Structure of the nucleotide exchange factor eIF2B reveals mechanism of memory-enhancing molecule

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 A drug-like inhibitor of the integrated stress response bridges tetrameric subcomplexes of eIF2B, assembling active holoenzyme. INTRODUCTION 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. RATIONALE 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. RESULTS 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. CONCLUSION 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. ISRIB’s action as an assembly-promoting enzyme activator provides a plausible model for its ability to ameliorate the inhibitory effects of eIF2α phosphorylation. Understanding the different modes of regulation of this vital translational control point will be of particular importance in the nervous system where ISRIB has been shown to have a range of effects, and will further enable ISRIB’s development as a promising therapeutic agent in combating cognitive disorders. ISRIB bound to human eIF2B. View of cryo-EM density for eIF2B(αβγδε)2, colored in distinct shades for each subunit copy: (red, α; blue, β; green, γ; gold, δ; gray, ε). Density assigned to ISRIB is depicted in CPK coloring (red, O; blue, N; green, Cl) and artistically contrasted from its target protein. 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, thereby inhibiting translation. ISRIB, a drug-like eIF2B activator, reverses the effects of eIF2 phosphorylation, and in rodents it enhances cognition and corrects cognitive deficits after brain injury. To determine its mechanism of action, we solved an atomic-resolution structure of ISRIB bound in a deep cleft within decameric human eIF2B by cryo–electron microscopy. Formation of fully active, decameric eIF2B holoenzyme depended on the assembly of two identical tetrameric subcomplexes, and ISRIB promoted this step by cross-bridging a central symmetry interface. Thus, regulation of eIF2B assembly emerges as a rheostat for eIF2B activity that tunes translation during the ISR and that can be further modulated by ISRIB.