Mini reviewThe mechanism of double-stranded DNA sensing through the cGAS-STING pathway
Introduction
Nucleic acids, such as DNA, RNA, or nucleotides, from viral or bacterial pathogens induce potent immune responses in the infected cells [1], [2], [3], [4], [5], [6], [7]. The detection of microbial nucleic acids is a central strategy by which the host senses infection and initiates protective immune responses [6], [7]. A number of innate sensors for microbial DNA or RNA have been identified [3], [4], [8], [9]. For example, toll-like receptor TLR9 recognizes CpG DNA, TLR3 recognizes dsRNA, and the RIG-I like receptors recognize dsRNA. Microbial DNA in the cytosol has long been known to induce potent innate immune responses by inducing the expression of type I interferons [10], [11], [12]. The search for cytosolic DNA sensors first leads to the discovery of STING (also known as MITA, ERIS, MPYS, and TMEM173), an adaptor protein located in the ER membrane, which mediates the signaling to cytosolic DNA and bacterial cyclic dinucleotides such as c-di-GMP and c-di-AMP [13], [14], [15], [16], [17], [18] (Fig. 1). Although STING serves as a direct sensor of cyclic dinucleotides, it is not a direct sensor for cytosolic DNA and exhibits very low affinity for dsDNA [7]. Another key cytosolic DNA sensor, AIM2, was also identified in this search as well [19], [20], [21]. AIM2 mediates the inflammatory response by activating caspase-1 through the AIM2 inflammasome and inducing the maturation of proinflammatory cytokines IL-1β and IL-18. Although the response mediated by the AIM2 inflammasome is important for host defense to microbial infection, the dominant response to cytosolic DNA is mediated by the transcriptional regulation of type I interferons [5].
In a search for the cytosolic DNA sensor mediating the induction of type I interferons, Zhijian Chen and his colleagues used brute force biochemical approaches and identified the enzyme cyclic GMP-AMP synthase (cGAS) as the dsDNA sensor upstream of STING [22]. cGAS is activated by dsDNA and catalyze the synthesis of a noncanonical cyclic dinucleotide 2′,5′ cGAMP (referred to as cGAMP hereafter) from ATP and GTP [23], [24], [25], [26] (Fig. 1). cGAMP serves as a endogenous second messenger to stimulate the induction of type I interferons via STING. Ligand binding by STING induces the recruitment of the protein kinase TBK1 and transcription factor IRF3 to the signaling complex [27] (Fig. 1). Phosphorylation of IRF3 by TBK1 at the signaling complex promotes the oligomerization of IRF3 and its translocation into the nucleus, where it activates the transcription of the IFN-β gene together with NF-κB [27], [28], [29] (Fig. 1).
Recent structural studies of human, mouse, and porcine cGAS catalytic domains in isolation and in complex with dsDNA provided critical insights into the mechanism of cGAS activation by dsDNA and the catalytic mechanism of the enzyme [24], [30], [31], [32], [33], [34]. Although the conclusions from these studies are different from each other, the emerging picture, supported by extensive biochemical, biophysical, and in vivo characterization of cGAS mutants, is that cGAS is activated by dsDNA induced oligomerization [30], [32], [35]. In addition, the structures of STING in isolation and in complex with various cyclic dinucleotides including cGAMP have also been determined as well [23], [36], [37], [38], [39], [40], [41]. In this review, we will give a brief overview of the mechanisms of cGAS activation by dsDNA, the structural basis of STING activation by cGAMP, and discuss several unresolved questions about the mechanism of cytosolic DNA sensing via the cGAS-STING pathway.
Section snippets
cGAS is activated by dsDNA and catalyze the synthesis of cGAMP, a high affinity ligand for STING
The antiviral activity of cGAS (also known as C6orf150) was first described by Charles Rice's group in a systematic screening of the antiviral activities of interferon inducible genes (ISGs) [42]. However, the mechanism underpinning the antiviral activity of cGAS was not established in this study. In a search of the cytosolic dsDNA sensor, Zhijian Chen's group used classical biochemical fractioning technique and identified cGAMP as the type I interferon inducing molecule in cells stimulated
The structures of DNA free cGAS. Why it is not active?
The crystal structures of DNA free human, mouse, and porcine cGAS catalytic domains (residues 157–522 for hcGAS) have been determined recently [24], [30], [31], [32], [33], [34]. The overall structures of cGAS from different species are similar to each other [30]. The structure of cGAS catalytic domain also shows significant structural similarities to the structure of the dsRNA activated enzyme OAS1 (rmsd of 3.1 Å between hcGAS and OAS1). The cGAS catalytic domain exhibits a bi-lobed structure (
cGAS is activated by dsDNA induced oligomerization
A number of structures of cGAS catalytic domains bound to dsDNA have been determined recently [24], [30], [31], [32]. However, the mechanisms of cGAS activation by dsDNA proposed in these studies are different from each other. In the first two reports on the structures of mouse and porcine cGAS:dsDNA complexes [24], [31], the authors proposed that cGAS binds to dsDNA with a 1:1 stoichiometry and interacts with dsDNA through a single binding site in a similar manner as how OAS1 binds dsRNA [47].
