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The molecular basis of tight nuclear tethering and inactivation of cGAS

Abstract

Nucleic acids derived from pathogens induce potent innate immune responses1,2,3,4,5,6. Cyclic GMP–AMP synthase (cGAS) is a double-stranded DNA sensor that catalyses the synthesis of the cyclic dinucleotide cyclic GMP–AMP, which mediates the induction of type I interferons through the STING–TBK1–IRF3 signalling axis7,8,9,10,11. cGAS was previously thought to not react with self DNA owing to its cytosolic localization2,12,13; however, recent studies have shown that cGAS is localized mostly in the nucleus and has low activity as a result of tight nuclear tethering14,15,16,17,18. Here we show that cGAS binds to nucleosomes with nanomolar affinity and that nucleosome binding potently inhibits its catalytic activity. To elucidate the molecular basis of cGAS inactivation by nuclear tethering, we determined the structure of mouse cGAS bound to human nucleosome by cryo-electron microscopy. The structure shows that cGAS binds to a negatively charged acidic patch formed by histones H2A and H2B via its second DNA-binding site19. High-affinity nucleosome binding blocks double-stranded DNA binding and maintains cGAS in an inactive conformation. Mutations of cGAS that disrupt nucleosome binding alter cGAS-mediated signalling in cells.

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Fig. 1: Tight nucleosome binding inactivates cGAS.
Fig. 2: Cryo-EM structure of mouse cGAS catalytic domain bound to human nucleosome.
Fig. 3: Interactions between cGAS and the nucleosome.
Fig. 4: Mutations at the cGAS–nucleosome interface affect nucleosome binding, dsDNA binding, cGAS activity and cGAS-mediated signalling.

Data availability

The three-dimensional cryo-EM density maps are deposited into the Electron Microscopy Data Bank (EMDB) under accession numbers EMD-22046, EMD-22206 and EMD-22047. The coordinates were deposited in the Protein Data Bank (PDB) with accession numbers 6X59, 6XJD and 6X5ASource data are provided with this paper.

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Acknowledgements

We acknowledge University of Texas, McGovern Medical School in Houston for providing access to their cryo-EM facility for data collection. We thank V. Mallampalli, G. Fan and I. Serysheva for assistance with cryo-EM data collection and Z. Cui at Texas A&M University for assistance with cryo-EM data processing. This research was supported in part by the Welch Foundation (Grants A-1931-20170325 to P.L. and A-1715 to W.R.L.) and National Institutes of Health (Grants R01 AI145287 to P.L., R01 GM121584 and R01 GM127575 to W.R.L.). A.P.W. and Y.L. were supported by NIH grant R01 HL148153 and award W81XWH-17-1-0052 through the Office of the Assistant Secretary of Defense for Health Affairs, Peer Reviewed Medical Research Programs.

Author information

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Authors

Contributions

P.L. conceived the study. B.Z. and P.X. expressed proteins, conducted binding studies and determined the structures. C.M.R. prepared the reconstituted nucleosome. T.J. conducted the binding studies and generated and purified the cGAS mutants. O.S. purified the cGAS mutants. Y.L. and A.P.W. studied the nuclear localization of cGAS. B.Z., P.X., W.R.L. and P.L. wrote the paper.

Corresponding authors

Correspondence to Wenshe Ray Liu or Pingwei Li.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature thanks Yuan He, Jae U Jung and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 cGAS binds tightly with nucleosome.

a, Gel-filtration chromatography and SDS–PAGE analyses of nucleosomes purified from HEK293T cells. b, Gel-filtration chromatography and SDS–PAGE analyses of the in vitro reconstituted nucleosomes. c, Gel-filtration chromatography showing that mouse cGAS catalytic domain binds to nucleosomes. d, SDS–PAGE analysis of nucleosome and mouse cGAS catalytic domain complex purified by gel-filtration chromatography. e, Polyacrylamide gel EMSA showing the interactions between mouse cGAS catalytic domain with reconstituted nucleosomes and nucleosomes purified from HEK293T cells. cGAS is mixed with nucleosomes at a molar ratio of 6:1. f, Agarose gel electrophoresis of the dsDNA from purified mononucleosomes and oligonucleosomes. The histones have been digested with proteinase K. g, SDS–PAGE analyses of purified mononucleosomes and oligonucleosomes. h, Polyacrylamide gel EMSA showing that mouse cGAS catalytic domain binds to mononucleosomes and oligonucleosomes (left panel). In the mixtures, the molar ratio of cGAS/mononucleosome is 3:1 and cGAS/oligonucleosome is 6:1. SDS–PAGE analyses of the input samples for EMSA were shown in the right panel. i, SDS–PAGE analyses of biotin-Avi-His6-SUMO fusion of human and mouse cGAS full length and catalytic domain proteins used for nucleosome binding studies.

