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bioRxiv · 10.1101/2024.05.08.593097

Filament formation activates protease and ring nuclease activities of CRISPR SAVED-Lon

Smalakyte, D.·Ruksenaite, A.·Sasnauskas, G.·Tamulaitiene, G.·Tamulaitis, G.
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Abstract

To combat phage infection, type III CRISPR-Cas systems utilize cyclic oligoadenylates (cAn) signaling to activate various auxiliary effectors, including the CRISPR-associated SAVED-Lon protease CalpL, which forms a tripartite effector system together with an anti-{sigma} factor, CalpT, and an ECF-like {sigma} factor, CalpS. Here we report the characterization of the Candidatus Cloacimonas acidaminovorans CalpL-CalpT-CalpS. We demonstrate that cA4 binding triggers CalpL filament formation and activates it to cleave CalpT within the CalpT-CalpS dimer. This cleavage exposes the CalpT C-degron, which targets it for further degradation by cellular proteases. Consequently, CalpS is released to bind to RNA polymerase, causing growth arrest in E. coli. Furthermore, the CalpL-CalpT-CalpS system is regulated by the SAVED domain of CalpL, which is a ring nuclease that cleaves cA4 in a sequential three-step mechanism. These findings provide key mechanistic details for the activation, proteolytic events, and regulation of the signaling cascade in the type III CRISPR-Cas immunity.

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Smalakyte, D., Ruksenaite, A., Sasnauskas, G., Tamulaitiene, G., Tamulaitis, G.. 2024-05-08. Filament formation activates protease and ring nuclease activities of CRISPR SAVED-Lon. https://doi.org/10.1101/2024.05.08.593097

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