Stimulator of interferon genes (STING) is a receptor in human cells that senses foreign cyclic dinucleotides released during bacterial infection and endogenous cyclic GMP–AMP signaling during viral infection and antitumor immunity 1 – 5 . STING shares no structural homology with other known signaling proteins 6 – 9 , limiting functional analysis and preventing explanation for the origin of cyclic dinucleotide signaling in mammalian innate immunity. Here we discover functional STING homologues encoded within prokaryotic defense islands and reveal a conserved mechanism of signal activation. Crystal structures of bacterial STING define a minimal homodimeric scaffold that selectively responds to c-di-GMP synthesized by a neighboring cGAS/DncV-like nucleotidyltransferase (CD-NTase) enzyme. Bacterial STING domains couple cyclic dinucleotide recognition with protein filament formation to drive TIR effector domain oligomerization and rapid NAD + cleavage. We reconstruct the evolutionary events following acquisition of STING into metazoan innate immunity and determine the structure of a full-length TIR-STING fusion from the Pacific oyster C. gigas . Comparative structural analysis demonstrates how metazoan-specific additions to the core STING scaffold enabled a switch from direct effector function to regulation of antiviral transcription. Together, our results explain the mechanism of STING-dependent signaling and reveal conservation of a functional cGAS-STING pathway in prokaryotic bacteriophage defense.

Highlights d Cap4 proteins are a major family of nucleotide second messenger receptors in bacteria d Cap4 receptors degrade DNA and mediate phage resistance by CBASS operons d The Cap4 ligand-binding SAVED domain evolved from CRISPR CARF proteins d As in cGAS-STING signaling, bacteria use 2 0 -5 0 -linked signals for antiviral immunity

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f IpaH proteins are bacterium-specific E3 enzymes that function as type three secretion system (T3SS) effectors in Salmonella, Shigella, and other Gram-negative bacteria. IpaH enzymes recruit host substrates for ubiquitination via a leucine-rich repeat (LRR) domain, which can inhibit the catalytic domain in the absence of substrate. The basis for substrate recognition and the alleviation of autoinhibition upon substrate binding is unknown. Here, we report the X-ray structure of Salmonella SspH1 in complex with human PKN1. The LRR domain of SspH1 interacts specifically with the HR1b coiled-coil subdomain of PKN1 in a manner that sterically displaces the catalytic domain from the LRR domain, thereby activating catalytic function. SspH1 catalyzes the ubiquitination and proteasome-dependent degradation of PKN1 in cells, which attenuates androgen receptor responsiveness but not NF-B activity. These regulatory features are conserved in other IpaH-substrate interactions. Our results explain the mechanism whereby substrate recognition and enzyme autoregulation are coupled in this class of bacterial ubiquitin ligases.