Abstract
Cyclic GMP–AMP synthase (cGAS) is a pattern recognition receptor critical for the innate immune response to intracellular pathogens, DNA damage, tumorigenesis and senescence. Binding to double-stranded DNA (dsDNA) induces conformational changes in cGAS that activate the enzyme to produce 2′-3′ cyclic GMP–AMP (cGAMP), a second messenger that initiates a potent interferon (IFN) response through its receptor, STING. Here, we combined two-state computational design with informatics-guided design to create constitutively active, dsDNA ligand-independent cGAS (CA-cGAS). We identified CA-cGAS mutants with IFN-stimulating activity approaching that of dsDNA-stimulated wild-type cGAS. DNA-independent adoption of the active conformation was directly confirmed by X-ray crystallography. In vivo expression of CA-cGAS in tumor cells resulted in STING-dependent tumor regression, demonstrating that the designed proteins have therapeutically relevant biological activity. Our work provides a general framework for stabilizing active conformations of enzymes and provides CA-cGAS variants that could be useful as genetically encoded adjuvants and tools for understanding inflammatory diseases.
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References
Gao, P. et al. Cyclic [G(2′,5′)pA(3′,5′)p] is the metazoan second messenger produced by DNA-activated cyclic GMP–AMP synthase. Cell 153, 1094–1107 (2013).
Wu, J. et al. Cyclic GMP–AMP is an endogenous second messenger in innate immune signaling by cytosolic DNA. Science 339, 826–830 (2013).
Sun, L., Wu, J., Du, F., Chen, X. & Chen, Z. J. Cyclic GMP–AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway. Science 339, 786–791 (2013).
Ablasser, A. et al. cGAS produces a 29–59-linked cyclic dinucleotide second messenger that activates STING. Nature 398, 380–384 (2013).
Motwani, M., Pesiridis, S. & Fitzgerald, K. A. DNA sensing by the cGAS-STING pathway in health and disease. Nat. Rev. Genet. 20, 657–674 (2019).
Decout, A., Katz, J. D., Venkatraman, S. & Ablasser, A. The cGAS-STING pathway as a therapeutic target in inflammatory diseases. Nat. Rev. Immunol. 21, 548–569 (2021).
Le Naour, J., Zitvogel, L., Galluzzi, L., Vacchelli, E. & Kroemer, G. Trial watch: STING agonists in cancer therapy. Oncoimmunology 9, 1777624 (2020).
Wu, J.-J., Zhao, L., Hu, H.-G., Li, W.-H. & Li, Y.-M. Agonists and inhibitors of the STING pathway: potential agents for immunotherapy. Med. Res. Rev. 40, 1117–1141 (2020).
Larkin, B. et al. Cutting Edge: Activation of STING in T cells induces type I IFN responses and cell death. J. Immunol. 199, 397–402 (2017).
Bulcha, J. T., Wang, Y., Ma, H., Tai, P. W. L. & Gao, G. Viral vector platforms within the gene therapy landscape. Signal Transduct. Target Ther. 6, 53 (2021).
Buschmann, M. D. et al. Nanomaterial delivery systems for mRNA vaccines. Vaccines 9, 65 (2021).
Volkman, H. E., Cambier, S., Gray, E. E. & Stetson, D. B. Tight nuclear tethering of cGAS is essential for preventing autoreactivity. eLife 8, e47491 (2019).
Corrales, L. et al. Direct activation of STING in the tumor microenvironment leads to potent and systemic tumor regression and immunity. Cell Rep. 11, 1018–1030 (2015).
Sivick, K. E. et al. Magnitude of therapeutic STING activation determines CD8+ T cell-mediated anti-tumor immunity. Cell Rep. 25, 3074–3085.e5 (2018).
Francica, B. J. et al. TNFα and radioresistant stromal cells are essential for therapeutic efficacy of cyclic dinucleotide STING agonists in nonimmunogenic tumors. Cancer Immunol. Res. 6, 422–433 (2018).
Holleufer, A. et al. Two cGAS-like receptors induce antiviral immunity in Drosophila. Nature 597, 114–118 (2021).
Slavik, K. M. et al. cGAS-like receptors sense RNA and control 3′2′-cGAMP signalling in Drosophila. Nature 597, 109–113 (2021).
