Structure of human cGAS reveals a conserved family of second-messenger enzymes in innate immunity - PubMed (original) (raw)

Structure of human cGAS reveals a conserved family of second-messenger enzymes in innate immunity

Philip J Kranzusch et al. Cell Rep. 2013.

Abstract

Innate immune recognition of foreign nucleic acids induces protective interferon responses. Detection of cytosolic DNA triggers downstream immune signaling through activation of cyclic GMP-AMP synthase (cGAS). We report here the crystal structure of human cGAS, revealing an unanticipated zinc-ribbon DNA-binding domain appended to a core enzymatic nucleotidyltransferase scaffold. The catalytic core of cGAS is structurally homologous to the RNA-sensing enzyme, 2'-5' oligo-adenylate synthase (OAS), and divergent C-terminal domains account for specific ligand-activation requirements of each enzyme. We show that the cGAS zinc ribbon is essential for STING-dependent induction of the interferon response and that conserved amino acids displayed within the intervening loops are required for efficient cytosolic DNA recognition. These results demonstrate that cGAS and OAS define a family of innate immunity sensors and that structural divergence from a core nucleotidyltransferase enables second-messenger responses to distinct foreign nucleic acids.

Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

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Figures

Fig. 1

Fig. 1. Structure of Human cGAS

(A) Cartoon schematic of the human cGAS primary sequence. (B) Overall structure of human cGAS with the N-terminal helical extension, NTase core scaffold, and carboxy-terminal domain (C-domain) shown in blue. A unique zinc-ribbon insertion is shown in magenta, and the zinc ion in yellow. (C) Structural overlay of cytosolic nucleic acid sensors, human cGAS (blue) and human OAS (pink). (D) Electrostatic surface potential of cGAS (left); a conserved positively charged nucleic-acid binding cleft equivalent to the OAS dsRNA-binding site as observed in the structure of an OAS–dsRNA complex (right, PDB code 4IG8). See also Figures S2 and S3.

Fig. 2

Fig. 2. In Vitro Reconstitution of cGAS Dinucleotide Signaling

(A,B) Thin layer chromatography analysis of cGAS cyclic dinucleotide synthesis. Purified full-length cGAS was incubated with substrate nucleotides and immune-stimulatory DNA (ISD DNA) as indicated. An E225A/D227A mutation to the cGAS active site (Mut) ablates cyclic dinucleotide production. Dotted radioactive spots corresponding to UV-shadowed AMP and 3′–5′ linked cGAMP markers demonstrate the product of cGAS activity is a noncanonical dinucleotide product (see also fig. S4). (C) cGAS activity is strictly dependent on dsDNA activation. (D) Fluorescence anisotropy analysis of cGAS and related Mab21L2 NTase binding to dsDNA. See also Figure S4.

Fig. 3

Fig. 3. cGAS Zinc-Ribbon Domain Is Essential for Interferon Signaling

(A) Sequence alignment of human and murine cGAS and OAS cytosolic sensors. The unique cGAS zinc-ribbon insertion domain and coordinating residues are indicated. (B) Structural details of the zinc coordination site. Highly conserved amino acids are labeled. (C) Reconstitution of STING-dependent cGAS signaling in cells. Luciferase production under control of the interferon-β (IFNβ) promoter demonstrates that DNA-stimulated cGAS signaling only activates the interferon pathway in the presence of STING; cGAS E225A/D227A contains two point mutations in the enzymatic active site. (D) Mutational analysis of the cGAS zinc-ribbon motif. Substitutions to the zinc coordination motif (H390A, C396A, C397A, C404A) abolish cGAS activity when expressed at low levels (10 ng), and overexpression demonstrates only H390A retains weakened signaling potential (150 ng). Error bars represent the SD from the mean of at least three independent experiments.

Fig. 4

Fig. 4. cGAS Zinc-Ribbon and Positive DNA-binding Cleft Are Essential for DNA Recognition and Catalytic Activity

(A) Reconstitution of STING-dependent cGAS signaling in cells as described in Figure 3. Single alanine and glutamine mutations to conserved positively charged amino acids within the DNA-binding cleft demonstrate K173, K384, K407 and K414 are required for efficient cytosolic DNA detection. (B) In vitro reconstitution of cGAS dinucleotide synthesis using purified components as described in Figure 2 (*P < 0.001). Mutations to the catalytic active site (E225A/D227A), zinc-coordination motif (C396A) and conserved DNA-binding cleft (K394A, K394E, K407A, K407E) all abolish DNA-stimulated enzymatic activity. (C) The ability of mutant cGAS enzymes to engage a 45 bp ISD dsDNA substrate was measured by fluorescence polarization using 2 μM of purified protein as described in Figure 2D (*P < 0.001). Mutations to the catalytic active (E225A/D227A site do no disrupt DNA interactions while disruption of the zinc-coordination motif drastically inhibits the ability of cGAS to interact with dsDNA. Error bars represent the SD from the mean of at least three independent experiments. See also Figure S1C.

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