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 cA 4 in both pre-cleavage and transient intermediate states. Sso2081 harbors a unique helical insert that encloses cA 4 in the central cavity. Two free phosphates symmetrically bind the catalytic site of apo Sso2081 and overlap with the two scissile phosphates of cA 4 , supporting a bilaterally symmetrical cleavage. The structure of transient intermediate state captured by Ser11Ala mutation immediately illustrates a stepwise cleavage of cA 4 by Sso2081. Our study establishes atomic mechanisms of cA 4 recognition and degradation by the type III CRISPR ring nuclease Crn1/Sso2081.