title: 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.
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.
authors.affiliation: Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322.
authors.forename: S
authors.initials: S
authors.lastname: Sunita
authors.affiliation: Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322.
authors.forename: Samantha L
authors.initials: SL
authors.lastname: Schwartz
authors.affiliation: Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322. Electronic address: gconn@emory.edu.
authors.forename: Graeme L
authors.initials: GL
authors.lastname: Conn
mesh_terms: D000595:Amino Acid Sequence; D004789:Enzyme Activation; D017353:Gene Deletion; D006801:Humans; D008969:Molecular Sequence Data; D022661:RNA, Untranslated / Q000502:physiology*; D017386:Sequence Homology, Amino Acid; D019892:eIF-2 Kinase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*
chemical_list: D022661:RNA, Untranslated; D019892:eIF-2 Kinase
keywords: RNA-protein interaction; eukaryotic initiation factor 2 (eIF2); innate immunity; protein kinase RNA-activated (PKR); translation control; viral non-coding RNA