Crystal structures of porcine STINGCBD-CDN complexes reveal the mechanism of ligand recognition and discrimination of STING proteins

The cyclic dinucleotide (CDN)-stimulator of interferon genes (STING) pathway plays an important role in the detection of viral and bacterial pathogens in animals. Previous studies have shown that the metazoan second messenger cyclic [G(2 ',5 ')pA(3 ',5 ')p] (2 ',3 '-cGAMP) generated by cyclic GMP-AMP synthase cGAS binds STING with high affinity compared with bacterial CDNs such as c-di-GMP, c-di-AMP, and 3 ',3 '-cGAMP. Despite recent progress indicating that the CDN-binding domain (CBD) of dimeric STING binds asymmetric 2 ',3 '-cGAMP preferentially over symmetric 3 ',3 '-CDNs, it remains an open question whether STING molecules, such as human STING, adopt a symmetric dimeric conformation to efficiently engage its asymmetric ligand. Here, structural studies of the CBD from porcine STING (STING(CBD)) in complex with CDNs at 1.76-2.6 angstrom resolution revealed that porcine STING(CBD), unlike its human and mouse counterparts, can adopt an asymmetric ligand-binding pocket to accommodate the CDNs. We observed that the extensive interactions and shape complementarity between asymmetric 2 ',3 '-cGAMP and the ligand-binding pocket make it the most preferred ligand for porcine STING and that geometry constraints limit the binding between symmetric 3 ',3 '-CDN and porcine STING. The ligand-discrimination mechanism of porcine STING observed here expands our understanding of how the CDN-STING pathway is activated and of its role in antiviral defense.