The catalytic mechanism of cGAS
Based on extensive structural studies of cGAS bound to ATP, GTP, and various reaction products or intermediates and biochemical studies of cGAS catalyzed reaction with various kinds of substrates, two groups proposed similar catalytic mechanisms for cGAS [24], [25]. cGAS is highly specific when both ATP and GTP are used as substrates. In the first step of the cGAS catalyzed reaction, the triphosphate group of ATP binds to the active site (Fig. 4). The 2′-OH of GTP attack the α-phosphate of ATP,
The mechanism of STING activation by exogenous and endogenous ligands
The critical role of STING as a direct sensor of exogenous cyclic dinucleotide was first demonstrated by the landmark studies of the Vance group [14]. In this work, they expressed the cytosolic domain of STING and confirmed its interaction with c-di-GMP by in vitro binding studies [14]. The first glimpse into the mechanism of cyclic dinucleotide sensing by STING was demonstrated by the structural studies of human STING (hSTING) ligand binding domain in isolation and in complex with c-di-GMP
New developments and future directions
The major components of the cGAS-STING pathway have been identified in the last few years. The mechanism of how these molecules mediate the signaling of the cGAS-STING pathway is emerging. However, details of the cGAS-STING pathway are still not completely clear. For example, the mechanism of how full-length cGAS recognizes dsDNA is still not clear. The contribution of the N-terminal regions of cGAS to dsDNA sensing and cGAS activation remains to be established. The detailed mechanism of cGAS
Acknowledgements
This study is supported by the National Institute of Health (Grant AI 087741 to P. Li) and the Welch Foundation (Grant A-1816 to P. Li). This work is dedicated to the memory of my dad Changrong Li (1938–2014, of P. Li).
Chang Shu is a postdoc research associate in the Department of Biochemistry and Biophysics of Texas A&M University. He obtained his Ph.D. from the College of Life Sciences of Peking University in 2009. His current research focuses on the structural mechanism of cytosolic DNA sensing in innate immunity. He determined the crystal structures of STING, TBK1, and cGAS.
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Chang Shu is a postdoc research associate in the Department of Biochemistry and Biophysics of Texas A&M University. He obtained his Ph.D. from the College of Life Sciences of Peking University in 2009. His current research focuses on the structural mechanism of cytosolic DNA sensing in innate immunity. He determined the crystal structures of STING, TBK1, and cGAS.
Xin Li obtained his Ph.D. in 2012 from the College of Veterinary Medicine of China Agricultural University. He studied the structures of MHC I and CD8 from cattle. Now he is a postdoctoral research associate in the Department of Biochemistry & Biophysics at Texas A&M University. His research focuses on the mechanism of cyclic GMP-AMP synthase (cGAS) activation by dsDNA.
Pingwei Li was born in 1967 in the suburb of Xi’an, China. He obtained his Ph.D. from Peking University in 1996, majoring in X-ray crystallography. During his graduate studies, he determined the crystal structure of Cu, Zn SOD from duck erythrocytes. He came to the United States in the spring of 1998 and did his first post-doc with Roland Strong in the Division of Basic Science at the Fred Hutchinson Cancer Research Center in Seattle. He determined the structures of the noncanonical MHC class I like molecules MICA, RAE-1β and their complexes with the natural killer cell and γδ T cell receptor NKG2D. He moved on to work in the Department of Molecular Biology at Princeton University as a staff scientist in the fall of 2001, studying the structures of the UBP family deubiquitinating enzymes HAUSP (USP7) and USP14 in the laboratory of Yigong Shi. He moved to the California Institute of Technology in the summer of 2003 and did another post-doc with Pamela Bjorkman. His research focuses on the structural studies of a monoclonal antibody (MW1) against polyglutamine. He cloned the VH and VL genes of the antibody, generated the Fv fragment by refolding, and determined the structure of MW1 Fv in isolation and in complex with a polyglutamine containing peptide GQ10G. He started his own lab in the Department of Biochemistry and Biophysics at Texas A&M University in the fall of 2005. His current research focuses on the structural basis of microbial nucleic acid sensing in innate immunity. His lab determined the structures of LGP2 and RIG-I C-terminal domains bound to dsRNA, the structure of STING bound to c-di-GMP and cGAMP, and the structure of TBK1. In his recent studies, his lab determined the structures of cGAS in isolation and in complex with dsDNA and elucidated the mechanism of cGAS activation by dsDNA.
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These authors contibuted equally to this work.