai et al. report the visualization of eIF2B bound to ISRIB at near-atomic resolution by cryo–electron microscopy. Biochemical studies revealed that ISRIB is a “molecular staple” that promotes assembly of the fully active form of eIF2B. Zyryanova et al. report similar structures together with information on the binding of ISRIB analogs and their effects on protein translation.

Science, this issue p. eaaq0939, p. 1533


The integrated stress response (ISR) is a conserved translational and transcriptional program affecting metabolism, memory, and immunity. The ISR is mediated by stress-induced phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) that attenuates the guanine nucleotide exchange factor eIF2B. A chemical inhibitor of the ISR, ISRIB, reverses the attenuation of eIF2B by phosphorylated eIF2α, protecting mice from neurodegeneration and traumatic brain injury. We describe a 4.1-angstrom-resolution cryo–electron microscopy structure of human eIF2B with an ISRIB molecule bound at the interface between the β and δ regulatory subunits. Mutagenesis of residues lining this pocket altered the hierarchical cellular response to ISRIB analogs in vivo and ISRIB binding in vitro. Our findings point to a site in eIF2B that can be exploited by ISRIB to regulate translation.

The integrated stress response (ISR) has homeostatic functions that increase fitness. However, in some pathological circumstances, benefit arises from attenuated signaling in the ISR (1). A search for ISR inhibitors led to the discovery of the integrated stress response inhibitor, ISRIB (2), a small molecule efficacious in mouse models of neurodegeneration (3) and traumatic brain injury (4).

ISRIB action converges on eukaryotic translation initiation factor 2B (eIF2B), a protein complex with guanine nucleotide exchange factor (GEF) activity toward eIF2 (5) that is inhibited by phosphorylated eIF2 (6, 7). Addition of ISRIB accelerates eIF2B GEF activity in vitro, and targeting eIF2B’s δ regulatory subunit can impart ISRIB resistance (8, 9). However, known ISRIB-resistant mutations in eIF2B cluster at a distance from both the regulatory site engaged by eIF2(αP) and the catalytic site engaged by eIF2γ (10). Thus, although the bulk of the evidence suggests that ISRIB binds eIF2B to regulate its activity, indirect modes of action are not excluded.

ISRIB stabilized the eIF2B complex from HeLa cells (fig. S1), as expected (9). Thus, we added a fluorescently labeled derivative of ISRIB (AAA2-101) (fig. S2A) to purified eIF2B and observed increased fluorescence polarization (FP) (Fig. 1A, left panel). Unlabeled ISRIB competed for eIF2B in the FP assay with a half-maximal effective concentration (EC50) in the nanomolar range (Fig. 1A, right panel). As observed for ISRIB action in cells, less-active analogs competed less successfully (fig. S2, B and C).

Fig. 1 Biophysical and structural analysis of ISRIB binding to human eIF2B.

(A) Fluorescence polarization (FP) assays showing binding of ISRIB to human eIF2B. (Left) A plot of the FP signal arising from fluorescein-labeled ISRIB analog (AAA2-101) (2.5 nM) as a function of the concentration of eIF2B in the sample. (Right) A plot of the relative FP signal arising from samples with fluorescein-labeled AAA2-101 (2.5 nM) bound to purified human eIF2B (30 nM) in the presence of the indicated concentration of unlabeled trans-ISRIB introduced as a competitor. Concentrations of eIF2B and ISRIB on respective plots are represented on a log10 scale. Curve fitting and EC50 were generated using agonist versus response function on GraphPad Prism; shown are values of three independently acquired measurements. (B) Representative views of the cryo-EM map of the ISRIB-bound decameric human eIF2B complex. Density is colored according to the subunit architecture indicated in the cartoons: α, blue; β, cyan; δ, green; γ, gold; ε, pink; ISRIB, orange. (C) Ribbon representation of ISRIB-bound human eIF2B “central” view of the (βδ)2 dimer interface with a single molecule of ISRIB. (D) Close-up of the “central” view showing the ISRIB-binding site. An ISRIB molecule is docked into the cavity at the (βδ)2 dimer interface. Residues contacting ISRIB in the central part of the pocket from the β (blue) and δ (green) subunits are indicated. ISRIB is represented in orange sticks.

