cGAS Dimerization Entangles DNA Recognition
2013; Cell Press; Volume: 39; Issue: 6 Linguagem: Inglês
10.1016/j.immuni.2013.11.012
ISSN1097-4180
AutoresPhilip J. Kranzusch, Russell E. Vance,
Tópico(s)RNA and protein synthesis mechanisms
ResumoDetection of foreign DNA in the cell cytosol triggers potent antiviral responses. In this issue of Immunity, Li et al., 2013Li X. Shu C. Yi G. Chaton C.T. Shelton C.L. Diao J. Zuo X. Kao C.C. Herr A.B. Li P. Immunity. 2013; 39 (this issue): 1019-1031Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar provide new structural and biochemical data indicating that a cytosolic DNA sensor, cyclic GMP-AMP synthase (cGAS), is activated by DNA-induced dimerization. Detection of foreign DNA in the cell cytosol triggers potent antiviral responses. In this issue of Immunity, Li et al., 2013Li X. Shu C. Yi G. Chaton C.T. Shelton C.L. Diao J. Zuo X. Kao C.C. Herr A.B. Li P. Immunity. 2013; 39 (this issue): 1019-1031Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar provide new structural and biochemical data indicating that a cytosolic DNA sensor, cyclic GMP-AMP synthase (cGAS), is activated by DNA-induced dimerization. Detection of foreign nucleic acids is a major strategy by which the innate immune system senses infection. Endocytosed nucleic acids, including DNA and RNA, are detected by Toll-like receptors (TLRs), and several distinct receptors detect nucleic acids in the cytosol. For example, double-stranded RNA (dsRNA) is detected by multiple sensors—including protein kinase R (PKR), 2′,5′ oligoadenylate synthase 1 (OAS1), and RIG-I-like receptors (RLRs)—that together activate diverse antiviral responses. In contrast, cytosolic dsDNA activates inflammasome responses via the AIM2 receptor, and in addition, stimulates production of type I interferons (IFNs), a family of antiviral cytokines. However, until recently, the mechanism linking cytosolic dsDNA to IFN production remained mysterious. In a landmark discovery late last year, James Chen and colleagues found that the enzyme cyclic GMP-AMP synthase (cGAS) functions as a cytosolic dsDNA sensor (Sun et al., 2013Sun L. Wu J. Du F. Chen X. Chen Z.J. Science. 2013; 339: 786-791Crossref PubMed Scopus (2508) Google Scholar). Upon engagement of cytoplasmic dsDNA, cGAS synthesizes a unique second messenger, a cyclic GMP-AMP dinucleotide (cGAMP), which binds and activates a downstream endoplasmic reticulum adaptor called STING. Once activated, STING initiates a TBK1-IRF3-dependent innate immune signaling cascade that culminates in transcription of type I IFNs and other antiviral genes. Since the discovery of cGAS, efforts have been underway to determine the mechanisms of DNA-dependent activation and cGAMP production and to understand how cGAS relates to the known pantheon of cellular nucleic acid sensors. A flurry of reports earlier this year described crystal structures of mouse (Gao et al., 2013Gao P. Ascano M. Wu Y. Barchet W. Gaffney B.L. Zillinger T. Serganov A.A. Liu Y. Jones R.A. Hartmann G. et al.Cell. 2013; 153: 1094-1107Abstract Full Text Full Text PDF PubMed Scopus (614) Google Scholar), porcine (Civril et al., 2013Civril F. Deimling T. de Oliveira Mann C.C. Ablasser A. Moldt M. Witte G. Hornung V. Hopfner K.P. Nature. 2013; 498: 332-337Crossref PubMed Scopus (445) Google Scholar), and human (Kranzusch et al., 2013Kranzusch P.J. Lee A.S. Berger J.M. Doudna J.A. Cell Rep. 2013; 3: 1362-1368Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar) enzymes, revealing a bilobed cGAS architecture comprising N-terminal nucleotidyltransferase and C-terminal dsDNA recognition domains. These cGAS structures bear remarkable similarity to the cytosolic dsRNA sensor OAS1, indicating that OAS and cGAS proteins constitute a new evolutionarily related family of OAS-like receptors (OLRs) involved in host defense (Civril et al., 2013Civril F. Deimling T. de Oliveira Mann C.C. Ablasser A. Moldt M. Witte G. Hornung V. Hopfner K.P. Nature. 2013; 498: 332-337Crossref PubMed Scopus (445) Google Scholar, Kranzusch et al., 2013Kranzusch P.J. Lee A.S. Berger J.M. Doudna J.A. Cell Rep. 2013; 3: 1362-1368Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). Surprisingly, early biochemical follow-up to the discovery of cGAS showed that its cyclic dinucleotide product contains a specific combination of 2′,5′ and 3′,5′ glycosidic linkages (cyclic[G(2′,5′)pA(3′,5′)p]; hereafter called 2′,5′ cGAMP) (Ablasser et al., 2013Ablasser A. Goldeck M. Cavlar T. Deimling T. Witte G. Röhl I. Hopfner K.P. Ludwig J. Hornung V. Nature. 