Ancient Origin of cGAS-STING Reveals Mechanism of Universal 2′,3′ cGAMP Signaling

Abstract

Summary In humans, the cGAS-STING immunity pathway signals in response to cytosolic DNA via 2′,3′ cGAMP, a cyclic dinucleotide (CDN) second messenger containing mixed 2′–5′ and 3′–5′ phosphodiester bonds. Prokaryotes also produce CDNs, but these are exclusively 3′ linked, and thus the evolutionary origins of human 2′,3′ cGAMP signaling are unknown. Here we illuminate the ancient origins human cGAMP signaling by discovery of a functional cGAS-STING pathway in Nematostella vectensis, an anemone species >500 million years diverged from humans. Anemone cGAS appears to produce a 3′,3′ CDN that anemone STING recognizes through nucleobase-specific contacts not observed in human STING. Nevertheless, anemone STING binds mixed-linkage 2′,3′ cGAMP indistinguishably from human STING, trapping a unique structural conformation not induced by 3′,3′ CDNs. These results reveal that human mixed-linkage cGAMP achieves universal signaling by exploiting a deeply conserved STING conformational intermediate, providing critical insight for therapeutic targeting of the STING pathway.

Analysis of Drosophila STING Reveals an Evolutionarily Conserved Antimicrobial Function

“…Interestingly, a functional cGAS-STING pathway exists in the sea anemone Nematostella vectensis . However, purified sea anemone cGAS is not active in vitro but will function in human cells, suggesting that there are additional co-factors that are required for cGAS activity (Kranzusch et al, 2015). Our experiments using dsDNA virus infection in flies containing P elements in CG7194, a putative cGAS ortholog in Drosophila (Wu et al, 2014), further suggest the lack of a cGAS-STING axis in Drosophila since we did not observe the induction of defense response genes or the dependence of dmSTING on survival to IIV6 infection.…”

Versatile modes of cellular regulation via cyclic dinucleotides

“…These data suggest that potent metazoan 2′,3′-cGAMP-STING activation possibly evolved through the exploitation of a highly conserved STING conformational intermediate that is not typically sampled by prokaryotic CDNs 94 .…”

“…In particular, structures of the human protein show a relatively splayed dimer 88,91,95 , whereas mouse STING crystallizes in a closed conformation with the two protomers rotated and brought in together relative to the 2-fold symmetry axis 93 . Interestingly, an ancient anemone STING homolog was crystallized in both an ‘open’ and a ‘closed’ state suggesting that the two likely represent alternatively sampled conformations rather than species-specific differences among homologs 94 .…”

“…The latter forms a ‘lid’ that caps the top of the CDN binding pocket and further restricts solvent access 92,94,96 . Apart from the pivotal role of Tyr 167 , the dinucleotide is further stabilized by stacking interactions with Arg 238 , which is itself buttressed by another lid’ residue, Tyr 240 92,96 .…”

“…Importantly, although anemone STING recognizes a 3′-3′-linked cGAMP signal naturally and binds the dinucleotide in yet a different splayed conformation, its mode of 2′,3′-cGAMP coordination is virtually identical to those of the human and mouse variants 94 . These data suggest that potent metazoan 2′,3′-cGAMP-STING activation possibly evolved through the exploitation of a highly conserved STING conformational intermediate that is not typically sampled by prokaryotic CDNs 94 .…”