ZBP1’s Inability to Convert Unmodified RNAs to the Z-form Underlies a Balanced Mechanism of RNA Recognition with ADAR1

Z-DNA Binding Protein 1 (ZBP1) is a critical pattern recognition receptor within the innate immune response to viral infection. ZBP1 senses foreign nucleic acids in the unusual, left-handed Z-conformation via binding through its N-terminal Zα1 and Zα2 domains and activates downstream pro-pyroptotic, -apoptotic, and -necroptotic pathways to initiate cell death and allow for viral clearance. Both dsDNA and dsRNA can adopt the Z-conformation, however, the conformational change is energetically expensive, especially for dsRNA, and typically requires chemical modifications or protein binding to induce a right-to-left-handed conversion and stabilization. ZBP1 has been previously shown to bind and convert B-DNA to the Z-conformation and was assumed to be able to convert A-RNA as well, despite the lack of experimental validation. Here, we use a variety of Nuclear Magnetic Resonance (NMR) and other biophysical and biochemical experiments to characterize the Z-DNA and Z-RNA binding properties of ZBP1’s Zα1 and Zα2 domains. While ZBP1’s Zα domains are able to convert and stabilize unmodified dsDNA in the Z-conformation, both domains are incapable of flipping unmodified A-conformation dsRNA. We show that ZBP1’s Zα domains require dsRNAs with Z-promoting chemical modification in order for them to bind and stabilize the Z-conformation. These results contrast with the Zα domain from Adenosine Deaminase Acting on RNA 1 (ADAR1), which can bind and flip both dsDNA and dsRNA into the Z-conformation, potentially indicating finely tuned competition between ADAR1 and ZBP1 for pro-survival and pro-death outcomes, respectively. This work highlights the functional variability of Zα domains and narrows down the potential physiological substrates of ZBP1 in infection and disease.