Hornung, V., Hartmann, R., Ablasser, A. & Hopfner, K.-P. OAS proteins and cGAS: unifying concepts in sensing and responding to cytosolic nucleic acids. Nat. Rev. Immunol. 14, 521–528 (2014).
Civril, F. et al. Structural mechanism of cytosolic DNA sensing by cGAS. Nature 498, 332–337 (2013).
Zhang, X. et al. The cytosolic DNA sensor cGAS forms an oligomeric complex with DNA and undergoes switch-like conformational changes in the activation loop. Cell Rep. 6, 421–430 (2014).
Li, X. et al. Cyclic GMP–AMP synthase is activated by double-stranded DNA-induced oligomerization. Immunity 39, 1019–1031 (2013).
Zhou, W. et al. Structure of the human cGAS–DNA complex reveals enhanced control of immune surveillance. Cell 174, 300–311.e11 (2018).
Andreeva, L. et al. cGAS senses long and HMGB/TFAM-bound U-turn DNA by forming protein–DNA ladders. Nature 549, 394–398 (2017).
Xie, W. et al. Human cGAS catalytic domain has an additional DNA-binding interface that enhances enzymatic activity and liquid-phase condensation. Proc. Natl Acad. Sci. USA 116, 11946–11955 (2019).
Hall, J. et al. The catalytic mechanism of cyclic GMP–AMP synthase (cGAS) and implications for innate immunity and inhibition. Protein Sci. 26, 2367–2380 (2017).
Huang, P.-S., Boyken, S. E. & Baker, D. The coming of age of de novo protein design. Nature 537, 320–327 (2016).
Kuhlman, B. et al. Design of a novel globular protein fold with atomic-level accuracy. Science 302, 1364–1368 (2003).
Leaver-Fay, A., Jacak, R., Stranges, P. B. & Kuhlman, B. A generic program for multistate protein design. PLoS ONE 6, e20937 (2011).
Leaver-Fay, A. et al. ROSETTA3: an object-oriented software suite for the simulation and design of macromolecules. Methods Enzymol. 487, 545–574 (2011).
Krissinel, E. & Henrick, K. Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions. Acta Crystallogr. D. Biol. Crystallogr. 60, 2256–2268 (2004).
Richter, V. et al. Structural and functional analysis of mid51, a dynamin receptor required for mitochondrial fission. J. Cell Biol. 204, 477–486 (2014).
Remmert, M., Biegert, A., Hauser, A. & Söding, J. HHblits: lightning-fast iterative protein sequence searching by HMM–HMM alignment. Nat. Methods 9, 173–175 (2011).
Whiteley, A. T. et al. Bacterial cGAS-like enzymes synthesize diverse nucleotide signals. Nature 567, 194–199 (2019).
Kranzusch, P. J., Lee, A. S.-Y., Berger, J. M. & Doudna, J. A. Structure of human cGAS reveals a conserved family of second-messenger enzymes in innate immunity. Cell Rep. 3, 1362–1368 (2013).
Humphreys, I. R. et al. Computed structures of core eukaryotic protein complexes. Science 374, eabm4805 (2021).
Tunyasuvunakool, K. et al. Highly accurate protein structure prediction for the human proteome. Nature 596, 590–596 (2021).
Du, M. & Chen, Z. J. DNA-induced liquid phase condensation of cGAS activates innate immune signaling. Science 1022, 704–709 (2018).
Lau, Y.-T. K. et al. Discovery and engineering of enhanced SUMO protease enzymes. J. Biol. Chem. 293, 13224–13233 (2018).
Ishikawa, H., Ma, Z. & Barber, G. N. STING regulates intracellular DNA-mediated, type I interferon-dependent innate immunity. Nature 461, 788–792 (2009).
Gall, A. et al. Autoimmunity initiates in nonhematopoietic cells and progresses via lymphocytes in an interferon-dependent autoimmune disease. Immunity 36, 120–131 (2012).
Snyder, A. G. et al. Intratumoral activation of the necroptotic pathway components RIPK1 and RIPK3 potentiates antitumor immunity. Sci. Immunol. 4, eaaw2004 (2019).
Liu, H. et al. Nuclear cGAS suppresses DNA repair and promotes tumorigenesis. Nature 563, 131–136 (2018).
