Molecular basis for PKR activation by PACT or dsRNA.
The mammalian protein kinase PKR is a critical component of the innate immune response against virus infection. Its cellular actions are mediated by modulating cell signaling and translational regulation. To be enzymatically active, latent PKR needs to be activated by binding to one of its activators, dsRNA or PACT protein. Although the structures of the N-terminal dsRNA-binding domain and the C-terminal kinase domain of PKR have been separately determined, the mode of activation of the enzyme remains unknown. To address this problem, we used biochemical, genetic, and NMR analyses to identify the PACT-binding motif (PBM) located in the kinase domain and demonstrated an intramolecular interaction between PBM and dsRNA-binding domain. This interaction is responsible for keeping PKR in an inactive conformation, because its disruption by point mutations of appropriate residues produced constitutively active PKR. Furthermore, a short decoy peptide, representing PBM, was able to activate PKR by interfering with the intramolecular interaction. These observations suggest a model for PKR activation upon binding of dsRNA or PACT.
Li
Shoudong
S
Department of Molecular Genetics/NE20, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA.
Peters
Gregory A
GA
Ding
Keyang
K
Zhang
Xiaolun
X
Qin
Jun
J
Sen
Ganes C
GC
D020816:Amino Acid Motifs / Q000235:genetics; D002478:Cells, Cultured; D004789:Enzyme Activation; D006801:Humans; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D009687:Nuclear Proteins / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary / Q000235:genetics; D012330:RNA, Double-Stranded / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D019892:eIF-2 Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D009687:Nuclear Proteins; C104647:PRKRA protein, human; D010455:Peptides; C501499:RBMY1A1 protein, human; D012330:RNA, Double-Stranded; D016601:RNA-Binding Proteins; D019892:eIF-2 Kinase