Biochemical and structural analyses of the ring nuclease Cad1 reveal its substrate binding specificity for ATP and a sequential activation process by cyclic oligoadenylates.
Structural and biochemical analyses reveal that cA4 binding to Cad1 induces a twist-and-squeeze conformational change that allosterically activates its adenosine deaminase domain.
The recently identified CARF (CRISPR-associated Rossman-fold) family of proteins play a critical role in prokaryotic defense, mediating cOA (cyclic oligoadenylate)-stimulated ancillary immune responses in the type III CRISPR-Cas systems. Whereas most previously characterized CARF proteins contain nucleic acids or protein degradation effectors, a subset of the family, including the CARF-fused adenosine deaminase (ADA) (Cad1), has recently been shown to convert ATP to ITP. The enzymatic mechanism and the activation process of Cad1, however, remain incompletely understood. Here we present biochemical and structural analyses of a ring nuclease Cad1, revealing its substrate binding specificity and a sequential activation process by cOAs. Despite an overall structural similarity to canonical ADA enzymes, the ADA domain of Cad1 possesses unique structural features that confer a specificity for ATP. Supported by mutational analysis, our structural work demonstrates an allosteric link between the cOA-binding CARF and the ADA domain through a protein network within the hexameric enzyme assembly. Binding of a cA4 molecule to paired CARF domains induces a twisting of the linked ADA domains around one another, which remodels their active sites and alters interactions with neighboring ADA domains, thereby driving a sequential conformational activation mechanism.
Whyms et al. (Fri,) reported a other. Biochemical and structural analyses of the ring nuclease Cad1 reveal its substrate binding specificity for ATP and a sequential activation process by cyclic oligoadenylates.