PKR (protein kinase R) is induced by interferon and is a key component of the innate immunity antiviral pathway. Upon binding dsRNA, PKR undergoes autophosphorylation reactions that activate the kinase, leading it to phosphorylate eIF2alpha, thus inhibiting protein synthesis in virally infected cells. PKR contains a dsRNA-binding domain (dsRBD) and a kinase domain. The dsRBD is composed of two tandem dsRNA-binding motifs. An autoinhibition model for PKR has been proposed, whereby dsRNA binding activates the enzyme by inducing a conformational change that relieves the latent enzyme of the inhibition that is mediated by the interaction of the dsRBD with the kinase. However, recent biophysical data support an open conformation for the latent enzyme, where activation is mediated by dimerization of PKR induced upon binding dsRNA. We have probed the importance of interdomain contacts by comparing the relative stabilities of isolated domains with the same domain in the context of the intact enzyme using equilibrium chemical denaturation experiments. The two dsRNA-binding motifs fold independently, with the C-terminal motif exhibiting greater stability. The kinase domain is stabilized by about 1.5 kcal/mol in the context of the holenzyme, and we detect low-affinity binding of the kinase and dsRBD constructs in solution, indicating that these domains interact weakly. Limited proteolysis measurements confirm the expected domain boundaries and reveal that the activation loop in the kinase is accessible to cleavage and unstructured. Autophosphorylation induces a conformation change that blocks proteolysis of the activation loop. Study holds ProTherm entries: 23304, 23305, 23306, 23307, 23308, 23309, 23310, 23311, 23312, 23313, 23314, 23315, 23316, 23317 Extra Details: Native to Intermediate. 0.1 mM EDTA and 0.1 mM TCEP are added in the experiment. dsRBM1, dsRNA-binding motif 1 consisting of PKR residues 6?79; PKR(protein kinase R), binding dsRNA, kinase domain, conformation change, proteolysis
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:54 p.m.
|Number of data points||28|
|Proteins||Eukaryotic translation initiation factor 2 subunit alpha ; Eukaryotic translation initiation factor 2 subunit alpha|
|Assays/Quantities/Protocols||Experimental Assay: m ; Experimental Assay: dG_H2O ; Experimental Assay: m ; Experimental Assay: dG_H2O ; Experimental Assay: m ; Experimental Assay: dG_H2O ; Experimental Assay: m prot_conc:0.1 mg/mL, 0.15 mg/mL ; Experimental Assay: dG_H2O prot_conc:0.1 mg/mL, 0.15 mg/mL ; Experimental Assay: m prot_conc:0.15 mg/mL ; Experimental Assay: dG_H2O prot_conc:0.15 mg/mL ; Experimental Assay: m ; Experimental Assay: dG_H2O|
|Libraries||Mutations for sequence A:GSSHCRFYENKYPEIDDIVMVNVQQIAEMGAYVKLLEYDNIEGMILLSELSRRRIRSIQKLIRVGKNDVAVVLRVDKEKGYIDLSKRRVSSEDIIKCEEKYQKSKTVHSILRYCAEKFQIPLEELYKTIAWPLSRKFGHAYEAFKLSIIDETVWEGIEPPSKDVLDELKNYISKR/B:GAHTVDKRFGMDFKEIELIGSGGFGQVFKAKHRIDGKTYVIKRVKYNNEKAEREVKALAKLDHVNIVHYNGCWDGFDYDPETSSKNSSRSKTKCLFIQMEFCDKGTLEQWIEKRRGEKLDKVLALELFEQITKGVDYIHSKKLINRDLKPSNIFLVDTKQVKIGDFGLVTSLKNDGKRTRSKGTLRYMSPEQISSQDYGKEVDLYALGLILAELLHVCDTAFETSKFFTDLRDGIISDIFDKKEKTLLQKLLSKKPEDRPNTSEILRTLTVWKKSPEKNERHTA/C:GAHTVDKRFGMDFKEIELIGSGGFGQVFKAKHRIDGKTYVIKRVKYNNEKAEREVKALAKLDHVNIVHYNGCWDGFDYDPETSSKNSSRSKTKCLFIQMEFCDKGTLEQWIEKRRGEKLDKVLALELFEQITKGVDYIHSKKLINRDLKPSNIFLVDTKQVKIGDFGLVTSLKNDGKRTRSKGTLRYMSPEQISSQDYGKEVDLYALGLILAELLHVCDTAFETSKFFTDLRDGIISDIFDKKEKTLLQKLLSKKPEDRPNTSEILRTLTVWKKSPEKNERHTA|