A unique Z-shaped tetramer mediates the autoinhibition of waterfowl STING.

Stimulator of interferon genes (STING) is a central player of innate immunity, coordinating host defense against viral infection and cancer. While the canonical architectures of apo-STING and ligand-bound STING have been established, current knowledge is limited to a subset of species, and a comprehensive cross-species, ligand-resolved structural atlas remains incomplete. Here, we determined the high-resolution crystal structures of duck and bovine STING ligand-binding domains (LBDs) bound to 2'3'-cGAMP and of duck, bovine, and human STING LBDs bound to the non-nucleotide agonist diABZI3. In the 2'3'-cGAMP complexes, the lid regions were ordered and completely covered the ligand-binding pocket, whereas in the diABZI3 complexes, the lid regions were completely disordered. In human and bovine STING, 2'3'-cGAMP induced a more closed dimer conformation than diABZI3, while duck STING exhibited minimal differences in closure between the two ligands. Strikingly, non-reducing SDS-PAGE revealed a distinct disulfide-linked tetramer in duck STING, which is abolished by the C195S mutation. Within the crystal lattice of the duck STING LBD-2'3'-cGAMP complex, we observed a unique Z-shaped tetramer stabilized by an interfacial disulfide bond between C195 and a network of polar interactions. Disrupting this interface, either by the C195S mutation or by ligand stimulation with 2'3'-cGAMP or diABZI3, relieved the tetrameric constraint and amplified STING signaling, establishing this tetramer as a duck-specific autoinhibitory assembly. These findings expand the structural repertoire of STING oligomeric assemblies, fill the structural gap for duck STING, and provide a comparative structural framework for species-specific STING regulation.