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.

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 (dsRBD) and a C-terminal kinase domain (KD), 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 poly(rI:rC) dsRNA activation profiles and binding affinities for adenoviral 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 (rPKR) 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 rPKR, are exploited by viral non-coding RNAs to accomplish efficient inhibition of PKR.