The tandem repetition of RNA-binding domains (RBDs)—often connected by intrinsically disordered sequences—is widely found in RNA-binding proteins (RBPs). However, whether such flexible arrangements can adopt defined conformations essential for function, and how these conformations contribute to their biological roles, remain poorly understood.

In a study published in PNAS, a research team led by Prof. LIU Jiaquan from the Center for Excellence in Molecular Cell Science (Shanghai Institute of Biochemistry and Cell Biology) of the Chinese Academy of Sciences, in collaboration with a team led by Prof. CHEN Lingling from the same center, uncovered a new mechanism of protein kinase R (PKR) activation.

By integrating single-molecule fluorescence imaging, AlphaFold structure prediction, and biochemical and cell-biological functional validation, the researchers found that the two double-stranded RNA-binding domains (dsRBDs) of PKR form a unique conformation through intramolecular interactions.

This architecture enables PKR to discriminate between substrates, promotes kinase dimerization, and enhances its ability to competitively recognize double-stranded RNA (dsRNA) in the presence of other RNA-binding proteins, collectively driving PKR activation.

These findings provide a new theoretical basis for understanding the functional advantages of tandem RBDs and for developing intervention strategies targeting aberrant PKR activation.

PKR is a key dsRNA sensor in innate immunity. Upon recognizing virus-derived dsRNA, PKR undergoes kinase dimerization and autophosphorylation, which in turn leads to the phosphorylation of its downstream substrate eIF2α, triggering translation inhibition and ultimately suppressing viral replication and spread.

To ensure the specificity of antiviral responses, PKR's phosphorylation level must be precisely coupled to its RNA-binding activity. However, how PKR's two dsRBDs cooperatively recognize RNA, and what functional advantages this tandem arrangement confers on the regulation of PKR activation, have remained unclear.

To investigate this mechanism, the research team used single-molecule fluorescence imaging to track PKR's dynamic behavior on dsRNA in real time. They found that PKR recognizes its substrate through a "scan-and-trap" mechanism: PKR first scans along the dsRNA; subsequently, dsRBD2 engages in an intramolecular interaction with dsRBD1, anchoring PKR to the dsRNA and shifting it from a dynamic, transient "sampling" mode to stable binding. This switch in binding mode markedly promotes dimerization of the PKR kinase domain, thereby driving strong kinase activation.

Notably, this cooperative dsRBD interaction fails to occur on most self-RNAs, yet occurs efficiently on immunogenic dsRNA. It also enhances PKR's ability to compete with other RNA-binding proteins for immunogenic RNA, enabling PKR to specifically recognize immunogenic dsRNA in the presence of a large excess of self-RNA. Disrupting the dsRBD1–dsRBD2 interaction interface severely impairs PKR function both in vitro and at the cellular level.

These results indicate that the functional advantage of the tandem dsRBDs does not simply arise from an increased number of binding sites, but depends on the binding-mode switch mediated by the interaction interface.

According to the researchers, this study reveals a new mechanism by which tandem RBDs function in RNA-binding proteins, expanding our understanding of how multiple domains in RBPs cooperate to regulate function.

Furthermore, disrupting the dsRBD1–dsRBD2 interaction interface markedly suppresses PKR activation, suggesting that this interface may serve as a potential drug target for modulating aberrant PKR activation and providing a basis for developing potential therapies for PKR-related diseases.