Second Messenger cA₄ Formation within the Composite Csm1 Palm Pocket of Type III-A CRISPR-Cas Csm Complex and Its...

Target RNA binding to crRNA-bound type III-A CRISPR-Cas multi-subunit Csm surveillance complexes activates cyclic-oligoadenylate (cA_n) formation from ATP subunits positioned within the composite pair of Palm domain pockets of the Csm1 subunit. The generated cA_n second messenger in turn targets the CARF domain of trans-acting RNase Csm6, triggering its HEPN domain-based RNase activity. We have undertaken cryo-EM studies on multi-subunit Thermococcus onnurineus Csm effector ternary complexes, as well as X-ray studies on Csm1-Csm4 cassette, both bound to substrate (AMPPNP), intermediates (pppA_n), and products (cA_n), to decipher mechanistic aspects of cA_n formation and release. A network of intermolecular hydrogen bond alignments accounts for the observed adenosine specificity, with ligand positioning dictating formation of linear pppA_n intermediates and subsequent cA_n formation by cyclization. We combine our structural results with published functional studies to highlight mechanistic insights into the role of the Csm effector complex in mediating the cA_n signaling pathway.