Dimeric Structure of Pseudokinase RNase L Bound to 2-5A Reveals a Basis for Interferon-Induced Antiviral Activity

RNase L is an ankyrin repeat domain-containing dual endoribonuclease-pseudokinase that is activated by unusual 2,′5′-oligoadenylate (2-5A) second messengers and which impedes viral infections in higher vertebrates. Despite its importance in interferon-regulated antiviral innate immunity, relatively little is known about its precise mechanism of action. Here we present a functional characterization of 2.5 Å and 3.25 Å X-ray crystal and small-angle X-ray scattering structures of RNase L bound to a natural 2-5A activator with and without ADP or the nonhydrolysable ATP mimetic AMP-PNP. These studies reveal how recognition of 2-5A through interactions with the ankyrin repeat domain and the pseudokinase domain, together with nucleotide binding, imposes a rigid intertwined dimer configuration that is essential for RNase catalytic and antiviral functions. The involvement of the pseudokinase domain of RNase L in 2-5A sensing, nucleotide binding, dimerization, and ribonuclease functions highlights the evolutionary adaptability of the eukaryotic protein kinase fold. [Display omitted] •Structural basis for RNase L regulation by 2-5A and nucleotide (ADP or ATP) binding•Recognition of 2-5A is mediated by both ankyrin repeat and protein kinase domains•Nucleotide enforces a closed conformation of the kinase domain•Nucleotide binding to the pseudokinase domain is essential for RNA cleavage function RNase L is an ankyrin repeat domain containing dual endoribonuclease-pseudokinase that is activated by 2′,5′-oligoadenylate (2-5A) second messengers and which impedes viral infections in higher vertebrates. Huang et al. revealed the structure of RNase L bound to 2-5A activator with and without the ATP mimetic AMP-PNP.