RNA Binding Mode Regulates PKR Activation

Abstract

The antiviral kinase PKR is activated by RNA. The enzyme contains two dsRNA-binding domains, dsRBD1 and dsRBD2, and a kinase domain. Co-localization of the kinase domains upon binding of two or more PKRs to a single dsRNA enhances dimerization and subsequent activation. However, multiple lines of evidence indicate that the mode of RNA binding regulates PKR activation beyond simply modulating the overall binding affinity. dsRBD2 binds RNA weakly and engagement of this domain by an RNA is correlated with enzymatic activation. In order to define the role of dsRBD2 in PKR activation we have systematically mutated residues in this domain that typically mediate dsRBD-RNA interactions and probed the consequences for PKR activation and overall binding affinity. Several mutations inhibit PKR activation and reduce RNA binding affinity in parallel. However, mutation of outward-facing residues on helix 1 that typically engages the RNA minor groove dramatically reduce PKR activation while only slightly decreasing overall RNA binding affinity. Steady-state fluorescence anisotropy analysis of PKR constructs containing a single tryptophan in either dsRBD1 or dsRBD2 indicate that both domains undergo a significant decrease in mobility upon RNA binding. These anisotropy changes are not markedly affected by the helix 1 mutations, implying that the interaction of dsRBD2 with RNA persists. We propose that instead of interacting with RNA, helix 1 in dsRBD2 engages in an intramolecular interaction that regulates kinase activity based on RNA binding mode.