2013; 498: 380-384Crossref PubMed Scopus (923) Google Scholar, Diner et al., 2013Diner E.J. Burdette D.L. Wilson S.C. Monroe K.M. Kellenberger C.A. Hyodo M. Hayakawa Y. Hammond M.C. Vance R.E. Cell Rep. 2013; 3: 1355-1361Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar, Gao et al., 2013Gao P. Ascano M. Wu Y. Barchet W. Gaffney B.L. Zillinger T. Serganov A.A. Liu Y. Jones R.A. Hartmann G. et al.Cell. 2013; 153: 1094-1107Abstract Full Text Full Text PDF PubMed Scopus (614) Google Scholar, Zhang et al., 2013Zhang X. Shi H. Wu J. Zhang X. Sun L. Chen C. Chen Z.J. Mol. Cell. 2013; 51: 226-235Abstract Full Text Full Text PDF PubMed Scopus (627) Google Scholar). 2′,5′ cGAMP is a more broadly potent activator of STING than is canonical 3′,5′ cGAMP (Ablasser et al., 2013Ablasser A. Goldeck M. Cavlar T. Deimling T. Witte G. Röhl I. Hopfner K.P. Ludwig J. Hornung V. Nature. 2013; 498: 380-384Crossref PubMed Scopus (923) Google Scholar, Diner et al., 2013Diner E.J. Burdette D.L. Wilson S.C. Monroe K.M. Kellenberger C.A. Hyodo M. Hayakawa Y. Hammond M.C. Vance R.E. Cell Rep. 2013; 3: 1355-1361Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar, Zhang et al., 2013Zhang X. Shi H. Wu J. Zhang X. Sun L. Chen C. Chen Z.J. Mol. Cell. 2013; 51: 226-235Abstract Full Text Full Text PDF PubMed Scopus (627) Google Scholar). Crystal structures of mammalian cGAS bound to dsDNA and dinucleotide (Civril et al., 2013Civril F. Deimling T. de Oliveira Mann C.C. Ablasser A. Moldt M. Witte G. Hornung V. Hopfner K.P. Nature. 2013; 498: 332-337Crossref PubMed Scopus (445) Google Scholar, Gao et al., 2013Gao P. Ascano M. Wu Y. Barchet W. Gaffney B.L. Zillinger T. Serganov A.A. Liu Y. Jones R.A. Hartmann G. et al.Cell. 2013; 153: 1094-1107Abstract Full Text Full Text PDF PubMed Scopus (614) Google Scholar) detail conformational changes induced by dsDNA that activate the enzyme core. However, these complexes include short 14–17 basepair dsDNA fragments that do not induce maximal stimulation, and a detailed picture of the complex pathway of 2′,5′ cGAMP synthesis remains incomplete. In this issue of Immunity, Li et al., 2013Li X. Shu C. Yi G. Chaton C.T. Shelton C.L. Diao J. Zuo X. Kao C.C. Herr A.B. Li P. Immunity. 2013; 39 (this issue): 1019-1031Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar reveal a new dimerization interface within cGAS that is critical for enzyme activation. Through careful structural analysis of both human cGAS alone and mouse cGAS bound to dsDNA, Li et al., 2013Li X. Shu C. Yi G. Chaton C.T. Shelton C.L. Diao J. Zuo X. Kao C.C. Herr A.B. Li P. Immunity. 2013; 39 (this issue): 1019-1031Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar identify protein dimerization interactions and a second DNA binding surface. These findings extend our understanding of cGAS enzymatic activation and suggest a further layer of control over DNA-dependent innate immune signaling. Previous structures of mouse and porcine cGAS bound to dsDNA focused on a primary DNA-binding platform formed by a long N-terminal helical extension in cGAS and a zinc-ribbon insertion within the C-terminal domain (Civril et al., 2013Civril F. Deimling T. de Oliveira Mann C.C. Ablasser A. Moldt M. Witte G. Hornung V. Hopfner K.P. Nature. 2013; 498: 332-337Crossref PubMed Scopus (445) Google Scholar, Gao et al., 2013Gao P. Ascano M. Wu Y. Barchet W. Gaffney B.L. Zillinger T. Serganov A.A. Liu Y. Jones R.A. Hartmann G. et al.Cell. 2013; 153: 1094-1107Abstract Full Text Full Text PDF PubMed Scopus (614) Google Scholar). The new interactions characterized by Li et al., 2013Li X. Shu C. Yi G. Chaton C.T. Shelton C.L. Diao J. Zuo X. Kao C.C. Herr A.B. Li P. Immunity. 2013; 39 (this issue): 1019-1031Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar now include a second DNA-binding surface and protein-dimerization interactions located on the opposite face of the cGAS enzyme within the C-terminal domain. The secondary DNA-binding site, involving conserved charged residues, covers a large area of the cGAS enzyme. As an indication that these interactions are not simply a consequence of unique crystallization conditions, Li et al., 2013Li X. Shu C. Yi G. Chaton C.T. Shelton C.L. Diao J. Zuo X. Kao C.C. Herr A.B. Li P. Immunity. 