Abstract
Cyclic-oligonucleotide-based antiphage signaling systems (CBASS) are diverse and abundant in bacteria. Here, we present the biochemical and structural characterization of two CBASS systems, composed of CdnG and Cap5, from Asticcacaulis sp. and Lactococcus lactis. We show that CdnG from Asticcacaulis sp. synthesizes 3′,2′-cGAMP in vitro, and 3′,2′-cGAMP is the biological signaling molecule that activates Cap5 for DNA degradation. Crystal structures of Cap5, together with the SAVED domain in complex with 3′,2′-cGAMP, provide insight into the architecture of Cap5 as well as molecular recognition of 3′,2′-cGAMP by the SAVED domain of Cap5. Amino acid conservation of the SAVED domain of Cap5, together with mutational studies, led us to propose a mechanism of Back-to-Front stacking of two SAVED domains, mediated by 3′,2′-cGAMP, to activate HNH nuclease domain for DNA degradation. This study of the most abundant CBASS system provides insights into the mechanisms employed by bacteria in their conflicts against phage.
Highlights
Cyclic-oligonucleotide-based antiphage signaling systems (CBASS) are diverse and abundant in bacteria
Because Sorek and colleagues employed short E2-like proteins (PF14457) for their classification of CBASS Type II12, the three-gene operons depicted in Fig. 1a appear to represent a new family of Type II system because both proteins encoded by the third genes belong to a new protein family without a Pfam number
We have reported the biochemical characterization of a member of the G clade of CD-NTases, and evidence for the enzymatic synthesis of 3′,2′-cGAMP in bacteria
Summary
Cyclic-oligonucleotide-based antiphage signaling systems (CBASS) are diverse and abundant in bacteria. We present the biochemical and structural characterization of two CBASS systems, composed of CdnG and Cap[5], from Asticcacaulis sp. Synthesizes 3′,2′-cGAMP in vitro, and 3′,2′-cGAMP is the biological signaling molecule that activates Cap[5] for DNA degradation. DncV-CapV represents the first characterized antiphage defense system that shares a similar mechanism to the eukaryotic cGAS-STING system. Recent studies by Sorek and colleagues indicate that the antiphage systems similar to DncV-CapV are abundant in bacteria, and these systems are collectively named cyclic-oligonucleotide-based antiphage signaling systems (CBASS)[11,12]. We report biochemical and structural characterization of two Cap[5] effectors composed of SAVED and HNH domains as well as their cognate CdnG proteins that synthesize the signaling molecule. Crystal structures of Cap[5], as well as SAVED domain in complex with 3′,2′-cGAMP provide molecular insight into activation of Cap[5] by 3′,2′-cGAMP
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