Acknowledgements
We thank M. Lajoie, M. Pepper and D. Baker for discussions; D. Mannikko and S. Stoll for related investigations; J. Woodward for information and related investigations; R. Krishnamurty for project management; and members of the King laboratory for comments on the manuscript. Figure 5b was created using BioRender.com. This work was funded by a grant from the Bill & Melinda Gates Foundation (OPP1156262) to N.P.K. and D.B.S. D.B.S. is a Howard Hughes Medical Institute Faculty Scholar. X-ray data were collected at the Advanced Light Source at LBNL, supported by the Howard Hughes Medical Institute and grants from NIH (P30 GM124169-01, ALS-ENABLE P30 GM124169 and S10OD018483), NCI SBDR (CA92584) and DOE-BER IDAT (DE-AC02-05CH11231). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Authors and Affiliations
Contributions
Q.M.D., E.E.G., D.B.S. and N.P.K. conceived the study. Q.M.D. designed CA-cGAS. Q.M.D. and S.O. performed bioinformatics analyses. E.E.G., H.E.V. and S.C. performed ISRE, cellular cGAMP concentration and in vivo assays. M.R.J. performed in vivo assays. Q.M.D. performed biochemical analyses. Q.M.D. and A.K. performed crystal screens and optimization. B.S. collected the crystallographic data. A.K.B. and M.J.B. analyzed and processed the crystallography data. All authors analyzed data. Q.M.D., D.B.S. and N.P.K. wrote and revised the manuscript.
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Competing interests
Q.M.D., E.E.G., D.B.S. and N.P.K. are inventors on a patent application (PCT/US22/24255) related to constitutively active cGAS proteins. The King laboratory has received unrelated sponsored research agreements from Pfizer and GSK. The remaining authors declare no competing interests.
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Nature Structural & Molecular Biology thanks Philip Kranzusch, John Wilson and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Florian Ullrich, in collaboration with the Nature Structural & Molecular Biology team.
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Extended data
Extended Data Fig. 1 Schematic of the ISRE assay.
a, CA-cGAS plasmids are generated, combined with hSTING and ISRE-Luciferase plasmids, and b, transfected into 293 T cells. c, Cells were incubated for 4–16 hours, lysed, and luciferase activity measured. d, Luciferase activity of the CA-cGAS-04 variant compared to WT, K395A/K399A, and K395M/K399M cGAS.
Extended Data Fig. 2 Bifurcation in unique sequences used to find E-value cutoff.
The number of sequences in multiple sequence alignments constructed using cGAS (orange) or MiD-51 (blue) input sequences, as well as the number of unique sequences found only in the cGAS or MiD51 alignment but not both (grey), and the total number of sequences (black) plotted as a function of the E-value cutoff used to generate the alignment. The ratio of unique sequences to total sequences (green) is plotted on the secondary y axis.
Extended Data Fig. 3 CA-cGAS activity and reaction products in vitro.
a, CA-cGAS activity by ISRE assay increases over time post transfection. b, Chromatogram of CA-cGAS purification by nickel affinity chromatography. The high absorbance at 260 nm relative to 280 nm indicates co-purificiation of significant amounts of nucleic acid. c, Chromatogram of CA-cGAS purification by heparin affinity chromatography monitored by absorbance at 280 nm and 260 nm. Note the much lower relative absorbance at 260 nm compared to (b). d, UV-vis spectrum of nickel-, heparin-, SEC-purified CA-cGAS and the ratio of absorbance to 260 nm to 280 nm, indicating pure protein. e, 2′,3′-cGAMP was measured by competition ELISA after in vitro reactions of wild-type cGAS with dsDNA, or CA-cGAS-41 and CA-cGAS-50 without dsDNA and either 100 mM ATP and 100 mM GTP, or 200 mM ATP only. f, In vitro reactions failed to produce detectable 3′,3′-cGAMP.
Extended Data Fig. 4 DNA binding and oligomerization of WT and CA-cGAS variants.
a, Agarose gel electrophoresis of dsDNA mixed with increasing concentrations of CA-cGAS or WT cGAS. Top row stained with SYBR Safe. Bottom row is the same gel stained with coomassie. Molar ratios are, left to right, 1:0, 0:1, 1:0.1, 1:0.316, 1:1, 1:3.16, and 1:10 dsDNA to CA-cGAS. The increasing molar ratio is indicated by the triangle above the gel images. Electrophoretic mobility shift assays were performed at least three times for each sample with similar results. Representative images are shown. The assay was performed three times with similar results b, SEC chromatogram of WT or CA-cGAS with or without incubation with dsDNA oligomers. WT cGAS, CA-cGAS-41, and CA-cGAS-50 all elute as monomers in the absence of dsDNA, shifting to earlier elution volumes in the presence of excess DNA. c, Activity in cells is not affected by truncating the unstructured N-terminal domain (compare to Fig. 3b).
