Extended Data Fig. 1: Sequence and structural analysis of hMB21D2 and Tc-cGLR.

a, Structure guided sequence alignment of the catalytic domain of hcGAS (PDB: 4KM5)¹², hMB21D2 and Tc-cGLR. Strict secondary structure conservation further supports conserved structural homology despite primary sequence divergence. The [D/E]hD[X_50–90]D catalytic triad is highlighted with a red outline and the human Zn-ribbon insertion that is absent in other cGLRs is denoted with a red dashed outline. hMB21D2 contains an additional 61 residues that are not resolved in the crystal structure and are absent from the alignment. b, c, Zoomed-in cutaways of the hMB21D2 (b) and Tc-cGLR (c) crystal structures highlighting positioning of conserved catalytic residues in the nucleotidyltransferase active site. In hcGAS, the analogous residues coordinate two Mg²⁺ metal ions to control synthesis of 2′3′-cGAMP (inset, middle; PDB: 6CTA)¹⁴. The hMB21D2 structure is in an inactive state distinguished by misaligned catalytic residues and occlusion of the active site by an extended Gly-Gly activation loop, indicating that catalytic activation is probably controlled by a conformational rearrangement. d, e, TLC analysis of in vitro tests for potential activating ligands of hMB21D2. No nucleotide products were identified upon stimulation with 40-nt or 40-bp nucleic acid ligands (d) or ligands known to activate mammalian Toll-like receptors (e). Data shown are representative of n = 3 independent experiments. f, Z-score structural similarity plot showing homology between hMB21D2 and Tc-cGLR with representative structures in the PDB (PDB90). Increasing Z-score indicates greater homology, confirming the close relationship between animal cGLR enzymes and mammalian cGAS, and more distant similarity to cGAS/DncV-like nucleotidyltransferases (CD-NTases) in bacterial antiphage defence systems and human oligoadenylate synthase 1 (refs. ^{9,15,45,47,48,49,50}). Z-score cut-offs are 13 and 15 for hMB21D2 and Tc-cGLR, respectively.