We purified endogenous eIF2B from HeLa cell lysates in the presence of ISRIB and determined the structure of the complex by single-particle cryo–electron microscopy (cryo-EM) at an overall resolution of 4.1 Å (Fig. 1B, figs. S1A and S3, and table S6). Within the β and δ regulatory core, protein side chains were clearly resolved, resulting in a near-complete atomic model of this region (Fig. 1C and fig. S4, A and B). The resolution of the γ and ε human catalytic subcomplex was lower compared with that of the regulatory core (fig. S4A), and the catalytically important C-terminal HEAT domain of the ε subunit remained unresolved in the cryo-EM map.

Owing to a lack of sufficient high-quality cryo-EM images of an apo-eIF2B complex, we were unable to calculate a difference map of eIF2B with and without ISRIB. However, a nearly continuous density with a shape and size of a single ISRIB molecule was conspicuously present at the interface of the β and δ regulatory subunits (Fig. 1B, “central view,” and fig. S4C). The ISRIB-binding pocket was located at the plane of symmetry between the β and δ subunits. In the central part of the pocket, the side chain of βH188 was positioned in the vicinity of the essential carbonyl moiety of ISRIB (11), and βN162 was poised to stabilize the diaminocyclohexane moiety of ISRIB through hydrogen-bonding interactions (Fig. 1D). More distally, the side chains of δL179, δF452, δL485, δL487, βV164, βI190, βT215, and βM217 formed the hydrophobic end of the symmetrical pocket that accommodated the aryl groups of ISRIB, with βI190 and δL179 located within van der Waals interaction distance to the aryl group (fig. S4D). A hamster Eif2b4L180F mutation (δL179 in the human eIF2B) disrupts ISRIB action in cells (8), which is consistent with a potential loss of these interactions as well as a clash between the bulkier side chain of phenylalanine and the bound ISRIB molecule (Fig. 1D).

Overall, the human structure is highly similar to that of the published Schizosaccharomyces pombe eIF2B structure (10) (root mean square deviation of 2.57 Å over 3049 α carbons, fig. S4E). However, there is no density in the corresponding region in the S. pombe eIF2B map, indicating that the density found in the human structure was that of the bound ISRIB.

To test these features of ISRIB binding, we used CRISPR-Cas9 to randomize residues lining the ISRIB-binding pocket (Eif2b2N162, Eif2b2H188, or Eif2b2I190) and correlated amino acid substitutions to ISRIB activity in the mutagenized cells. Histidinol, an agent that activates the eIF2α kinase GCN2 and induces the ISR, normally activates a CHOP::GFP (green fluorescent protein) reporter gene, whereas ISRIB represses the reporter (8). Fluorescence-activated cell sorting (FACS) of histidinol-treated, mutagenized cells segregated them into ISRIB-sensitive [ISRIBSEN (CHOP::GFP inhibited)] and ISRIB-resistant [ISRIBRES (CHOP::GFP activated)] classes (Fig. 2A, left and right panels, respectively).

Fig. 2 Structure-directed chemogenetic analysis of ISRIB and its analogs’ binding to eIF2B.

(A) Histograms of the ISR-responsive CHOP::GFP fluorescent reporter activity, induced by histidinol (HIS+, 0.5 mM) in ISRIB-sensitive (ISRIBSEN) (left panels) and ISRIB-resistant (ISRIBRES) (right panels) pools of CHO-K1 cells, selected for their responsiveness to ISRIB (200 nM), following CRISPR-Cas9–induced random mutagenesis of the indicated codon of Eif2b2. DMSO, dimethyl sulfoxide. (B) Bar graph of the distribution of residues identified at the indicated positions of mutagenized Eif2b2, analyzed by the next-generation sequencing. Shown is the number of sequenced reads in ISRIBSEN pools (orange bars) or ISRIBRES pools (purple bars) encoding each amino acid (*, stop codon; X, ambiguous sequence). Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr. (C) Graphs showing inhibition of the ISR-activated CHOP::GFP reporter [induced as in (A)] by ISRIB or two related analogs, compound AAA1-075B (075B) and compound AAA1-084 (084), in ISRIBSEN (left) and ISRIBRES (right) mutant pools of Eif2b2H188X. Shown is a representative from three independent experiments for each of the compounds. Concentration of inhibitor is represented on a log10 scale. Curve fitting and EC50 were generated using agonist versus response function on GraphPad Prism.

