Structure-Guided Reprogramming of Human cGAS Dinucleotide Linkage Specificity

Cyclic dinucleotides (CDNs) play central roles in bacterial pathogenesis and innate immunity. The mammalian enzyme cGAS synthesizes a unique cyclic dinucleotide (cGAMP) containing a 2ʹ–5ʹ phosphodiester linkage essential for optimal immune stimulation, but the molecular basis for linkage specificity is unknown. Here, we show that the Vibrio cholerae pathogenicity factor DncV is a prokaryotic cGAS-like enzyme whose activity provides a mechanistic rationale for the unique ability of cGAS to produce 2ʹ–5ʹ cGAMP. Three high-resolution crystal structures show that DncV and human cGAS generate CDNs in sequential reactions that proceed in opposing directions. We explain 2ʹ and 3ʹ linkage specificity and test this model by reprogramming the human cGAS active site to produce 3ʹ–5ʹ cGAMP, leading to selective stimulation of alternative STING adaptor alleles in cells. These results demonstrate mechanistic homology between bacterial signaling and mammalian innate immunity and explain how active site configuration controls linkage chemistry for pathway-specific signaling. [Display omitted] •Vibrio cholerae DncV is a structural and functional homolog of human cGAS•DncV and human cGAS cyclic AMP-GMP formation proceeds in opposing reaction orders•Structures of catalytically trapped DncV explain eukaryotic 2ʹ–5ʹ linkage specificity•Reprogrammed human cGAS produces 3ʹ–5ʹ cGAMP and pathway-specific signaling A comparative structure-function analysis of the mammalian innate immunity enzyme cGAS and its homolog in bacteria, DncV, reveals the molecular basis of the unique 2ʹ–5ʹ phosphodiester linkage formed by the former.