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

Abstract

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.

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

(A) Fluorescence polarization (FP) assays showing binding of ISRIB to human eIF2B. Left panel: 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 panel: 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 was generated using agonist vs. 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.

Figure 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 (ISRIB^SEN) (left panels) and ISRIB-resistant (ISRIB^RES) (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. (B) Bar graph of the distribution of residues identified at the indicated positions of mutagenized Eif2b2, analyzed by the next generation sequencing (NGS). Shown is the number of sequenced reads in ISRIB^SEN pools (orange bars) or ISRIB^RES pools (plum bars) encoding each amino acid (* - stop codon, X – ambiguous sequence). (C) Graphs showing inhibition of the ISR-activated CHOP::GFP reporter (induced as in panel “A”) by ISRIB or two related analogs, compound AAA1-075B (075B) and compound AAA1-084 (084), in ISRIB^SEN (left) and ISRIB^RES (right) mutant pools of EIf2b2^H188X. 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 was generated using agonist vs. response function on GraphPad Prism.

Figure 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 ISRIB^RES, ISRIB^SEN, compound 075B^SEN and compound 084^SEN sub-pools, selected for their responsiveness to ISRIB or its analogs (2.5 µM) from a population of originally ISRIB^RES Eif2b2^H188X 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 ISRIB^RES (plum), ISRIB^SEN (orange), compound 075B^SEN (blue) and compound 084^SEN (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 was generated using “agonist vs. 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 wildtype (wt) or mutant eIF2B (δ^L180F or β^H188K) in the sample. Shown are mean ± SD (n=3). Concentrations of eIF2B are represented on a log₁₀ scale.