Structural insights into signaling promiscuity of the CBASS anti-phage defense system from a radiation-resistant...

Radiation-resistant bacteria are of great application potential in various fields, including bioindustry and bioremediation of radioactive waste. However, how radiation-resistant bacteria combat against invading phages is seldom addressed. Here, we present a series of crystal structures of a sensor and an effector of the cyclic oligonucleotide-based anti-phage signaling system (CBASS) from a radioresistant bacterium Deinococcus wulumuqiensis. We found that the sensor CD-NTase enzyme, DwCdnB, can bind all four ribonucleotides and synthesize a variety of cyclic di-nucleotides, including the novel second messenger 3'3'-cyclic di-CMP. Crystal structures of DwCdnB in complex with ATP and dATP provide structural explanations for specific recognition of ribonucleotides via metal coordination with ribose 2'-OH. Crystal structures of DwCdnB in complex with purine and/or pyrimidine nucleotides in the presence of Mg²⁺ revealed similar binding modes; however, in the presence of Mn²⁺, the UTP/CTP rotates and flips into the donor pocket and make extensive contacts with additional five residues, suggesting essential role of Mn²⁺ for catalytic production of cyclic di-pyrimidines. Finally, structural analysis of the downstream effector DwCap5 further provides a structural explanation for its non-specific recognition of a broad range of cyclic di-nucleotides. In sum, this work provides key structural insights into the immune mechanisms of radioresistant bacteria.