Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Structural mechanism of cytosolic DNA sensing by cGAS

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.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Crystal Structure of cGASMab21.
Figure 2: The cGASMab21–DNA–GTP–ATP complex.
Figure 3: Platform and Zn thumb are involved in dsDNA-dependent activity.
Figure 4: NTase and DNA-induced structural switch.
Figure 5: Model for DNA sensing by cGAS.

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

References

  1. Rathinam, V. A. K. & Fitzgerald, K. A. Cytosolic surveillance and antiviral immunity. Curr. Opin. Virol. 1, 455–462 (2011)

    Article  CAS  Google Scholar 

  2. Takeuchi, O. & Akira, S. Pattern recognition receptors and inflammation. Cell 140, 805–820 (2010)

    Article  CAS  Google Scholar 

  3. Keating, S. E., Baran, M. & Bowie, A. G. Cytosolic DNA sensors regulating type I interferon induction. Trends Immunol. 32, 574–581 (2011)

    Article  CAS  Google Scholar 

  4. Krug, A. Nucleic acid recognition receptors in autoimmunity. Handb. Exp. Pharmacol. 183, 129–151 (2008)

    Article  CAS  Google Scholar 

  5. Takaoka, A. et al. DAI (DLM-1/ZBP1) is a cytosolic DNA sensor and an activator of innate immune response. Nature 448, 501–505 (2007)

    Article  CAS  ADS  Google Scholar 

  6. Bürckstümmer, T. et al. An orthogonal proteomic-genomic screen identifies AIM2 as a cytoplasmic DNA sensor for the inflammasome. Nature Immunol. 10, 266–272 (2009)

    Article  Google Scholar 

  7. Fernandes-Alnemri, T., Yu, J. W., Datta, P., Wu, J. & Alnemri, E. S. AIM2 activates the inflammasome and cell death in response to cytoplasmic DNA. Nature 458, 509–513 (2009)

    Article  CAS  ADS  Google Scholar 

  8. Hornung, V. et al. AIM2 recognizes cytosolic dsDNA and forms a caspase-1-activating inflammasome with ASC. Nature 458, 514–518 (2009)

    Article  CAS  ADS  Google Scholar 

  9. Ablasser, A. et al. RIG-I-dependent sensing of poly(dA:dT) through the induction of an RNA polymerase III-transcribed RNA intermediate. Nature Immunol. 10, 1065–1072 (2009)

    Article  CAS  Google Scholar 

  10. Chiu, Y. H., Macmillan, J. B. & Chen, Z. J. RNA polymerase III detects cytosolic DNA and induces type I interferons through the RIG-I pathway. Cell 138, 576–591 (2009)

    Article  CAS  Google Scholar 

  11. Yang, P. et al. The cytosolic nucleic acid sensor LRRFIP1 mediates the production of type I interferon via a β-catenin-dependent pathway. Nature Immunol. 11, 487–494 (2010)

    Article  CAS  Google Scholar 

  12. Kim, T. et al. Aspartate-glutamate-alanine-histidine box motif (DEAH)/RNA helicase A helicases sense microbial DNA in human plasmacytoid dendritic cells. Proc. Natl Acad. Sci. USA 107, 15181–15186 (2010)

    Article  CAS  ADS  Google Scholar 

  13. Zhang, Z. et al. The helicase DDX41 senses intracellular DNA mediated by the adaptor STING in dendritic cells. Nature Immunol. 12, 959–965 (2011)

    Article  CAS  Google Scholar 

  14. Unterholzner, L. et al. IFI16 is an innate immune sensor for intracellular DNA. Nature Immunol. 11, 997–1004 (2010)

    Article  CAS  Google Scholar 

  15. Rathinam, V. A. & Fitzgerald, K. A. Innate immune sensing of DNA viruses. Virology 411, 153–162 (2011)

    Article  CAS  Google Scholar 

  16. Ishikawa, H., Ma, Z. & Barber, G. N. STING regulates intracellular DNA-mediated, type I interferon-dependent innate immunity. Nature 461, 788–792 (2009)

    Article  CAS  ADS  Google Scholar 

  17. Ishikawa, H. & Barber, G. N. STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling. Nature 455, 674–678 (2008)

    Article  CAS  ADS  Google Scholar 

  18. Wu, J. et al. Cyclic GMP-AMP is an endogenous second messenger in innate immune signaling by cytosolic DNA. Science 339, 826–830 (2013)

    Article  CAS  ADS  Google Scholar 

  19. Abe, T. et al. STING recognition of cytoplasmic DNA instigates cellular defense. Mol. Cell 50, 5–15 (2013)

    Article  CAS  Google Scholar 

  20. Burdette, D. L. et al. STING is a direct innate immune sensor of cyclic di-GMP. Nature 478, 515–518 (2011)

    Article  CAS  ADS  Google Scholar 

  21. McWhirter, S. M. et al. A host type I interferon response is induced by cytosolic sensing of the bacterial second messenger cyclic-di-GMP. J. Exp. Med. 206, 1899–1911 (2009)

    Article  CAS  Google Scholar 

  22. Woodward, J. J., Iavarone, A. T. & Portnoy, D. A. c-di-AMP secreted by intracellular Listeria monocytogenes activates a host type I interferon response. Science 328, 1703–1705 (2010)

    Article  CAS  ADS  Google Scholar 

  23. 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)

    Article  CAS  ADS  Google Scholar 

  24. Kuchta, K., Knizewski, L., Wyrwicz, L. S., Rychlewski, L. & Ginalski, K. Comprehensive classification of nucleotidyltransferase fold proteins: identification of novel families and their representatives in human. Nucleic Acids Res. 37, 7701–7714 (2009)