Extended Data Fig. 5 Similarities and differences between CA-cGAS crystal structures and the active conformation of mcGAS.
The crystallographic asymmetric units of a, CA-cGAS-41 and b, CA-cGAS-50 with 2Fo-Fc maps after model building and refinement. The activation loop in chain A of the asymmetric unit does not adopt the inactive (PDB ID 4K8V) or active (PDB ID 4K97) conformation in c, CA-cGAS-41 or d, CA-cGAS-50. e, Details of the CA-cGAS-41 mutations (labeled and colored blue). f, CA-cGAS-41 crystallographic contacts between chains A and B within the asymmetric unit. g, Details of the CA-cGAS-50 mutations (labeled and colored blue). h, CA-cGAS-50 crystallographic contacts between copies of the A chain in one asymmetric unit and the B chain in another asymmetric unit. All maps are 2Fo-Fc maps contoured at 1.0 sigma.
Extended Data Fig. 6 ISRE activity assays of additional mcGAS and hCGAS variants.
a, The activity of bioinformatics consensus mutations applied to the break in the spine helix in a CA-cGAS-41 background. b, Based on the available activity data, computational, and bioinformatics analysis, we can infer a series of mutations that may enhance the activity of CA-cGAS. Specifically, T197V variants are more active than variants without that mutation, variants with T197L are inactive, here T197I decreased the activity of CA-cGAS-41, suggesting that valine is the largest hydrophobic residue allowed at this position. R158S adds a capping residue on the carbonyl end of the N-terminal segment of the spine helix but does not increase activity. Y200I instead of Y200F makes CA-cGAS-41 more like CA-cGAS-50, testing the interchangeability of the activating mutations. Mutations at position 207 are designed to destabilize the inactive conformation of the active site loop. S207G and S207V are as active as CA-cGAS-41, but S207I is inactive. c, Recent work suggests phosphorylation at Y201 retains cGAS in the cytosol42. However, in the active conformation this residue is well packed; phosphorylation would likely inhibit activation, but it is unclear how phosphorylation might affect CA-cGAS activity. To mimic phosphorylation at residue 201, we introduced the mutation Y201E. We also made Y201F or Y201W mutations to prevent phosphorylation at that site. Y201E completely knocks out activity in WT cGAS and significantly lowers it in some, but not all, CA-cGAS variants. Mutating Y201 to phenylalanine or tryptophan had little effect on cGAS activity. d, The activity of CA-cGAS-22, -41, -42, and -50 mutations in an hcGAS background.
Extended Data Fig. 7 Schematic depiction of multi-state design framework and characterization pipeline.
The key steps of the design and characterization pipeline are depicted schematically. The steps at which each CA-cGAS variant was discovered are indicated.
Supplementary information
Supplementary Table 1-2
Supplementary Table 1: All cGAS designs screened by the ISRE assay and their activity. Supplementary Table 2: Sequences of CA-cGAS variants.
Source data
Source Data Fig. 1
Alpha-carbon distances between active and inactive states, computational design metrics, and ISRE raw data.
Source Data Fig. 2
MiD51 and cGAS hit scores, residue probability table.
Source Data Fig. 3
ISRE data, fluorescence intensity measurements, and slopes.
Source Data Fig. 5
cGAMP measurements and statistical source data for tumor measurements.
Source Data Extended Data Fig. 2
Sequence classification as a function of MSA permissiveness.
Source Data Extended Data Fig. 3
Chromatograms and spectra, cGAMP measurements.
Source Data Extended Data Fig. 4
Chromatograms and ISRE data.
Source Data Extended Data Fig. 4
Unprocessed gel images.
Source Data Extended Data Fig. 6
ISRE data.
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Dowling, Q.M., Volkman, H.E., Gray, E.E. et al. Computational design of constitutively active cGAS. Nat Struct Mol Biol 30, 72–80 (2023). https://doi.org/10.1038/s41594-022-00862-z
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DOI: https://doi.org/10.1038/s41594-022-00862-z
