Extended Data Fig. 9: Structural analysis of metazoan TIR–STING homologues.

a, Structure-guided alignment of the TIR domain in oyster TIR–STING with reference bacterial and metazoan TIR-domain-containing proteins. SARM1 is an example of a human TIR domain that catalyses NAD⁺ cleavage, and MyD88 is an example of a human TIR domain that signals through protein–protein interaction. The catalytic glutamate responsible for supporting NAD⁺ cleavage is conserved at the same spatial position among bacterial and oyster TIRs but is mutated in MyD88 (green box). However, it is not currently possible to predict from structure or sequence alone whether TIR domains have enzymatic activity. b, Distinct from other TIR domain structures, the TIR domain in oyster TIR–STING contains a proline-rich loop region at the interface, suggesting a specific role in dimer stabilization. c, Superposition of a homology model of the SfSTING TIR domain with the TIR domain of oyster TIR–STING shows the predicted catalytic glutamates for both proteins occupy distinct locations in the TIR fold. d, Superposition of a homology model of the SfSTING TIR domain compared to the crystal structure of human SARM1 bound to ribose implies that different NAD⁺ binding pockets may exist between bacterial and eukaryotic TIRs, as previously suggested²¹. e, Superposition of a homology model of the SfSTING TIR domain with the bacterial TIR domain from Paracoccus denitrificans shows a high degree of similarity. No crystal structures are available for bacterial TIR domains in an active state, preventing identification of a specific mechanism of catalytic activation. f, EMSA analysis of oyster TIR–STING and mouse STING demonstrates a wide preference for cyclic dinucleotide interactions and clear ability to recognize the mammalian cGAS product 2′,3′-cGAMP. Data are representative of three independent experiments. g, h, Oyster TIR–STING, which binds all tested cyclic dinucleotides, does not exhibit NAD⁺ cleavage activity even at 10× the protein and ligand concentrations used to achieve robust activity with bacterial TIR–STING. We tested four other oyster TIR–STING homologues and observed no cyclic-dinucleotide-stimulated NAD⁺ cleavage activity. These results support a potential switch in TIR-dependent protein–protein interactions to control downstream signalling similar to the TIR domain in human MyD88. Data are representative of two independent experiments.