cGAS and CD-NTase enzymes: structure, mechanism, and evolution

Abstract

Cyclic GMP-AMP synthase (cGAS) is a signaling enzyme in human cells that controls immune-sensing of cytosolic DNA. The recent discoveries of diverse structural homologs of cGAS in animals and bacteria reveal that cGAS-like signaling is surprisingly ancient and widespread in biology. Together with the Vibrio cholerae protein dinucleotide cyclase in Vibrio (DncV), cGAS and DncV homologs comprise a family of cGAS/DncV-like nucleotidyltransferase (CD-NTase) enzymes that synthesize noncanonical RNA signals including cyclic dinucleotides, cyclic trinucleotides, and linear oligonucleotides. Structural and biochemical breakthroughs provide a framework to understand how CD-NTase signaling allows cells to respond to changing environmental conditions. The CD-NTase family also includes uncharacterized human genes like MB21D2 and Mab21L1, highlighting emerging functions of cGAS-like signaling beyond innate immunity.

Figure 1

CD-NTase signaling in animals and bacteria. (a) Cartoon schematic of the human cGAS-STING signaling pathway. cGAS binds to cytosolic double-stranded DNA and is activated to produce the second messenger 2′3′-cGAMP. 2′3′-cGAMP is recognized by STING, and initiates a downstream IRF3/NF-κB-dependent immune response. (b) Overview of Type I and Type II CD-NTase Signaling. Type I CD-NTase pathways like human cGAS-STING rely on detection of an activating ligand (e.g. cytosolic DNA) depicted here as a star. In contrast, Type II CD-NTase pathways like Vibrio DncV-CapV are active in the absence of ligand, and may rely on depletion of a ligand (e.g. folate-like metabolites) that represses enzyme activation. In either case, activated CD-NTases catalyze multi-turnover synthesis of a nucleotide second messenger (e.g. 2′3′-cGAMP or 3′3′-cGAMP) to amplify signaling and initiate downstream effector responses.

Figure 2

Structural anatomy of cGAS and CD-NTase enzymes. (a) Structural overview of the human cGAS–DNA complex. CD-NTase enzymes adopt a conserved cage-like structure where a long α-helix ‘spine’ braces an N-terminal NTase core and C-terminal helix bundle. Note, a 1:1 unit of cGAS–DNA is shown for clarity, but the minimally active complex is a 2:2 unit. (b) The cage-like architecture creates a deep cavity for nucleotide substrate coordination and enzymatic catalysis. Unlike classical nucleotidyltransferases (NTases) like DNA polymerase-β that require a nucleic acid template (orange, right) to dictate nucleotide substrate specificity, amino-acid side chains in the CD-NTase active-site lid (orange, left) directly control nucleotide interactions. (c) Structural overview of the ligand binding surfaces in the cGAS–DNA, OAS–RNA, and DncV–folate complexes. Structures are shown rotated 180° from the orientation in (a). Activating and inactivating CD-NTase ligands (yellow) bind on the back of the enzyme along the α-helix spine. Insertion of a zinc-ribbon (Zn-Ribbon) along the ligand-binding surface in part explains evolution of cGAS specificity for DNA interactions. (d) CD-NTase active sites contain a highly conserved h[QT]GS, DE[h[DE]h, h[DE]h catalytic triad and distinct ‘acceptor’ and ‘donor’ nucleotide pockets. Each CD-NTase catalyzes a multi-step reaction that proceeds through a defined reaction path (e.g. the cGAS intermediate product is pppG[2′–5′]pA).

Figure 3

Conservation and evolution of the CD-NTase enzyme family. (a, b) Overview of shared conservation of animal (top, magenta) and bacterial (bottom, green) CD-NTases. Distinct branches of individual animal CD-NTase proteins may have arisen from ancestral enzymes rooted in bacteria. Known structures are shown as schematics, and the hypothesized horizontal transfer events are indicated as a dashed line.