b, Zoomed-in cutaways highlighting structural insertions and deletions unique to each cGLR. The purple and green boxes indicate cutaways in b. Structural comparison with the human cGAS (hcGAS)–DNA complex (Protein Data Bank (PDB): 6CTA) 14 reveals that cGLRs have a conserved architecture with a nucleotidyltransferase signalling core and a shared primary ligand-binding surface (dashed lines). Together, these results establish the existence of cGLRs in animals and demonstrate that remodelling of a primary ligand-binding surface enables the recognition of divergent molecular patterns.Ī, Crystal structures and surface electrostatics of hMB21D2 and Tc-cGLR. castaneum XP_969398.1 is a close homologue of mammalian cGAS and is distinct from previously characterized RNA sensors including oligoadenylate synthase 1 (ref. Despite exhibiting a clear difference in ligand specificity, analysis of all structures in the Protein Data Bank confirmed that T. castaneum XP_969398.1 is activated to synthesize a nucleotide product upon recognition of double-stranded RNA (dsRNA) (Fig. 1a) and we therefore tested this enzyme with candidate DNA and RNA ligands. castaneum XP_969398.1 shares highly conserved basic residues with human cGAS (Fig. In contrast to hMB21D2, the surface of T. The hMB21D2 surface is overall neutral with no obvious capacity to bind nucleic acids, and no enzymatic activity was detected with a panel of potential activating ligands (Extended Data Fig. We hypothesized that the remodelling of this groove controls the detection of distinct ligands. 1a), but is notably distinguished by the absence of a Zn-ribbon and the insertion of a C-terminal α-helix in hMB21D2 (Fig. A conserved groove is present in both the hMB21D2 and the T. In human cGAS, the primary ligand-binding surface is a long groove on the back of the enzyme formed by the α-helix spine and a Zn-ribbon motif that is essential for recognition of double-stranded DNA 3, 11, 12, 13, 14. castaneum XP_969398.1 structures each reveal close homology to human cGAS with a shared bi-lobed architecture, a caged nucleotidyltransferase core, a Gly- activation loop and a putative catalytic triad (Fig. Despite divergence in the primary sequence, the hMB21D2 and T. To define the function of cGAS-like enzymes in animals, we screened predicted cGAS homologues for suitability in structural analysis and determined a 2.4 Å crystal structure of the human protein MB21D2 (hMB21D2 encoded by C3orf59) and a 1.6 Å crystal structure of a protein from the beetle species Tribolium castaneum (GenBank XP_969398.1) (Supplementary Table 1). Similar to radiation of Toll-like receptors in pathogen immunity, our results establish cGLRs as a diverse family of metazoan pattern recognition receptors. A crystal structure of Drosophila stimulator of interferon genes (dSTING) in complex with 3′2′-cGAMP explains selective isomer recognition, and 3′2′-cGAMP induces an enhanced antiviral state in vivo that protects from viral infection. We show that RNA recognition activates Drosophila cGLR1 to synthesize the novel product cGpAp (3′2′-cGAMP). We demonstrate that surface remodelling of cGLRs enables altered ligand specificity and used a forward biochemical screen to identify cGLR1 as a double-stranded RNA sensor in the model organism Drosophila melanogaster. Crystal structures of human and insect cGLRs reveal a nucleotidyltransferase signalling core shared with cGAS and a diversified primary ligand-binding surface modified with notable insertions and deletions. Here we show that cGAS-like receptors (cGLRs) are innate immune sensors that are capable of recognizing divergent molecular patterns and catalysing synthesis of distinct nucleotide second messenger signals. Animal genomes typically encode multiple proteins with predicted homology to cGAS 6, 7, 8, 9, 10, but the function of these uncharacterized enzymes is unknown.
Cyclic GMP–AMP synthase (cGAS) is a cytosolic DNA sensor that produces the second messenger cGpAp (2′3′-cGAMP) and controls activation of innate immunity in mammalian cells 1, 2, 3, 4, 5.