Structural and functional characterization of the bacterial Cap3 enzyme in deconjugation and regulation of the cyclic dinucleotide transferase CD-NTase

The Cyclic GMP-AMP synthase (cGAS) and cGAS/DncV-like nucleotidyltransferase (CD-NTase) enzymes belong to the key components of the innate immune sensor system that generates cyclic dinucleotide molecules in response to danger signals. Recently, it was discovered that CD-NTase in bacteria can undergo conjugation to protein substrates via an E1/E2 enzyme-mediated process, resembling ubiquitin modification system. Subsequently, these CD-NTase conjugated molecules will be hydrolyzed by the Cap3 enzyme in the same gene cluster. However, the experimental structure of bacterial CD-NTase recognized by Cap3 is unknown. Here, we first determined the crystal structure of the Cap3 enzyme in complex with the C-terminal tail of CD-NTase. Our structural and enzymatic analysis revealed that the C-terminal tail of CD-NTase is both necessary and sufficient for the Cap3-mediated hydrolysis of CD-NTase from its substrates. Interestingly, we further observed that after the hydrolysis reaction, the terminal glycine residue of the CD-NTase C-terminal tail was sequentially removed by Cap3, indicating that Cap3 might play a role in quenching the CD-NTase conjugation reaction. Our work provides experimental evidence elucidating the interaction between Cap3 and CD-NTase, and suggests a potential role for Cap3 in the bacterial Cyclic-oligonucleotide-based anti-phage signaling system (CBASS). •The first experimental structural data of the Cap3 protease bound to the C-terminal regions of the CD-NTase protein has been obtained.•The C-terminal tail of CD-NTase is sufficient for recognition and cleavage by Cap3.•The potential Cap3 cleavage site of CD-NTase conjugates mirrors the dynamic coupling between cleavage and conjugation reactions.