Mechanistic link between PKR dimerization, autophosphorylation, and elF2α substrate recognition

The antiviral protein kinase PKR inhibits protein synthesis by phosphorylating the translation initiation factor eIF2 alpha on Ser51. Binding of double-stranded RNA to the regulatory domains of PKR promotes dimerization, autophosphorylation, and the functional activation of the kinase. Herein, we identify mutations that activate PKR in the absence of its regulatory domains and map the mutations to a recently identified dimerization surface on the kinase catalytic domain. Mutations of other residues on this surface block PKR autophosphorylation and eIF2 alpha phosphorylation, while mutating Thr446, an autophosphorylation site within the catalytic-domain activation segment, impairs eIF2 alpha phosphorylation and viral pseudosubstrate binding. Mutational analysis of catalytic-domain residues preferentially conserved in the eIF2 alpha kinase family identifies helix alpha G as critical for the specific recognition of eIF2 alpha. We propose an ordered mechanism of PKR activation in which catalytic-domain climerization triggers Thr446 autophosphorylation and specific eIF2 alpha substrate recognition.