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. 2013 Jun 20;498(7454):332-7.
doi: 10.1038/nature12305. Epub 2013 May 30.

Structural mechanism of cytosolic DNA sensing by cGAS

Filiz Civril  1 , Tobias Deimling, Carina C de Oliveira Mann, Andrea Ablasser, Manuela Moldt, Gregor Witte, Veit Hornung, Karl-Peter Hopfner
Affiliations

Structural mechanism of cytosolic DNA sensing by cGAS

Filiz Civril et al. Nature. .

Abstract

Cytosolic DNA arising from intracellular bacterial or viral infections is a powerful pathogen-associated molecular pattern (PAMP) that leads to innate immune host defence by the production of type I interferon and inflammatory cytokines. Recognition of cytosolic DNA by the recently discovered cyclic-GMP-AMP (cGAMP) synthase (cGAS) induces the production of cGAMP to activate the stimulator of interferon genes (STING). Here we report the crystal structure of cGAS alone and in complex with DNA, ATP and GTP along with functional studies. Our results explain the broad DNA sensing specificity of cGAS, show how cGAS catalyses dinucleotide formation and indicate activation by a DNA-induced structural switch. cGAS possesses a remarkable structural similarity to the antiviral cytosolic double-stranded RNA sensor 2'-5'oligoadenylate synthase (OAS1), but contains a unique zinc thumb that recognizes B-form double-stranded DNA. Our results mechanistically unify dsRNA and dsDNA innate immune sensing by OAS1 and cGAS nucleotidyl transferases.

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Figures

Figure 1
Figure 1. Crystal Structure of Mab21 cGASMab21
a) Activity assays of human and porcine cGASMab21 alone or in presence of dsDNA. B. subtilis DisA, a c-di-AMP synthase is used as positive control. The di-nucleotide products are indicated with asterisks. b) Side and top views of cGASMab21. The model is shown as ribbon representation with annotated domains and secondary structure (blue α-helices, yellow β-strands). c) Close up view of the ‘zinc-thumb’.
Figure 2
Figure 2. The cGASMab21:DNA:GTP:ATP complex
a). Side and top views of cGASMab21 (color code of Fig. 1b) in complex with dsDNA (brown), GTP and ATP (ruby stick models). DNA binds along the platform between spine and Zn-thumb. b) Close up view of the DNA binding site with selected annotated residues. DNA is bound mainly via the minor groove. A notable exception is the Zn-thumb near the major groove. c) Schematic representation of DNA:cGAS contacts.
Figure 3
Figure 3. Platform and zinc thumb are involved in dsDNA-dependent activity
a) NTase assays performed with different cGASMab21 mutants (2µM) in presence of 3µM dsDNA (50mer). Human WT cGASMab21 (positive control) synthesizes di-nucleotide, DNA binding site mutant K173A+R176A show reduced activity. K407A+K411A DNA-binding site mutant, C396A+C397A Zn-thumb mutant, Zn-thumbless, L174N structural switch mutant, active site mutants E200Q+D202N of porcine cGASMab21 and E225A+ D227A and G212A+S213A of human cGASMab21 are inactive. The asterisk indicates the di-nucleotide product. b) IFN-β stimulation of cGAS mutants in HEK293T cells stably expressing murine STING. HEK293T cells were transfected with plasmids encoding indicated constructs along with the IFN-β promoter reporter plasmid pIFN-β-GLUC. Luciferase activity is plotted: mean ± sd (n=3). Both full-length and the crystallized region (cGASMab21 human 155–522) induce IFN-β promoter transactivation. Active site mutations (G212A+S213A and E225Q/A+D227N/A) abolish IFN-β stimulation. DNA-binding site mutants (K173A+R176A, K407A+K411A), Zn -thumb mutants (C396A+C397A, Zn-thumbless) and structural switch mutant (L174N) either reduce or abolish IFN-β stimulation. Empty vector was used as negative control while cyclic-di-GMP synthase (cdG syn) expressing vector was used as positive control. Inset: Western blot showing WT and mutant protein levels with β-actin as loading control. c) Electrophoretic mobility shift analysis of 50mer dsDNA (0.2 µM) bound to cGASMab21 mutants at indicated concentrations. Plotted bars: mean±sd (n=3). While K407A+K411A DNA-binding site mutant and C396A+C397A Zn-thumb mutant show slightly reduced but not impaired affinity to dsDNA no detectable binding change was observed with the other mutants.
Figure 4
Figure 4. NTase and DNA induced structural switch
a) Close-up view of the NTase active site. Selected residues that are implicated in binding and catalysis are annotated. Both base moieties partially stack to each other and are further bound by stacking to Y413 and recognition by R353. E200 (mutated to Q in our structure) and D202 (mutated to N in our structure) bind an active site magnesium that coordinates phosphates of nucleotide 2. The attacking OH of nucleotide 1 is activated by D296 for nucleophilic attack on the α-phosphate of nucleotide 2 (arrow). b) Close up view of DNA backbone phosphate binding at the spine. c) This DNA phosphate binding triggers a change in the spine helix (*), which allows a closure of the active site cleft (**) and repositioning of the substrate binding loop for Mg2+-coordination of E200 (***).
Figure 5
Figure 5. Model for DNA sensing by cGAS
a) Superposition of cGAS�DNA (blue) with OAS1�RNA (grey) shows key elements for nucleic duplex selectivity. Both enzymes bind one DNA (brown) / RNA (black) backbone at the same protein site (*). The Zn-thumb specifically recognizes the position of the second DNA strand in B-form (**). However, it would clash with A-form RNA/DNA. b) Unified activation model for cytosolic double-stranded nucleic acid sensing by cGAS and OAS1 NTases by a ligand induced structural switch.
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