Domain stabilities in protein kinase R (PKR): evidence for weak interdomain interactions.


Abstract

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

Submission Details

ID: tp5dKZdL3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:54 p.m.

Version: 1

Publication Details
Anderson E;Cole JL,Biochemistry (2008) Domain stabilities in protein kinase R (PKR): evidence for weak interdomain interactions. PMID:18393532
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1QU6 1999-07-08T00:00:00+0000 0 STRUCTURE OF THE DOUBLE-STRANDED RNA-BINDING DOMAIN OF THE PROTEIN KINASE PKR REVEALS THE MOLECULAR BASIS OF ITS DSRNA-MEDIATED ACTIVATION
2A19 2005-06-19T00:00:00+0000 2.5 PKR kinase domain- eIF2alpha- AMP-PNP complex.
2A1A 2005-06-19T00:00:00+0000 2.8 PKR kinase domain-eIF2alpha Complex
3UIU 2011-11-06T00:00:00+0000 2.9 Crystal structure of Apo-PKR kinase domain
6D3K 2018-04-16T00:00:00+0000 2.6 Crystal structure of unphosphorylated human PKR kinase domain in complex with ADP
6D3L 2018-04-16T00:00:00+0000 3.1 Crystal structure of unphosphorylated human PKR
1Q46 2003-08-01T00:00:00+0000 2.86 crystal structure of the eIF2 alpha subunit from saccharomyces cerevisia
2A19 2005-06-19T00:00:00+0000 2.5 PKR kinase domain- eIF2alpha- AMP-PNP complex.
2A1A 2005-06-19T00:00:00+0000 2.8 PKR kinase domain-eIF2alpha Complex
3J81 2014-08-29T00:00:00+0000 4.0 CryoEM structure of a partial yeast 48S preinitiation complex

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Eukaryotic translation initiation factor 2 subunit alpha P20459 IF2A_YEAST
185.0 B,C Eukaryotic translation initiation factor 2 subunit alpha P19525 E2AK2_HUMAN