Structural basis for pseudokinase-mediated regulation of GCN2 in the integrated stress response.

The general control nonderepressible 2 (GCN2) is a conserved stress-responsive protein that plays a critical role in restoring cellular homeostasis in the integrated stress response (ISR). In response to amino acid starvation or ribosome stalling and collisions, GCN2 phosphorylates the translation initiation factor eIF2α, conferring translational control to alleviate stress. GCN2 is a multidomain protein, containing a tandem kinase domain (KD) and a catalytically inactive pseudokinase domain (ψKD). Stress-induced activation of the kinase domain requires allosteric regulation and dimerization mediated by its regulatory domains. While the pseudokinase domain is essential for GCN2 function in yeast, its mechanistic role remains unclear and underexplored in other organisms. Here, we present the first crystal structure of the human GCN2 ψKD, revealing its distinct structural features. The structure visualizes an insertion N-terminal to helix αC unique to the GCN2 ψKD that interacts with the pseudoactivation loop, stabilizing an inactive conformation. Further structural analysis shows that the ψKD forms a dimer in the crystal lattice via a network of hydrophobic and electrostatic interactions spanning both the N- and C-lobes. Mutations that disrupt the dimer interface reduced downstream ATF4 expression that is important for stress adaptation, underscoring the functional significance of the GCN2 ψKD dimer in regulating GCN2 activity. Complementary AI-guided structure predictions indicate that the dimeric GCN2 ψKD architecture is conserved across evolution. These results support the role of ψKD dimerization as a regulatory feature in GCN2-mediated ISR signaling.