9g9i
CryoEM structure of Enterococcus italicus Csm-crRNA-CTR2 complex bound to pNppA3 and AMPNPP
Structural highlights
FunctionCAS10_ENTI1 CRISPR (clustered regularly interspaced short palindromic repeat) is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements and conjugative plasmids). CRISPR clusters contain spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). The type III-A Csm effector complex binds crRNA and acts as a crRNA-guided RNase, DNase and cyclic oligoadenylate synthase; binding of target RNA cognate to the crRNA is required for all activities. In a heterologous host the appropriately targeted Csm effector complex prevents growth of dsDNA phage phiNM1-gamma6.[1] ssDNase activity is stimulated in the ternary Csm effector complex; binding of cognate target RNA activates the ssDNase, as the target RNA is degraded ssDNA activity decreases.[UniProtKB:A0A0A7HFE1] This subunit is a single-strand-specific deoxyribonuclease (ssDNase) which digests both linear and circular ssDNA; it has both exo- and endonuclease activity.[UniProtKB:B6YWB8] When associated with the ternary Csm effector complex (the crRNA, Cas proteins and a cognate target ssRNA) synthesizes cyclic oligoadenylates (cOA) from ATP, producing (mostly) cyclic hexaadenylate (cA6). cA6 synthesis occurs in the Csm effector complex and requires cognate target RNA and ATP; other NTPs are not incorporated. cOAs are second messengers that induce an antiviral state important for defense against invading nucleic acids.[2] Publication Abstract from PubMedType III clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems (type III CRISPR-Cas systems) use guide RNAs to recognize RNA transcripts of foreign genetic elements, which triggers the generation of cyclic oligoadenylate (cOA) second messengers by the Cas10 subunit of the type III effector complex. In turn, cOAs bind and activate ancillary effector proteins to reinforce the host immune response. Type III systems utilize distinct cOAs, including cyclic tri- (cA3), tetra- (cA4) and hexa-adenylates (cA6). However, the molecular mechanisms dictating cOA product identity are poorly understood. Here we used cryoelectron microscopy to visualize the mechanism of cA6 biosynthesis by the Csm effector complex from Enterococcus italicus (EiCsm). We show that EiCsm synthesizes oligoadenylate nucleotides in 3'-5' direction using a set of conserved binding sites in the Cas10 Palm domains to determine the size of the nascent oligoadenylate chain. Our data also reveal that conformational dynamics induced by target RNA binding results in allosteric activation of Cas10 to trigger oligoadenylate synthesis. Mutations of a key structural element in Cas10 perturb cOA synthesis to favor cA3 and cA4 formation. Together, these results provide comprehensive insights into the dynamics of cOA synthesis in type III CRISPR-Cas systems and reveal key determinants of second messenger product selectivity, thereby illuminating potential avenues for their engineering. Mechanistic determinants and dynamics of cA6 synthesis in type III CRISPR-Cas effector complexes.,Jungfer K, Moravcik S, Garcia-Doval C, Knorlein A, Hall J, Jinek M Nucleic Acids Res. 2025 Jan 11;53(2):gkae1277. doi: 10.1093/nar/gkae1277. PMID:39817514[3] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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