Extended Data Fig. 3: Biochemical analysis of c-di-GMP synthesis by bacterial STING-associated CD-NTases.

a, In addition to FsCdnE (Fig. 2b), CdnE homologues from three divergent STING-containing CBASS operons were purified and tested for cyclic-dinucleotide-synthesis specificity using α³²P-radiolabelled NTPs and thin-layer chromatography. Deconvolution experiments show a single major product requiring only GTP that migrates identically to c-di-GMP synthesized by the GGDEF enzyme WspR. All reactions were treated with alkaline phosphatase to remove exposed phosphates. Only two bacterial genomes encoding a STING-containing CBASS operon retain proteins with a predicted canonical GGDEF c-di-GMP signalling domain. The exceptions are chlorobi bacterium EBPR_Bin_190, which contains a single GGDEF domain that is fused to a SLATT domain and may be part of a CBASS-like system¹⁰, and a Lachnospiraceae bacterium RUG226 genome that encodes many GGDEF genes. The Lachnospiraceae bacterium RUG226 CdnE retains exclusive production of c-di-GMP suggesting the CdnE–STING system is sequestered in this bacterium or that an unknown mechanism may exist to prevent toxic STING activation. ReCdnE, Roseivirga ehrenbergii; CgCdnE, Capnocytophaga granulosa; LbCdnE, Lachnospiraceae bacterium; N, all four rNTPs; P_i, inorganic phosphate; ori., origin. Data are representative of two independent experiments. b, Nuclease treatment confirms that the FsCdnE enzymatic product contains only canonical 3′–5′ phosphodiester bonds. The [α³²P]GTP cyclic dinucleotide product is susceptible to cleavage by nuclease P1 resulting in release of GMP as a new species, which migrates further up the TLC plate. Further digestion with calf-intestinal phosphatase (CIP) removes all exposed phosphates, resulting in complete loss of a labelled product spot. DncV (Dinucleotide cyclase in Vibrio)-derived 3′,3′-cGAMP is similarly susceptible to complete digestion by P1 and CIP treatment, whereas 2′,3′-cGAMP synthesized by mouse cGAS is only partially digested owing to the presence of the non-canonical 2′–5′ bond. Data are representative of two independent experiments. c, High-resolution mass spectrometry analysis confirms the identity of the major FsCdnE enzymatic product as canonical c-di-GMP. Chemically synthesized c-di-GMP was used for direct spectral comparison. d, Sequence alignment and enlarged inset of the active-site of the RmCdnE structure in complex with nonhydrolyzable UTP and ATP analogues (PDB 6E0L), demonstrating a contact in the CD-NTase lid domain known to control nucleobase sequence specificity¹⁵. RmCdnE synthesizes cyclic UMP–AMP and uses N166 to specifically contact the uridine Watson–Crick edge. By contrast, FsCdnE and CgCdnE contain an aspartic acid substitution at this position and synthesize c-di-GMP, and V. cholerae DncV and human cGAS contain a serine substitution at this position and synthesize 3′,3′-cGAMP and 2′,3′-cGAMP. An aspartic acid at the FsCdnE D233 position is conserved among 93% of STING-associated CD-NTase enzymes (96 of 103), consistent with strict specificity of c-di-GMP as the nucleotide second messenger that controls bacterial STING activation. RmCdnE: PDB 6E0L¹⁵; V. cholerae DncV: PDB 4TY0⁵⁷; and human cGAS: PDB 6CTA (DNA omitted for clarity)³⁷. e, f, Mutational analysis of the importance of D233 in FsCdnE c-di-GMP synthesis activity. D233 substitutions do not disrupt the overall ability of FsCdnE to selectively synthesis c-di-GMP, but a D233A substitution causes a mild reduction in nucleobase selectivity and efficiency of c-di-GMP synthesis. These results are consistent with a role for D233 in nucleobase selection but demonstrate full selectively is achieved by additional contacts in the active site pocket. Data are representative of three independent experiments.