Structural mechanism of cGAS inhibition by the nucleosome

Abstract

histones in situ, which were partially lost upon aclarubicin treatment (Extended Data Fig. 1c, d). Thus, histones appear to dynamically engage cGAS in the nucleus.Consistent with prior work [13], functional analysis of cGAS in vitro enzymatic activity revealed that mononucleosomes (hereafter nucleosomes) inhibited DNA-induced cGAMP synthesis (Extended Data Fig. 1e). Likewise, compact chromatin fibres (12-mer nucleosome arrays) suppressed cGAS activity (Extended Data Fig. 1e). H2A-H2B dimers also had an inhibitory effect, but neither H2A or H2B monomers nor H3 or H4 monomers, respectively (Extended Data Fig. 1f, g). Thus, H2A-H2B dimers on their own can suppress cGAS (Extended Data Fig. 1h), albeit with weaker overall potency compared to fullassembled nucleosomes with additional features of nucleosomes in chromatin being necessary to exert maximal inhibition. Overall structure of the cGAS-NCP complexTo determine how cGAS interacts with nucleosomes, we pursued structural studies. A 1.5:1 molar mixture of human cGAS (residues 155 to 522) with a 147 bp 601 DNA nucleosome core particle (NCP) resulted in heterogenous particle distributions (Extended Data Fig. 2ad). To select for and stabilize more homogenous cGAS-NCP complexes, we combined gradient centrifugation with chemical crosslinking (GraFix) [15]. Both WT cGAS and cGAS K394E, a mutant impaired in dsDNA-mediated cGAS dimerisation [16], were used for structure determination. For the cGAS K394E mutant, we obtained a 4.1 Å reconstruction revealing two NCPs organized in a NCP 1 -cGAS 1 -cGAS 2 -NCP 2 sandwich arrangement with an expected molecular weight around 560 kDa, consistent with the most prominent peak fraction in multi-angle light scattering (MALS) (Fig. 1a, b, Extended Data Fig. 3, Supplementary Video 1, 2, and Extended Data Table 1a). The two individual nucleosomes are held together by two cGAS protomers. While the first cGAS protomer and its corresponding NCP (designated cGAS 1 and NCP 1 ) are well-resolved, the second nucleosome/cGAS pair (NCP 2 and cGAS 2 ) is less ordered (Extended Data Fig. 3e). In the dimeric NCP 1 -cGAS 1 -cGAS 2 -NCP 2 arrangement, each cGAS protomer interacts with the histone octamer of one NCP through histones H2A and H2B and the nucleosomal DNA (e.g. cGAS 1 and NCP 1 ), while contacting the second nucleosome (e.g. cGAS 1 and NCP 2 ) primarily through interactions with the nucleosomal DNA (Fig. 1a, b). In the WT cGAS structure, we observed a similar overall structural arrangement, with the NCP 1 -cGAS 1 -cGAS 2 -NCP 2 complex at 5.1Å and the focused NCP 1 -cGAS 1 structure at 4.7Å resolution (Extended Data

Keeping innate immune response in check: when cGAS meets the nucleosome

“…In parallel with this study, five other groups also reported on the cryo-EM structures of human and mouse cGAS-nucleosome complexes. [11][12][13][14][15] We note that the 2:2 and higher-order assemblies mediated by cGAS site C are observed in all three human cGAS-NCP structures 9,12,15 but not in their mouse counterparts. 11,13,14 Consistent with this distinction, site C has been shown to be an evolutionarily strengthened DNA-binding interface when proceeding from mouse to humans.…”

“…The studies discussed here 9,[11][12][13][14][15] provide compelling evidence of cGAS sequestration and inhibition by the nucleosome, with such nuclear tethering trapping cGAS in an inactive conformation. Fig.…”

Molecular and Structural Basis of DNA Sensors in Antiviral Innate Immunity

“…The structure of the cGAS catalytic domain bound to a nucleosome has been resolved by many groups, which reveals that cGAS inhibition in the nucleus is mediated by interaction through histone 2A-2B but not through nucleosomal DNA binding. The interaction between cGAS and histone embeds the cGAS DNA-binding site B, and prevents the formation of active cGAS dimers (108)(109)(110). Kujirai et al has reported a cryo-electron microscopy structure with two cGAS molecules bridging two nucleosome core particle (NCP).…”

Cisplatin Inhibits Bladder Cancer Proliferation Through cGAS-STING Pathway.

“…2E). The subcellular distribution of cGAS is closely related to its function [17][18][19][20][21] . At rst we con rmed that cGAS protein of T24 and TCCSUP cell lines was mainly located in nucleus (Fig.…”

“…Liu et al found that the nuclear translocation of cGAS in THP-1 and HeLa cells can affect its functions by suppressing homologous-recombination-mediated repair 16 . Studies have also shown that the binding capacity of cGAS to nucleosomes is much greater than that of cGAS to dsDNA [17][18][19][20][21] . This can explain why the cGAS-STING signal is not signi cantly activated during mitosis when a large amount of dsDNA is exposed to the cytoplasm due to the dissolved nuclear membrane.…”