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 the 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 founding member of CRISPR-associated ring nuclease 1 (Crn1) Sso2081 from Saccharolobus solfataricus, alone, bound to phosphate ions or cA4 in both pre-cleavage and cleavage intermediate states. These structures together with biochemical characterizations establish the molecular basis of cA4 recognition and catalysis by Sso2081. The conformational changes in the C-terminal helical insert upon the binding of phosphate ions or cA4 reveal a gate-locking mechanism for ligand binding. The critical residues and motifs identified in this study provide a new insight to distinguish between cOA-degrading and -nondegrading CARF domain-containing proteins.

Molecular mechanism of allosteric activation of the CRISPR ribonuclease Csm6 by cyclic tetra-adenylate

“…In the absence of ligand, this loop is partially disordered and adopt an open conformation, suggesting an induced-fit mechanism for ligand binding. Similar mechanisms have been observed previously in the standalone ring nucleases, such as Sso2081 (Du et al, 2023 ), Sis0455 (Molina et al, 2022 ), and AcrIII-1 (Athukoralage et al, 2020a ).…”

“…2A ; Appendix Fig. S2 ) (Du et al, 2023 ; Makarova et al, 2020 ). These results indicate that the mechanism by which TtCsm6 binds and cleaves cA 4 is largely conserved with the Crn1 standalone ring nucleases.…”

“…6 , step 3). Considering the structural similarity with Crn1 standalone nucleases such as Sso2081, TtCsm6 may employ a stepwise mechanism for cA 4 cleavage as observed for Sso2081(Du et al, 2023 ). Interestingly, a previous study has demonstrated that not only cA 4 but also its linear cleavage intermediate, A 4 > P, can activate the ribonuclease activity of TtCsm6 (Niewoehner et al, 2017 ).…”

“…While the cOA-dependent indiscriminate degradation of RNA and/or DNA results in the clearance of invaders, the prolonged activation of this signaling can lead to cell dormancy or cell death (Rostol and Marraffini, 2019 ). Recent studies have identified a group of cOA-degrading enzymes called ring nucleases that dedicate to bisect the cOA molecules into two linear products, providing an off-switch regulation for the cOA signaling (Athukoralage et al, 2019 ; Athukoralage et al, 2020a ; Athukoralage et al, 2020b ; Athukoralage et al, 2018 ; Brown et al, 2020 ; Du et al, 2023 ; Molina et al, 2022 ; Molina et al, 2021 ). The majority of ring nucleases are CARF domain-containing proteins, which include the Crn1 (CRISPR ring nuclease 1) proteins Sso2081 and Sso1393 from S. solfataricus (Athukoralage et al, 2018 ), as well as Sis0811 (Molina et al, 2021 ) and Sis0455 (Molina et al, 2022 ) from S. islandicus .…”

Filament formation activates protease and ring nuclease activities of CRISPR SAVED-Lon

“…For example, the dimeric CARF-fold Crn1 catalyze in-line nucleophilic attacks of two opposite cA4 2′-OH groups on the adjacent phosphodiester bonds, yielding A2>p products with 2',3'-cyclic phosphates. The reactions on the opposite sides of the cA4 ring are not concerted, but the linearized intermediate A4>p is not released from the active site [47][48][49] . A similar mechanism is employed by CARF domains of the self-limiting CARF effectors that cut cA4 or cA6 to A2>p and A3>p, respectively [26][27][28][29] .…”