    Article  CAS  Google Scholar 

  25. Cui, S. et al. The C-terminal regulatory domain is the RNA 5′-triphosphate sensor of RIG-I. Mol. Cell 29, 169–179 (2008)

    Article  CAS  Google Scholar 

  26. Witte, G., Hartung, S., Buttner, K. & Hopfner, K. P. Structural biochemistry of a bacterial checkpoint protein reveals diadenylate cyclase activity regulated by DNA recombination intermediates. Mol. Cell 30, 167–178 (2008)

    Article  CAS  Google Scholar 

  27. Stagno, J., Aphasizheva, I., Rosengarth, A., Luecke, H. & Aphasizhev, R. UTP-bound and apo structures of a minimal RNA uridylyltransferase. J. Mol. Biol. 366, 882–899 (2007)

    Article  CAS  Google Scholar 

  28. Xiong, Y. & Steitz, T. A. Mechanism of transfer RNA maturation by CCA-adding enzyme without using an oligonucleotide template. Nature 430, 640–645 (2004)

    Article  CAS  ADS  Google Scholar 

  29. Ablasser, A. et al. cGAS produces a 2′-5′-linked cyclic dinucleotide second messenger that activates STING Nature http://dx.doi.org/10.1038/nature12306 (30 May 2013)

  30. Hartmann, R., Justesen, J., Sarkar, S. N., Sen, G. C. & Yee, V. C. Crystal structure of the 2′-specific and double-stranded RNA-activated interferon-induced antiviral protein 2′-5′-oligoadenylate synthetase. Mol. Cell 12, 1173–1185 (2003)

    Article  CAS  Google Scholar 

  31. Donovan, J., Dufner, M. & Korennykh, A. Structural basis for cytosolic double-stranded RNA surveillance by human oligoadenylate synthetase 1. Proc. Natl Acad. Sci. USA 110, 1652–1657 (2013)

    Article  CAS  ADS  Google Scholar 

  32. 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)

    Article  CAS  Google Scholar 

  33. Cavlar, T., Deimling, T., Ablasser, A., Hopfner, K. P. & Hornung, V. Species-specific detection of the antiviral small-molecule compound CMA by STING. EMBO J. 32, 1440–1450 (2013)

    Article  CAS  Google Scholar 

  34. Kabsch, W. XDS. Acta Crystallogr. 66, 125–132 (2010)

    Article  CAS  Google Scholar 

  35. Vonrhein, C., Blanc, E., Roversi, P. & Bricogne, G. Automated structure solution with autoSHARP. Methods Mol. Biol. 364, 215–230 (2007)

    CAS  Google Scholar 

  36. Cowtan, K. The Buccaneer software for automated model building. 1. Tracing protein chains. Acta Crystallogr. 62, 1002–1011 (2006)

    Article  Google Scholar 

  37. Morris, R. J., Perrakis, A. & Lamzin, V. S. ARP/wARP’s model-building algorithms. I. The main chain. Acta Crystallogr. 58, 968–975 (2002)

    Google Scholar 

  38. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. 60, 2126–2132 (2004)

    Google Scholar 

  39. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. 66, 213–221 (2010)

    Article  CAS  Google Scholar 

  40. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007)

    Article  CAS  Google Scholar 

  41. Blanc, E. et al. Refinement of severely incomplete structures with maximum likelihood in BUSTER-TNT. Acta Crystallogr. 60, 2210–2221 (2004)

    Article  CAS  Google Scholar 

  42. Rao, F. et al. Enzymatic synthesis of c-di-GMP using a thermophilic diguanylate cyclase. Anal. Biochem. 389, 138–142 (2009)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank A. Butryn for comments on the manuscript. We thank the Max-Planck-Crystallization facility for initial crystal screening and the Swiss Light Source, European Synchrotron Radiation Facility and the German electron synchrotron Petra III for beam time and on-site assistance. This work was funded by the National Institutes of Health (U19AI083025), the European Research Council Advanced Grant 322869, and the Center for Integrated Protein Science Munich (CIPSM) to K.-P.H., by DFG grant 3717/2-1 to G.W., by GRK1721 to K.-P.H. and G.W., by DFG grant SFB670 and ERC grant 243046 to V.H.; C.C.O.M. is supported by GRK1721.

Author information

Authors and Affiliations

Authors

Contributions

F.C. crystallized and determined the structure of cGAS, performed biochemical assays, interpreted data and wrote the manuscript. T.D. crystallized and refined the DNA complex. C.C.O.M., A.A., T.D. and M.M. performed biochemical assays. G.W. performed biochemical assays, interpreted data and helped with structure determination. V.H. supervised the cell-based experiments and interpreted data. K.-P.H designed the research, helped with structure determination, interpreted data and wrote the manuscript.

Corresponding author

Correspondence to Karl-Peter Hopfner.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Additional information

Coordinates and structure factors have been deposited at the Protein Data Bank (4JLX, 4JLZ and 4KB6).

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1–6 and Supplementary Table 1. The figures show the activity both ‘in vitro’ and ‘in cells’, sequence conservation and secondary structure and the table summarizes the crystallographic data collection and model refinement statistics. (PDF 4253 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Civril, F., Deimling, T., de Oliveira Mann, C. et al. Structural mechanism of cytosolic DNA sensing by cGAS. Nature 498, 332–337 (2013). https://doi.org/10.1038/nature12305

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature12305

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing