Regulation by the integrated stress response (ISR) converges on the phosphorylation of translation initiation factor eIF2 in response to a variety of stresses. Phosphorylation converts eIF2 from a substrate to a competitive inhibitor of its dedicated guanine nucleotide exchange factor, eIF2B, inhibiting translation. ISRIB is a drug-like eIF2B activator that reverses the effects of eIF2 phosphorylation, enhances cognition, and corrects cognitive deficits after brain injury in rodents. Because ISRIB shows promise for treating neurological disorders a deeper understanding of its mechanism of action is crucial. Previous work identified eIF2B as a target of ISRIB and suggested that the molecule stabilizes and activates the enzyme. However, the molecule’s mode of binding and means of activation remain unknown.

To identify the binding site and mechanism of action of ISRIB, we used cryo–electron microscopy (cryo-EM) to determine an atomic-resolution structure of decameric human eIF2B bound to ISRIB. We validated the structural model using mutational analysis and the synthesis of ISRIB analogs. Combined with pre–steady-state kinetic analysis of eIF2B complex assembly, these findings enabled us to derive a functional model of ISRIB action.

A robust recombinant expression and purification protocol for all subunits of human eIF2B produced a stable eIF2B holoenzyme that sedimented as a decamer. Under conditions of elevated ionic strength, an eIF2Bα dimer [eIF2B(α₂)] dissociated from the remainder of the decamer, whereas ISRIB prevented disassembly. Sedimentation velocity experiments determined that in the absence of eIF2Bα, the remaining subunits form tetrameric complexes [eIF2B(βγδε)]. Loss of eIF2B(α₂) largely abolished eIF2B’s nucleotide exchange activity. To explain these findings, we determined a structure of human eIF2B bound to ISRIB at 2.8 Å average resolution. The structure revealed that ISRIB binds within a deep cleft at a two-fold symmetry interface between the eIF2Bβ and eIF2Bδ subunits in the decamer.

Greater resolution within the binding pocket enabled precise positioning of ISRIB, which we validated by probing with designed ISRIB analogs and mutational analysis. For example, stereospecific addition of a methyl group to ISRIB abrogated activity, whereas an eIF2B(δL179A) mutation accommodated this analog and restored activity. Further, a predicted C-H-π interaction between eIF2B(βH188) and ISRIB was confirmed by mutation of βH188 to other aromatic residues, which resulted in enhanced stability of the complex. To determine how ISRIB enhances incorporation of eIF2B(α₂) into the complex, we analyzed the eIF2B(βγδε) tetramer structurally and functionally. Cryo-EM imaging and analytical ultracentrifugation revealed that ISRIB staples two eIF2B(βγδε) tetramers together to form an octamer across its two-fold symmetry axis. The resulting octamer displays a composite surface for avid eIF2B(α₂) binding, explaining ISRIB’s mechanism of action. Consistent with this model, saturating half-binding sites in the tetramer with ISRIB prevented dimerization and failed to activate the enzyme. Additional loss-of-function and gain-of-function dimerization mutants produced complexes that were insensitive to ISRIB.

From this work, the regulation of eIF2B assembly from stable subcomplexes emerges as a rheostat for eIF2B activity that tunes translation during the ISR and can be further modulated by ISRIB acting as a “molecular staple.” As a two-fold symmetric small molecule, ISRIB bridges a central symmetry axis of the decameric eIF2B complex, stabilizing it in an activated state …