Stepwise Ligand Capture Primes PKR for Activation and RNA Discrimination

ABSTRACT Protein Kinase R (PKR) triggers the innate immune response upon detecting double-stranded RNAs (dsRNAs). Its kinase activity is controlled by two dsRNA-binding domains (dsRBDs), yet how these dsRBDs operate during RNA ligand recognition and downstream signaling remains unresolved. Here, we uncover an unexpected interplay between the dsRBDs that orchestrates a two-step ligand sampling, ensuring accurate and efficient PKR activation. dsRBD1 surveys all RNA duplexes through one-dimensional diffusion but dissociates within milliseconds to minimize background signaling, while dsRBD2-RNA binding plays a key role in fostering kinase-kinase interactions. Upon encountering foreign dsRNAs, dsRBD2 arrests the diffusive dsRBD1, thereby establishing a scaffold essential for kinase dimerization and phosphorylation. By contrast, prevalent bulges and large internal loops in self-RNA duplexes obstruct dsRBD2 from capturing dsRBD1, creating a steeply elevated energy landscape that enables PKR to discriminate foreign RNAs from abundant self-RNAs, thus evolving a delicate strategy to achieve both high selectivity and robust signaling.