A bacterial E2 enzyme regulates an antiviral cGAS through a peptide exchange-ligation-cleavage mechanism

Abstract

The cyclic oligonucleotide based anti phage signaling systems (CBASSs) are a family of defense systems in prokaryotes. Composed of a cyclic GMP-AMP synthase (cGAS) and CBASS-associated proteins, CBASSs utilize cyclic oligonucleotides to activate antiviral immunity. One major group of CBASS-associated proteins are homologs of eukaryotic E2 ubiquitin-conjugating enzymes. However, the function of E2 in CBASSs remains elusive. Here, we report that a bacterial E2 forms a covalent bond with cGAS, between the active site of E2 and a glycine near the C-terminus of cGAS. Unlike the eukaryotic ubiquitination system where E1 catalyzes covalent bond formation between E2 and ubiquitin, this covalent bond is directly formed between the “glycinated” cysteine at the E2 active site and the cGAS glycine residue, thereby releasing the C-terminal peptide of the cGAS. We further found that this link stabilizes cGAS, while it inhibits its synthetase activity. In the presence of danger signals such as ADP and PPi, the covalent E2-cGAS complex undergoes auto-proteolysis to release cGAS, which then produces 3',2'-cGAMP to activate antiviral defense. Our findings reveal a novel form of E2-mediated covalent modification, a negative regulatory role of E2 in CBASS, and an unexpected interplay between cGAS signaling and the ubiquitin system in bacteria.