A CRISPR-associated Rossman-fold Ring Nuclease and Adenosine Deaminase Fusion Allosterically Converts ATP to ITP

Whyms, C. T.; Zhao, Y.; Addo-Yobo, D.; He, H.; Whittington, A. C.; Trasanidou, D.; Salazar, C. R. P.; Staals, R. H. J.; Li, H.

doi:10.1101/2024.12.12.628194

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bioRxiv · 10.1101/2024.12.12.628194

A CRISPR-associated Rossman-fold Ring Nuclease and Adenosine Deaminase Fusion Allosterically Converts ATP to ITP

Whyms, C. T.·Zhao, Y.·Addo-Yobo, D.·He, H.·Whittington, A. C.·Trasanidou, D.·Salazar, C. R. P.·Staals, R. H. J.·Li, H.

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Abstract

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 comprises CARF-fused adenosine deaminase (ADA) (Cad1) and has a yet to be determined function. Here we present biochemical and structural analyses of a ring nuclease Cad1, revealing its unexpected role in deaminating adenosine-5'-triphosphate to inosine-5'-triphosphate in a cOA-dependent manner. Despite an overall structural similarity to canonical ADA enzymes, the ADA domain of Cad1 possesses unique structural features underlying its specificity for ATP. Supported by mutational analysis, we demonstrate an allosteric link between the cOA-binding CARF and the ADA domain, suggesting that Cad1 is a cOA-stimulated effector that influences cellular metabolic processes. HighlightsO_LITaqCad1 converts ATP to ITP in a magnesium- and cOA-dependent manner C_LIO_LICryo-EM structures reveal TaqCad1 forms a homohexamer C_LIO_LICryo-EM structures reveal how cA4 is degraded and modulates the ADA active site C_LI

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Whyms, C. T., Zhao, Y., Addo-Yobo, D., He, H., Whittington, A. C., Trasanidou, D., Salazar, C. R. P., Staals, R. H. J., Li, H.. 2024-12-17. A CRISPR-associated Rossman-fold Ring Nuclease and Adenosine Deaminase Fusion Allosterically Converts ATP to ITP. https://doi.org/10.1101/2024.12.12.628194

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