Sugar phosphate activation of the stress sensor eIF2B

Elucidation of the structure of proteins and protein complexes can unlock important information about functions. While X-ray crystallography is the most commonly used method For structure determination, the 3D structure of biological macromolecular complexes that cannot be easily crystallized because of size or complexity can now be determined to atomic resolution using cryo-EM. This paper shows how naturally occurring ligands can be identified and characterized using a combination of binding and activity studies followed by three dimensional structural analysis. Cryo-EM was used to solve the structure of the decameric eIF2B complex and to identify the sugar phosphates’ binding site in the evolutionarily conserved substrate binding pocket of subunit eIF2Bα. This suggested that sugar phosphate binding by the α subunit may enhance eIF2B decamer formation and enhance the eIF2B GEF activity. This was confirmed by other orthogonal experiments described in the paper.

The paper, published in Nature Communications concludes:

• “Motivated by the biochemical evidence that sugar phosphates are activators of eIF2B, [the authors] determined a 2.9 Å cryo-EM structure of eIF2B bound to F6P, the highest affinity identified ligand.”
• The cryo-EM structure showed that “F6P occupies an inter-domain cavity within each eIF2Bα monomer (chains G and H), which is the conserved metabolite binding pocket in the ancestral eIF2Bα/β/δ homologs.”
• “The 1′ position of F6P is exposed to solvent, suggesting a way by which bulkier ribonucleotides (e.g. UMP and AMP) could be accommodated in this site.”
• “We further show that sugar phosphates stabilize the decameric holoenzyme and enhance its GEF activity similar to synthetic eIF2B activators. However, these natural metabolites exert their effect by engaging the eIF2Bα2 dimer, rather than by bridging the eIF2B(β/δ)2 tetramer interface like ISRIB.”
• “Ultimately, understanding the role of ligand binding in control of eIF2B activity in vivo will be of utmost importance.”