Molecular basis of stepwise cyclic tetra-adenylate cleavage by the type III CRISPR ring nuclease Crn1/Sso2081

Abstract

The cyclic oligoadenylates (cOAs) act as second messengers of type III CRISPR immunity system through activating the auxiliary nucleases for indiscriminate RNA degradation. The cOA-degrading nucleases (ring nucleases) provide an off-switch regulation of the signaling, thereby preventing cell dormancy or cell death. Here, we describe the crystal structures of the CRISPR-associated ring nuclease 1 (Crn1) from Saccharolobus solfataricus (Sso) 2081 in its apo or bound to cA4 in both pre-cleavage and transient intermediate states. Sso2081 harbors a unique helical insert that encloses cA4 in the central cavity. Two free phosphates symmetrically bind the catalytic site of apo Sso2081 and overlap with the two scissile phosphates of cA4, supporting a bilaterally symmetrical cleavage. The structure of transient intermediate state captured by Ser11Ala mutation immediately illustrates a stepwise cleavage of cA4 by Sso2081. Our study establishes atomic mechanisms of cA4 recognition and degradation by the type III CRISPR ring nuclease Crn1/Sso2081.