Schematic of RNase L regulation by 2-5A and nucleotide binding

RNase L is an ankyrin repeat domain-containing dual endoribonuclease-pseudokinase that is activated by unusual 2,050-oligoadenylate (2-5A) second messengers and which impedes viral infections in higher vertebrates. Despite its importance in interferonregulated antiviral innate immunity, relatively little is known about its precise mechanism of action. Here   we present a functional characterization of 2.5 A ˚ and 3.25 A ˚ 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.

2-5A binding to RNase L through direct contact with the ANK domain and kinase N-lobe mediates molecular dimerization, while nucleotide binding to the kinase domain restrains the kinase domain to a single closed conformation required for the composition of the RNase active site.