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

Abstract

Summary 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 non-hydrolysable 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, impose a rigid intertwined dimer configuration that is essential for RNase catalytic and anti-viral 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.

Structural basis for the endoribonuclease activity of the type III-A CRISPR-associated protein Csm6

“…Taken together, these experiments show that TtCsm6 is a divalent metal-independent, ssRNA-specific, endoribonuclease. This is consistent with the catalytic activities of other HEPN-domain ribonucleases such as Ire1, RNase L, and the tRNA anticodon RNases PrrC and RloC, which are all metal-independent enzymes (Davidov and Kaufmann 2008;Lee et al 2008;Meineke and Shuman 2012;Han et al 2014;Huang et al 2014).…”

“…HEPN domains occur across all domains of life and are characterized by the presence of a conserved motif conforming to the consensus sequence R-X 4-6 -H (Anantharaman et al 2013). The domains are frequently found in ribonucleases and in several of these the R-X 4-6 -H motif has been shown to be required for catalytic activity (Davidov and Kaufmann 2008;Lee et al 2008;Han et al 2014;Huang et al 2014). The nuclease activity of Csm6 proteins has not been tested to date, although Pyrococcus furiosus Csx1 was previously shown to be a nucleic acid-binding protein (Kim et al 2013).…”

“…Collectively, these results confirm that the ribonuclease activity of TtCsm6 is mediated by the R-X 4-6 -H motif in the HEPN domain independently of the CARF domain. In an analogy with the catalytic mechanisms of Ire1 and RNase L (Lee et al 2008;Han et al 2014;Huang et al 2014), we propose that the dimerization of the HEPN domains and the resulting juxtaposition of the R-X 4-6 -H motifs lead to the formation of a composite symmetric active center that binds the substrate RNA asymmetrically (Supplemental Fig. 6B).…”

“…Site-directed mutagenesis studies have shown that the conserved R-X 4-6 -H motif is responsible for the catalytic activity in a number of dimeric HEPN domain-containing ribonucleases, including tRNA anticodon nucleases PrrC and RloC, as well as the eukaryotic pseudokinase-ribonuclease enzymes Ire1 and RNase L (Davidov and Kaufmann 2008;Lee et al 2008;Meineke and Shuman 2012;Han et al 2014;Huang et al 2014). The molecular architecture of the TtCsm6 HEPN domain dimer is highly similar to that of the kinase-ribonuclease Ire1 (Supplemental Fig.…”

“…3B; Supplemental Fig. 6), suggesting that Csm6 proteins use a similar mechanism of RNA hydrolysis (Lee et al 2008;Han et al 2014;Huang et al 2014). To confirm the catalytic function of the putative active site residues in TtCsm6, we probed the ribonuclease activity of TtCsm6 proteins in which the invariant R-X 4-6 -H motif residues (Arg 415, Asn416, and His422) as well as a nearby glutamate (Glu332) were substituted by alanine.…”