To determine if the phenotypically distinguished pools of mutagenized cells (Fig. 2A) were enriched in different mutations, we subjected genomic DNA derived from each population to deep--sequencing analysis (Fig. 2B and table S4). The ISRIBRES pool targeted at Eif2b2H188 diverged markedly from the parental sequence (Fig. 2B, middle panel). Of a total of 250,617 sequencing reads, histidine was present in only 6443 (2.6%), with arginine, glycine, leucine, lysine, and glutamine dominating (24, 21, 18, 8.2, and 6.2%, respectively). Histidine was preserved in the ISRIBSEN pool (269,253 of 328,113 reads, 82%). The ISRIBRES pool of cells targeted at Eif2b2I190 was dominated by tryptophan, methionine, and tyrosine (28, 24, and 15%, respectively), consistent with a role for these bulky side chains in occluding the ISRIB-binding pocket (Fig. 2B, bottom panel). Mutagenesis of Eif2b2N162 was less successful in generating a pool of strongly ISRIB-resistant cells; nonetheless, threonine was enriched in the ISRIBRES pool (26%) (Fig. 2B, top panel). Thus, residues lining the ISRIB-binding pocket play a role in ISRIB action.

Despite considerable allele diversity, the Eif2b2H188X ISRIBRES pool exhibited selective loss of sensitivity to ISRIB, while retaining a measure of responsiveness to certain ISRIB analogs (Fig. 2D). The residual response to analogs AAA1-075B (075B) and AAA1-084 (084) (albeit with much reduced affinity compared to the ISRIBSEN population) (Fig. 2C), pointed to a shift in the binding properties of the ISRIB pocket, induced by the mutations at β188.

Next, we exploited the diversity of ISRIBRES mutations in the Eif2b2H188X population to select for subpools that either acquired sensitivity to compounds AAA1-075B or AAA1-084, regained sensitivity to ISRIB, or retained ISRIB resistance (Fig. 3A) and sequenced their Eif2b2 alleles (Fig. 3B and table S5). As expected, sorting for ISRIB sensitivity enriched, by >20-fold, rare wild-type H188 alleles that persisted in the ISRIBRES Eif2b2H188X pool (Fig. 3B, compare purple and orange bars). H188 was also somewhat enriched in the pools of 075BSEN or 084SEN cells, but unlike the ISRIBSEN, these pools were also enriched for residues other than histidine (Fig. 3B and table S5). Notably, selecting for sensitivity to these ISRIB analogs enriched for different residues than those found in the original ISRIBRES pool: Arginine, glycine, and leucine were depleted and replaced by lysine, serine, alanine, and threonine (Fig. 3B, compare purple to blue and cyan bars). The correlation between mutations in residues lining the ISRIB-binding pocket and selective responsiveness to ISRIB analogs, observed in the pools, was confirmed in individual mutant clones (fig. S5).

Fig. 3 Sensitivity to ISRIB analogs selects for a divergent palette of mutations in codon 188 of Eif2b2.

(A) Histograms of the ISR-responsive CHOP::GFP fluorescent reporter activity, induced by histidinol (HIS+, 0.5 mM) in ISRIBRES, ISRIBSEN, compound 075BSEN, and compound 084SEN subpools, selected for their responsiveness to ISRIB or its analogs (2.5 μM) from a population of originally ISRIBRES Eif2b2H188X mutant cells. (B) Bar graph of the distribution of residues identified at Eif2b2 codon 188 in phenotypically divergent pools of CHO-K1 cells. The number of sequenced reads in ISRIBRES (purple), ISRIBSEN (orange), compound 075BSEN (blue), and compound 084SEN (cyan) pools encoding each amino acid (*, stop codon; X, ambiguous) is plotted. (C) Plot of the relative FP signal arising from samples with fluorescein-labeled AAA2-101 (2.5 nM) bound to purified hamster eIF2B (30 nM) in the presence of the indicated concentration of unlabeled ISRIB introduced as a competitor (represented on a log10 scale). Shown is a representative of two independent experiments. The fitting curve and EC50 were generated using “agonist versus response” function on GraphPad Prism. (D) A plot of the FP signal arising from fluorescein-labeled AAA2-101 (2.5 nM) as a function of the concentration of wild-type (wt) or mutant eIF2B (βH188K or δL180F) in the sample. Shown are mean ± SD (n = 3). Concentrations of eIF2B are represented on a log10 scale.