Source data

Extended Data Fig. 2 cGAS-nucleosome binding studies and activity assays of cGAS-nucleosome complex.

a, SPR binding studies show that full-length human cGAS binds to nucleosomes purified from HEK293T cells with nanomolar affinity. b, SPR binding studies show that human cGAS catalytic domain binds to reconstituted nucleosomes with nanomolar affinity. c, d, Bio-layer interferometry binding studies of full-length human cGAS and its catalytic domain with nucleosomes (HEK293T). e, f, SPR binding studies show that full-length mouse cGAS and its catalytic domain bind nucleosomes (HEK293T) with nanomolar affinities. g, h, Bio-layer interferometry binding studies of full-length mouse cGAS and its catalytic domain with nucleosomes (HEK293T). i, Gel-filtration chromatography (top) and SDS–PAGE (bottom) analyses of 45-bp ISD dsDNA, nucleosome and cGAS mixture show that the nucleosome efficiently competes with dsDNA to bind cGAS. j, Agarose gel electrophoresis of the salmon sperm DNA used in cGAS activity assays. k, cGAS activity assays by ion exchange chromatography show that oligonucleosome binding potently inhibits the activity of cGAS and ligand-free cGAS can be activated by the cGAS-oligonucleosome complex.

Source data

Extended Data Fig. 3 Cryo-EM analysis of mouse cGAS catalytic domain in complex with the reconstituted nucleosome.

a, Purification and SDS–PAGE analysis of nucleosomes from HEK293T cells in complex with mouse cGAS catalytic domain. b, Purification and SDS–PAGE analysis of reconstituted nucleosomes in complex with mouse cGAS catalytic domain. c, Representative micrograph of mouse cGAS -nucleosome complex in vitrified ice. Scale bar, 50 nm. d, 2D class averages of mouse cGAS-nucleosome complex particles. Scale bar, 10 nm. e, Flowchart of data processing; see Methods for details. f, Angular distribution of the mouse cGAS-nucleosome (1:1) particles included in the final reconstruction. g, Angular distribution of the mouse cGAS-nucleosome (2:1) particles included in the final reconstruction. h, Final 3D reconstruction of the mouse cGAS-nucleosome (1:1) complex, coloured according to the local resolution. i, Final 3D reconstruction of the mouse cGAS-nucleosome (2:1) complex, coloured according to the local resolution. j, Corrected Gold-standard Fourier shell correlation curves of the mouse cGAS-nucleosome (1:1) complex for the 3D electron microscopy reconstruction. k, Corrected Gold-standard Fourier shell correlation curves of the mouse cGAS-nucleosome (2:1) complex for the 3D electron microscopy reconstruction. l, Polyacrylamide gel shift assay (left) showing that one nucleosome can bind to two molecules of mouse cGAS catalytic domain. Nucleosome is mixed with mouse cGAS at molar ratio of 1:1, 1:2 and 1:3. SDS–PAGE analysis of the samples used for the gel shift assays was shown on right panel. m, Density map (contoured at 3σ) and structural model of mouse cGAS-nucleosome (2:1) complex at 6.8 Å resolution.

Source data

Extended Data Fig. 4 Density maps and structural models of cGAS-nucleosome (reconstituted, 1:1) complex.

af, The density maps (grey mesh) of histones H2A, H2B, H3, H4, part of mouse cGAS catalytic domain, and the Widom 601 nucleosome positioning sequence DNA contoured at 3σ. The protein and DNA structures fitted into the density map are shown by the stick models.

Extended Data Fig. 5 Cryo-EM analysis of mouse cGAS domain in complex with nucleosome purified from HEK293T cells.

a, Representative micrograph of mouse cGAS-nucleosome complex in vitrified ice. Scale bar, 50 nm. b, 2D class averages of mouse cGAS-nucleosome complex particles. Scale bar, 10 nm. c, Flowchart of data processing; see Methods for details. d, Angular distribution of particles included in the final reconstruction. e, Final 3D reconstruction, coloured according to the local resolution. f, Corrected Gold-standard Fourier shell correlation curves for the 3D electron microscopy reconstruction. g, Density map (contoured at 3σ) and structural model of cGAS-nucleosome (1:1) complex.

