Abstract
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 (STINGCBD) in complex with CDNs at 1.76-2.6 Å resolution revealed that porcine STINGCBD, 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.
Highlights
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 GMPAMP synthase cGAS binds STING with high affinity compared with bacterial CDNs such as c-di-GMP, c-di-AMP, and 3,3cGAMP
The binding affinity of porcine STING for 3Ј,3Ј-cGAMP ligand is the lowest with a Kd of 12.8 M in our microscale thermophoresis (MST) assay (Fig. 1D). These results show that, like STING from other species, porcine STING binds the endogenous ligand 2Ј,3Ј-cGAMP with the highest binding affinity
Summary
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 GMPAMP synthase cGAS binds STING with high affinity compared with bacterial CDNs such as c-di-GMP, c-di-AMP, and 3,3cGAMP. 2Ј,3Ј-cGAMP as the endogenous ligand has The STING protein consists of four transmembrane helices been shown previously to have higher binding affinity for STING in the N terminus that is followed by the cytosolic CDN-bindthan the bacterial second messengers [20, 21]. The full-length STING was solved by the cryo-EM method [23] It reveals that ligand binding induces these conformational changes mentioned above but results in the cytosolic domain half-turn rotation relative to the transmembrane region as well, which adds another layer of regulation of STING signaling.
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