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

• Published in: Biochemical and biophysical research communications Vol. 727; p. 150326
• Main Authors: Li, Fangqi, Ma, Cuiyan, Ding, Xiang, Niu, Lili, Guo, Linyue, Yang, Fuquan, Ma, Wenfu
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.10.2024
• Subjects: Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Cap2; Cap3; CBASS; CD-NTase; CD-NTase conjugation; cGAS; Crystallography, X-Ray; Hydrolysis; Models, Molecular; Nucleotidyltransferases - chemistry; Nucleotidyltransferases - genetics; Nucleotidyltransferases - metabolism; Protein Conformation; cGAS; CD-NTase; CBASS; CD-NTase conjugation; Cap3; Cap2
• ISSN: 0006-291X; 1090-2104; 1090-2104
• DOI: 10.1016/j.bbrc.2024.150326

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.