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Substrate selectivity and catalytic activation of the type III CRISPR ancillary nuclease Can2


Jungfer, Kenny; Sigg, Annina; Jinek, Martin (2024). Substrate selectivity and catalytic activation of the type III CRISPR ancillary nuclease Can2. Nucleic Acids Research, 52(1):462-473.

Abstract

Type III CRISPR-Cas systems provide adaptive immunity against foreign mobile genetic elements through RNA-guided interference. Sequence-specific recognition of RNA targets by the type III effector complex triggers the generation of cyclic oligoadenylate (cOA) second messengers that activate ancillary effector proteins, thus reinforcing the host immune response. The ancillary nuclease Can2 is activated by cyclic tetra-AMP (cA4); however, the mechanisms underlying cA4-mediated activation and substrate selectivity remain elusive. Here we report crystal structures of Thermoanaerobacter brockii Can2 (TbrCan2) in substrate- and product-bound complexes. We show that TbrCan2 is a single strand-selective DNase and RNase that binds substrates via a conserved SxTTS active site motif, and reveal molecular interactions underpinning its sequence preference for CA dinucleotides. Furthermore, we identify a molecular interaction relay linking the cA4 binding site and the nuclease catalytic site to enable divalent metal cation coordination and catalytic activation. These findings provide key insights into the molecular mechanisms of Can2 nucleases in type III CRISPR-Cas immunity and may guide their technological development for nucleic acid detection applications.

Abstract

Type III CRISPR-Cas systems provide adaptive immunity against foreign mobile genetic elements through RNA-guided interference. Sequence-specific recognition of RNA targets by the type III effector complex triggers the generation of cyclic oligoadenylate (cOA) second messengers that activate ancillary effector proteins, thus reinforcing the host immune response. The ancillary nuclease Can2 is activated by cyclic tetra-AMP (cA4); however, the mechanisms underlying cA4-mediated activation and substrate selectivity remain elusive. Here we report crystal structures of Thermoanaerobacter brockii Can2 (TbrCan2) in substrate- and product-bound complexes. We show that TbrCan2 is a single strand-selective DNase and RNase that binds substrates via a conserved SxTTS active site motif, and reveal molecular interactions underpinning its sequence preference for CA dinucleotides. Furthermore, we identify a molecular interaction relay linking the cA4 binding site and the nuclease catalytic site to enable divalent metal cation coordination and catalytic activation. These findings provide key insights into the molecular mechanisms of Can2 nucleases in type III CRISPR-Cas immunity and may guide their technological development for nucleic acid detection applications.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Department of Biochemistry
07 Faculty of Science > Department of Biochemistry
Dewey Decimal Classification:610 Medicine & health
570 Life sciences; biology
Scopus Subject Areas:Life Sciences > Genetics
Language:English
Date:11 January 2024
Deposited On:09 May 2024 10:37
Last Modified:30 Jun 2024 01:41
Publisher:Oxford University Press
ISSN:0305-1048
OA Status:Gold
Free access at:Publisher DOI. An embargo period may apply.
Publisher DOI:https://doi.org/10.1093/nar/gkad1102
PubMed ID:38033326
  • Content: Published Version
  • Language: English
  • Licence: Creative Commons: Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)