Structural basis for nucleosome-mediated inhibition of cGAS activity

Abstract

Activation of cyclic GMP-AMP synthase (cGAS) through sensing cytosolic double stranded DNA (dsDNA) plays a pivotal role in innate immunity against exogenous infection as well as cellular regulation under stress. Aberrant activation of cGAS induced by self-DNA is related to autoimmune diseases. cGAS accumulates at chromosomes during mitosis or spontaneously in the nucleus. Binding of cGAS to the nucleosome competitively attenuates the dsDNA-mediated cGAS activation, but the molecular mechanism of the attenuation is still poorly understood. Here, we report two cryo-electron microscopy structures of cGAS-nucleosome complexes. The structures reveal that cGAS interacts with the nucleosome as a monomer, forming 1:1 and 2:2 complexes, respectively. cGAS contacts the nucleosomal acidic patch formed by the H2A-H2B heterodimer through the dsDNA-binding site B in both complexes, and could interact with the DNA from the other symmetrically placed nucleosome via the dsDNA-binding site C in the 2:2 complex. The bound nucleosome inhibits the activation of cGAS through blocking the interaction of cGAS with ligand dsDNA and disrupting cGAS dimerization. R236A or R255A mutation of cGAS impairs the binding between cGAS and the nucleosome, and largely relieves the nucleosome-mediated inhibition of cGAS activity. Our study provides structural insights into the inhibition of cGAS activity by the nucleosome, and advances the understanding of the mechanism by which hosts avoid the autoimmune attack caused by cGAS.

Fig. 1. Nucleosome-mediated inhibition of cGAS activity against dsDNA.

a A schematic diagram showing domains of the full-length cGAS (cGAS-FL) and the truncated cGAS (cGAS-CD) used for cryo-EM analysis. b EMSA analysis for binding of cGAS-FL and cGAS-CD in dimer or monomer states to mononucleosomes. Each lane contains 1 pmol of nucleosome reacting with 1–4 pmol of cGAS protein. The molar ratio of cGAS to the nucleosome is indicated at the top of the gel. c A bar diagram showing dose-dependent inhibition of cGAS activity by mononucleosomes. The inhibitory effects of 0–3 μM purified nucleosomes on the enzymatic activity of 5 μM dimeric cGAS against 7 μM 45 bp dsDNA were examined. Data are represented as means ± SEM (n = 3). NCP nucleosome core particle. AUC absorbance area under the curve.

Fig. 2. Overall structure of cGAS–nucleosome complexes.

a High-resolution composite cryo-EM map of the 1:1 cGAS–nucleosome complex. b Cartoon representation of the 1:1 cGAS-nucleosome complex in different views. c High-resolution composite cryo-EM map of the 2:2 cGAS–nucleosome complex. d Cartoon representation of the 2:2 cGAS–nucleosome complex in different views.

Fig. 3. Interaction between the cGAS site B and the nucleosomal acidic patch formed by H2A–H2B heterodimer.

a Location of the cGAS binding site on the acidic nucleosomal surface. The histone octamer is shown as electrostatic surface representation. Red represents negative charge, and blue represents positive charge. b Overview of the cGAS site B bound to the H2A–H2B heterodimer. c–f Detailed view of the interaction between cGAS and H2A–H2B heterodimer formed by Loop 1 (c), Loop 2 (d), Loop 3 (e) and α5 (f) from cGAS site B. Residues at the interface are shown as sticks. Densities of the side chains of the residues from the interaction regions are shown as blue meshes based on our 3.8 Å map. The side chains of the residues from the helix α5 of cGAS that may form potential interactions are also indicated (f), although their densities could not be well resolved.

Fig. 4. Analysis of the cGAS Mutations at the site B interface.

a Pull-down assays showing the effects of cGAS-CD mutants on H2A-H2B binding. MBP-His-tagged WT cGAS-CD and cGAS-CD mutants in monomer state were incubated with no-tagged H2A-H2B and NI-beads for 2 h. The bound proteins were analyzed by coomassie blue staining. b EMSA data for binding of cGAS-CD mutants to mononucleosomes. The monomeric cGAS-CD proteins were used in the EMSA experiments. Each lane contains 1 pmol of nucleosome reacting with 1–4 pmol of cGAS protein. The molar ratio of cGAS to the nucleosome is indicated at the top of the gel. c The cGAMP production level of 5 μM WT cGAS-CD or cGAS-CD mutants in dimer state against 7 μM dsDNA with or without 3 μM mononucleosomes. Data are represented as means ± SEM (n = 3). NCP nucleosome core particle. AUC absorbance area under the curve.

Fig. 5. Interaction between the cGAS site C and the nucleosomal DNA.

a Overview of the cGAS site C bound to the DNA from the adjacent nucleosome at the opposite side. b Detailed view of the interaction between cGAS site C and the nucleosomal DNA. c The electrostatic surface representation of the cGAS site C interface. Red represents negative charge, and blue represents positive charge. d EMSA analysis for binding of the cGAS-CD mutants at site C to mononucleosomes. The monomeric cGAS-CD proteins were used in the EMSA experiments. Each lane contains 1 pmol of nucleosome reacting with 1–4 pmol of cGAS protein. The molar ratio of cGAS to the nucleosome is indicated at the top of the gel. NCP, nucleosome core particle.

Fig. 6. Higher-order cGAS–nucleosome complex.

a–d cryo-EM map and structure of higher-order cGAS-nucleosome complex in a 4:3 manner. The models of cGAS and nucleosome are fitted into the map using UCSF chimera. The model of the 1:1 cGAS–nucleosome complex on the top is colored blue, the model of the 2:1 cGAS–nucleosome complex in the middle is colored orange and the model of the 1:1 cGAS–nucleosome complex at the bottom is colored pink.