The HisRS-like domain of GCN2 is a pseudoenzyme that can bind uncharged tRNA

GCN2 is a stress response kinase that phosphorylates the translation initiation factor eIF2α to inhibit general protein synthesis when activated by uncharged tRNA and stalled ribosomes. The presence of a HisRS-like domain in GCN2, normally associated with tRNA aminoacylation, led to the hypothesis that eIF2α kinase activity is regulated by the direct binding of this domain to uncharged tRNA. Here we solved the structure of the HisRS-like domain in the context of full-length GCN2 by cryoEM. Structure and function analysis shows the HisRS-like domain of GCN2 has lost histidine and ATP binding but retains tRNA binding abilities. Hydrogen deuterium exchange mass spectrometry, site-directed mutagenesis and computational docking experiments support a tRNA binding model that is partially shifted from that employed by bona fide HisRS enzymes. These results demonstrate that the HisRS-like domain of GCN2 is a pseudoenzyme and advance our understanding of GCN2 regulation and function. [Display omitted] •GCN2 is a sensor of nutrient limitation•The complete structure of GCN2 has eluded determination for decades•We solved the structure of the HisRS-like domain of human GCN2•This domain is a pseudoenzyme that cannot bind histidine or ATP, but can bind tRNA Yin et al. solved the structure of the HisRS-like domain of human GCN2 by cryoEM. Canonical HisRS enzymes bind histidine, ATP, and tRNA. Structure and function analysis demonstrate that the HisRS-like domain of GCN2 is a pseudoenzyme that has lost the ability to bind histidine and ATP, but retains the ability to bind tRNA.