The Regulatory and Kinase Domains but Not the Interdomain Linker Determine Human Double-stranded RNA-activated Kinase (PKR) Sensitivity to Inhibition by Viral Non-coding RNAs

• Type of Material: Article
• Authors: S. Sunita, Emory University; Samantha L. Schwartz, Emory University; Graeme Conn, Emory University
• Language: English
• Date: 2015-11-20
• Publisher: American Society for Biochemistry and Molecular Biology
• Publication Version: Final Published Version
• Copyright Statement: © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
• Final Published Version (URL): http://dx.doi.org/10.1074/jbc.M115.679738
• Title of Journal or Parent Work: Journal of Biological Chemistry
• ISSN: 0021-9258
• Volume: 290
• Issue: 47
• Start Page: 28156
• End Page: 28165
• Grant/Funding Information: This work was supported in part by National Institute of Allergy and Infectious Diseases, National Institutes of Health Grant R21-AI097803 and a grant from the Emory University Research Council (URC 2010050).
• Keywords: PHOSPHORYLATION; DEPENDENT PROTEIN-KINASE; PURIFICATION; VIRUS; Life Sciences & Biomedicine; DIMERIZATION; Biochemistry & Molecular Biology; MEGAPRIMER PCR; MECHANISM; BINDING; RANDOM MUTAGENESIS LIBRARIES; Science & Technology; REVEALS
• Research Categories: Health Sciences, General; Chemistry, Biochemistry

Abstract

Double-stranded RNA (dsRNA)-activated protein kinase (PKR) is an important component of the innate immune system that presents a crucial first line of defense against viral infection. PKR has a modular architecture comprising a regulatory N-terminal dsRNA binding domain and a C-terminal kinase domain interposed by an unstructured ∼80-residue interdomain linker (IDL). Guided by sequence alignment, we created IDL deletions in human PKR (hPKR) and regulatory/kinase domain swap human-rat chimeric PKRs to assess the contributions of each domain and the IDL to regulation of the kinase activity by RNA. Using circular dichroism spectroscopy, limited proteolysis, kinase assays, and isothermal titration calorimetry, we show that each PKR protein is properly folded with similar domain boundaries and that each exhibits comparable polyinosinic-cytidylic (poly(rI:rC)) dsRNA activation profiles and binding affinities for adenoviral virus-associated RNA I (VA RNAI) and HIV-1 trans-activation response (TAR) RNA. From these results we conclude that the IDL of PKR is not required for RNA binding or mediating changes in protein conformation or domain interactions necessary for PKR regulation by RNA. In contrast, inhibition of rat PKR by VA RNAI and TAR RNA was found to be weaker than for hPKR by 7- and >300-fold, respectively, and each human-rat chimeric domain-swapped protein showed intermediate levels of inhibition. These findings indicate that PKR sequence or structural elements in the kinase domain, present in hPKR but absent in rat PKR, are exploited by viral non-coding RNAs to accomplish efficient inhibition of PKR.