Fig 1. Summary of domain interactions in Gcn2 that couple binding of uncharged tRNA to activation of kinase function in starved cells.

(A) Schematic diagram of the domain structure of yeast Gcn2 and interdomain interactions controlling kinase activity. The amino acid coordinates of the RWD/GI domain (pdb 2yz0), pseudokinase domain (YKD) [21], kinase domain (KD) [12], HisRS-like domain [14], and CTD [26] are derived from crystal structures (RWD, KD, CTD) or multiple sequence alignments (YKD and HisRS-like) of the respective domains. Stimulatory interactions are depicted with arrows, and involve domain interactions with Gcn1/Gcn20 (RWD) or uncharged tRNA (HisRS-like); or dimerization and ribosome-binding activities (CTD). The CTD also mediates autoinhibition of kinase activity, depicted with a bar. (B) Schematic summary of conformational rearrangements in the KD evoked by uncharged tRNA binding to the HisRS domain in amino acid-starved cells. The mode of dimerization and disposition of helix αC, β strand-3 (β3), and the activation loop, as well as key residues in the KD controlling ATP binding and catalysis (Lys-628, Asn-793, Glu-643, Arg-834, and Leu-856) are depicted for the inactive and active states of the Gcn2 KD that prevail in nonstarved or amino acid-starved cells, respectively. It is assumed that, in both states, Gcn2 dimerizes through self-interaction of the CTD and the schematic depicts only the disposition of the KDs within the full-length dimer. At low levels of uncharged tRNA in nonstarved cells (left), the KD exists in an equilibrium between two inactive conformations, with monomeric KDs (upper) or the KDs in an antiparallel dimer (lower). In both, ATP binding is hindered by a closed conformation of the N- and C-lobes and by Gln-793 (N793), which forms a flap over the ATP-binding pocket; and catalysis is blocked by hinge rigidity and rotation of αC to a nonproductive orientation that is stabilized by L856 and the inhibitory E643-R834 salt bridge. CTD/KD domain interaction also promotes this inactive conformation by an unknown mechanism. In amino acid-starved cells, binding of uncharged tRNA to the HisRS domain, along with stimulatory contributions of the RWD domain (engaged with Gcn1/Gcn20), the YKD domain, and the CTD, evokes a parallel mode of KD dimerization with αC properly oriented to form the stimulatory K628-E643 salt-bridge, and with ATP binding enhanced by a reduction in hinge rigidity that facilitates an open conformation of the N- and C-lobes and displaces the inhibitory N793 flap. Autophosphorylation of the activation loop ensues to produce a fully functional kinase locked into the active conformation. (See text for additional details; modified from Garriz et al [13].)