Antiviral signaling by a cyclic nucleotide activated CRISPR protease

Funding information: M.G. and J.L.S.B. are funded by the Deutsche Forschungsgemeinschaft under Germany’s Excellence Strategy–EXC2151–390873048. M.F.W. acknowledges a European Research Council Advanced Grant (grant number 101018608) and the China Scholarship Council (REF: 202008420207 to H.C.). G.H. is grateful for funding by the Deutsche Forschungsgemeinschaft (grant number HA6805/6-1). CRISPR defense systems such as the well-known DNA-targeting Cas9 and the RNA-targeting type III systems are widespread in prokaryotes1,2. The latter can orchestrate a complex antiviral response that is initiated by the synthesis of cyclic oligoadenylates (cOAs) upon foreign RNA recognition3-5. Among a large set of proteins that were linked to type III systems and predicted to bind cOAs6,7, a CRISPR associated Lon protease (CalpL) stood out to us. The protein contains a sensor domain of the SAVED (SMODS-associated and fused to various effector domains) family7, fused to a Lon protease effector domain. However, the mode of action of this effector was unknown. Here, we report the structure and function of CalpL and show that the soluble protein forms a stable tripartite complex with two further proteins, CalpT and CalpS, that are encoded in the same operon. Upon activation by cA4, CalpL oligomerizes and specifically cleaves the MazF-homolog CalpT, releasing the extracytoplasmic function (ECF) sigma factor CalpS from the complex. This provides a direct connection between CRISPR-based foreign nucleic acid detection and transcriptional regulation. Furthermore, the presence of a cA4-binding SAVED domain in a CRISPR effector reveals an unexpected link to the cyclic oligonucleotide-based antiphage signaling system (CBASS). Postprint