Cryo-EM structure of histidyl-tRNA synthetase-like domain reveals activating crossed helices at the core of GCN2

GCN2 is a conserved receptor kinase activating the integrated stress response (ISR) in eukaryotic cells. The ISR kinases detect accumulation of stress molecules and reprogram translation from basal tasks to preferred production of cytoprotective proteins. GCN2 stands out evolutionarily among all protein kinases due to the presence of a histidyl-tRNA synthetase-like (HRSL) domain, which arises only in GCN2 and is located next to the kinase domain (KD). How HRSL contributes to GCN2 signaling remains unknown. Here, we report a 3.2 Å cryo-EM structure of HRSL from thermotolerant yeast <i>Kluyveromyces marxianus</i>. This structure shows a constitutive symmetrical homodimer featuring a compact helical-bundle structure at the junction between HRSL and KDs, in the core of the receptor. Mutagenesis demonstrates that this junction structure activates GCN2 and indicates that our cryo-EM structure captures the active signaling state of HRSL. Based on these results, we put forward a GCN2 regulation mechanism, where HRSL drives the formation of activated kinase dimers. The remaining domains of GCN2 have the opposite role and in the absence of stress they help keep GCN2 basally inactive. This auto-inhibitory activity is relieved upon stress ligand binding. We propose that the opposing action of HRSL and additional GCN2 domains thus yields a regulated ISR receptor.