Extended Data Fig. 6 Mutations in the acidic patch of the nucleosome abolished cGAS binding.

a, Superposition of structures of ligand-free mouse cGAS (PDB, 4K8V), mouse cGAS in complex with dsDNA (PDB, 4LEY), and mouse cGAS bound to the nucleosome. b, Sequence alignment of human and mouse cGAS around the nucleosome binding site. The conserved basic residues around the nucleosome binding site are coloured red. Residues that abolish nucleosome binding when mutated are highlighted yellow. c, Superposition for the structures of ligand-free human cGAS (PDB, 4LEV), ligand-free mouse cGAS (PDB, 4K8V) and mouse cGAS bound to the nucleosome. d, Ni-NTA agarose pull-down assays of mouse cGAS catalytic domain by His-tagged H2A-H2B dimer. The 6A dimer contains mutations E61A, E64A, D90A, E91A, E92A of H2A and D51A of H2B. The 6K dimer contains mutations E61K, E64K, D90K, E91K, E92K of H2A and D51K of H2B. e, Sequence alignment of wild-type (WT) human H2A and human H2A.Bbd. The acidic patch residues of WT H2A are coloured red. f, Polyacrylamide gel shift assay (left) showing that the recombinant nucleosome variant (H2A.Bbd) does not bind to mouse cGAS catalytic domain. In this assay, mouse cGAS was mixed with nucleosomes at a molar ratio of 3:1. SDS–PAGE analysis of the samples for gel shift assay were shown on the right panel.

Extended Data Fig. 7 Characterization of mouse cGAS catalytic domain mutants and oligonucleosome binding studies.

a, SDS–PAGE analysis of mouse cGAS catalytic domain mutants used for the gel shift assays and enzyme activity assays. b, SDS–PAGE analysis of mouse cGAS and nucleosome mixture samples used for the gel shift assay. c, Polyacrylamide gel EMSA shows that mutations at the cGAS-nucleosome interface affect oligonucleosome binding by cGAS. In these samples, mouse cGAS was mixed with oligonucleosomes at a molar ratio of 6:1. The samples used for the binding studies were analysed by SDS–PAGE (right panel). d, Circular dichroism of mouse cGAS catalytic domain and its mutants used for gel shift assays and enzyme activity assays. cGAS mutants that have strong binding to nucleosomes are shown by the spectra on the left. cGAS mutants that have weak or no binding to nucleosomes are shown by the spectra on the right.

Source data

Extended Data Fig. 8 Mutations at the cGAS-nucleosome interface affect nucleosome binding by human cGAS.

a, SDS–PAGE analysis of biotin-labelled full-length human cGAS mutants used for the SPR binding studies. bn, SPR binding studies of full-length human cGAS mutants with reconstituted nucleosomes.

Source data

Extended Data Fig. 9 Mutations at the cGAS-nucleosome interface affect dsDNA binding, cGAS activity and  cGAS-mediated signalling.

a, Agarose gel shift assay shows that mutations at the nucleosome binding surface of human cGAS affect the binding of a 45-bp dsDNA. b, Enzyme activity assays of mouse cGAS catalytic domain mutants by ion exchange chromatography. In this assay, 2.5 μM mouse cGAS was incubated with 0.2 mg ml−1 salmon sperm DNA. Wild-type mouse cGAS and negative control without DNA are coloured in green and blue. Mutations that abolish nucleosome binding are coloured red. The negative control mutation K382E is coloured orange. c, Enzyme activity assays of full-length human cGAS mutants by ion exchange chromatography. In this assay, 2.5 μM human cGAS was incubated with 0.2 mg ml−1 salmon sperm DNA. WT human cGAS and negative control without DNA are coloured in green and blue. Mutations that abolish nucleosome binding are coloured red. The negative control mutation K394E is coloured orange. d, IFN-β luciferase reporter assays show that mutations of human cGAS affect signalling in HEK293T cells. Luciferase reporter signals from the cells transfected with 0.025 ng cGAS and 0.4 ng STING are indicated by the orange bars, from cells transfected with 0.00625 ng cGAS and 0.4 ng STING by the cyan bars, from cells transfected with 0.025 ng cGAS, 0.4 ng STING, or the vector control by the green, brown and purple bars, respectively. The data (mean ± s.e.m.) are representative of three independent experiments. Each dot represents a biological replicate (n = 3). Two-tailed Student’s t-test: *P < 0.05, **P < 0.01, ***P < 0.001; NS, not significant. e, Western blot shows that WT mouse cGAS and its mutants have similar expression level in the transfected HEK293T cells. f, Western blot shows WT human cGAS and its mutants have similar expression level in the transfected HEK293T cells.

Source data

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics

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Zhao, B., Xu, P., Rowlett, C.M. et al. The molecular basis of tight nuclear tethering and inactivation of cGAS. Nature 587, 673–677 (2020). https://doi.org/10.1038/s41586-020-2749-z

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