To directly address the effect of ISRIB-resistant mutations on ISRIB binding, we purified eIF2B from wild-type, Eif2b4L180F, and Eif2b2H188K Chinese hamster ovary (CHO) cells (fig. S6). The wild-type eIF2B gave rise to a concentration-dependent FP signal in the presence of a fluorescein-labeled AAA2-101 that was readily competed with unlabeled ISRIB (Fig. 3C). However, eIF2B purified from the mutant cells failed to give rise to an FP signal (Fig. 3D), thereby establishing a correlation between ISRIB resistance in cells and defective ISRIB binding in vitro.

The ISRIB-binding pocket, defined structurally and validated chemogenetically, straddles the twofold axis of symmetry of the core regulatory subcomplex, and a single molecule of ISRIB appears to engage the same residues from opposing protomers of the (βδ)2 dimer. These features fit with ISRIB’s own symmetry and could explain the ability of ISRIB to stabilize the eIF2B decamer, possibly increasing its abundance in ISRIB-treated cells. Our findings are also consistent with ISRIB’s ability to stabilize a rate-limiting assembly intermediate of the active decamer, as demonstrated biochemically in an accompanying manuscript (12). Indeed, comparison of the S. pombe (10) and ISRIB-bound human eIF2B argues against large domain movements associated with ISRIB binding. However, an important allosteric effect of ISRIB binding might easily have been overlooked, as the critical catalytic domain of the ε subunit is resolved in neither structure. Similar considerations apply to the potential effect of ISRIB on the inhibitory interaction between eIF2B with eIF2(αP). These might arise from subtle ISRIB-induced conformational changes propagated through the regulatory core to the eIF2(αP) binding cavity formed by the convergence of the tips of the α, β, and δ subunits (10, 1315). The lower resolution of the cryo-EM density in that region might have masked important allosteric changes. Although the relative contribution of accelerated assembly, enhanced stability, or allostery to ISRIB action remain to be resolved, it is intriguing to consider that endogenous ligands might engage the ISRIB-binding site to regulate eIF2B in yet-to-be-determined physiological states.

Supplementary Materials

Materials and Methods

Supplementary Text

Schemes S1 to S4

Figs. S1 to S6

Tables S1 to S6

References (1633)

References and Notes

Acknowledgments: We thank D. Barrett and C. Ortori (University of Nottingham) for measuring the ISRIB content of eIF2B; P. Sterk (University of Cambridge) for next-generation sequencing analysis; S. Chen, C. Savva, G. McMullan, T. Darling, and J. Grimmett (MRC Laboratory for Molecular Biology) for technical support with cryo-EM, movie data acquisition, and help with computing; S. Preissler (University of Cambridge) for editorial comments; and Diamond for access and support of A. Siebert and C. Hecksel from the cryo-EM facilities at the UK National Electron Bio-Imaging Centre (eBIC) (proposals EM-14606 and EM-17057), funded by the Wellcome Trust, MRC, and Biotechnology and Biological Sciences Research Council. Funding: We acknowledge funding by a Wellcome Trust Principal Research Fellowship (Wellcome 200848/Z/16/Z) to D.R.; a Specialist Programme from Bloodwise (12048), the UK Medical Research Council (MC_U105161083), and core support from the Wellcome Trust Medical Research Council Cambridge Stem Cell Institute to A.J.W.; and a Wellcome Trust Strategic Award to the Cambridge Institute for Medical Research (Wellcome 100140). Support from A Higher Committee for Education Development, Iraq, Scholarship (4241047) to A.A.A. and a Research Fellowship from Royal Commission for the Exhibition of 1851 to F.A. is greatly appreciated. Author contributions: A.F.Z. and D.R. designed and implemented the study and wrote the manuscript. F.W., A.F., and A.J.W. obtained and interpreted the structural data and edited the manuscript. A.A.A., C.F., and P.M.F. designed and synthesized compounds, interpreted the chemogenetic data, and edited the manuscript. A.C.-C., Y.S., and H.P.H. designed and implemented the somatic cell genetics, cell phenotyping, and deep sequencing and edited the manuscript. F.A. and L.P. analyzed NGS sequencing data and edited the manuscript. Competing interests: None declared. Data and materials availability: Cryo-EM density maps are deposited in the Electron Microscopy Data Bank (EMD-4162), atomic coordinates are deposited in the Protein Data Bank (6EZO), and raw sequencing reads are deposited in the BioProject database (PRJNA432684).
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