Structure and Function of Protein Kinase R

Protein Kinase R (PKR) is a central component of the innate immunity antiviral pathway and is activated by dsRNA. PKR contains two tandem dsRNA binding domains and a C-terminal kinase domain. In the canonical activation model, binding of multiple PKR monomers to dsRNA enhances dimerization of the kinase domain, leading to enzymatic activation. A minimal dsRNA length of 30 bp is required for activation. However, short (~15 bp) stem-loop RNAs containing flanking single stranded tails (ss-dsRNAs) are capable of activating PKR. Here, we characterize the structural features of ss-dsRNAs that contribute to activation. We have designed a model ss-dsRNA containing 15 nt single stranded tails and a 15 bp stem and made systematic truncations of the tail and stem regions. Activation potency and binding affinity decrease as the ssRNA tails are truncated and activation is abolished in cases where the binding affinity is strongly reduced. We demonstrate that isolated single-stranded RNAs bind to PKR with micromolar dissociation constants and can induce activation. Single-stranded RNAs also activate PKR constructs lacking the double-stranded RNA binding domain and bind to a basic region adjacent to the N-terminus of the kinase. The length of the duplex regions in several natural RNA activators of PKR is below the minimum of 30 bp required for activation and similar interactions with single stranded regions may contribute to PKR activation in these cases. We have also solved the crystal structure of the PKR kinase domain. PKR forms a unique oligomeric assembly of alternating front-to-front and back-to-back interfaces. Within the front-to-front interface activation segments from each protomer are exchanged and oriented toward the active site of the reciprocal protomer. Similar complexes have been reported for other kinases and are interpreted to represent a trans-autophosphorylation complex. This structure has profound implications for PKR activation models