Inhibition by ATP regulates the activity of a CBASS anti-phage nucleotide cyclase

The bacterial anti-phage immune system is complex, diverse and in several important cases ancestral to that found in eukaryotes, including humans. One example is CBASS (Cyclic oligonucleotide Based Anti-phage Signalling System), a widespread bacterial defence that signals phage presence in the cell via cyclic nucleotide second messengers, activating ancillary effectors to combat infection. CBASS is homologous and ancestral to the eukaryotic cGAS/STING pathway for antiviral defence. The heart of the system is a nucleotide cyclase known as a CD-NTase (cGAS/DncV-like nucleotidyltransferase) which is activated by phage infection. The mechanisms of activation of CBASS cyclases are diverse and in most cases not fully understood at a molecular level. Moreover, it is vital to keep these signal-generating enzymes fully inactive in the absence of phage infection, to avoid auto-toxicity. Here, we report a structural and mechanistic study of a CBASS cyclase from Bacillus cereus (BcCdnD). Using crystal structures of key reaction intermediates, coupled with kinetic analyses, we show that the substrate, ATP, plays a fundamental role in the inhibition of the non-activated form of the enzyme in vitro. We provide a molecular explanation for this regulation and explore the implications for regulation of these important defence systems in bacterial immunity.