2013; 39 (this issue): 1019-1031Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar point out that previously published structures of cGAS also exhibit C-terminal dimerization contacts, including a mouse cGAS-DNA structure that crystallized as two copies within the asymmetric unit of the crystal (Gao et al., 2013Gao P. Ascano M. Wu Y. Barchet W. Gaffney B.L. Zillinger T. Serganov A.A. Liu Y. Jones R.A. Hartmann G. et al.Cell. 2013; 153: 1094-1107Abstract Full Text Full Text PDF PubMed Scopus (614) Google Scholar). In Li et al., 2013Li X. Shu C. Yi G. Chaton C.T. Shelton C.L. Diao J. Zuo X. Kao C.C. Herr A.B. Li P. Immunity. 2013; 39 (this issue): 1019-1031Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar’s new crystal forms of cGAS, the potential dimerization contacts are observed between the zinc-ribbon and C-terminal domains of closely packed protein molecules. Crystal growth is inherently dependent on packing interactions between molecules, and therefore additional experimental support is required to determine whether these interactions are biologically relevant or merely the result of crystallization conditions. To verify DNA-induced cGAS dimerization, Li et al., 2013Li X. Shu C. Yi G. Chaton C.T. Shelton C.L. Diao J. Zuo X. Kao C.C. Herr A.B. Li P. Immunity. 2013; 39 (this issue): 1019-1031Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar first employ small-angle X-ray scattering (SAXS) to probe the size and shape of cGAS complexes in solution. SAXS data are particularly useful as an adjunct to crystallography, because the profile of elastic scattering of X-rays can be used to calculate a molecular envelope and distinguish the occurrence of potential crystallographic complexes in solution. Li et al., 2013Li X. Shu C. Yi G. Chaton C.T. Shelton C.L. Diao J. Zuo X. Kao C.C. Herr A.B. Li P. Immunity. 2013; 39 (this issue): 1019-1031Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar’s data indicate that a larger-than-monomeric complex forms upon DNA engagement, and the calculated molecular envelope agrees with the 2:2 dimeric cGAS-DNA complex observed in the crystal structure. As further support of dimerization in solution, Li et al., 2013Li X. Shu C. Yi G. Chaton C.T. Shelton C.L. Diao J. Zuo X. Kao C.C. Herr A.B. Li P. Immunity. 2013; 39 (this issue): 1019-1031Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar determine the molecular mass of cGAS-DNA complexes by using analytical ultracentrifugation. The calculated mass of sedimented cGAS complexes confirms enhanced dimer formation in the presence of DNA and additionally reveals potential formation of higher oligomeric species. What role does the secondary DNA-binding site and cGAS dimerization play in enzyme activation and innate immune signaling? To address this question, Li et al., 2013Li X. Shu C. Yi G. Chaton C.T. Shelton C.L. Diao J. Zuo X. Kao C.C. Herr A.B. Li P. Immunity. 2013; 39 (this issue): 1019-1031Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar purify mouse cGAS variants encoding key mutations designed to disrupt each site of DNA binding or the dimerization interface. In agreement with prior studies, residues in the primary DNA binding site, including conserved basic residues along the N-terminal helix and within the zinc-ribbon domain, are essential for enzyme activation (Civril et al., 2013Civril F. Deimling T. de Oliveira Mann C.C. Ablasser A. Moldt M. Witte G. Hornung V. Hopfner K.P. Nature. 2013; 498: 332-337Crossref PubMed Scopus (445) Google Scholar, Gao et al., 2013Gao P. Ascano M. Wu Y. Barchet W. Gaffney B.L. Zillinger T. Serganov A.A. Liu Y. Jones R.A. Hartmann G. et al.Cell. 2013; 153: 1094-1107Abstract Full Text Full Text PDF PubMed Scopus (614) Google Scholar, Kato et al., 2013Kato K. Ishii R. Goto E. Ishitani R. Tokunaga F. Nureki O. PLoS ONE. 2013; 8: e76983Crossref PubMed Scopus (48) Google Scholar, Kranzusch et al., 2013Kranzusch P.J. Lee A.S. Berger J.M. Doudna J.A. Cell Rep. 2013; 3: 1362-1368Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). However, Li et al., 2013Li X. Shu C. Yi G. Chaton C.T. Shelton C.L. Diao J. Zuo X. Kao C.C. Herr A.B. Li P. Immunity. 2013; 39 (this issue): 1019-1031Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar demonstrate that mutations to the secondary DNA binding site and dimerization interface also disrupt enzyme activation. Importantly, some mutations that disrupt enzyme dimerization do not impair 1:1 cGAS-DNA interaction, indicating that these mutations do not destabilize the overall protein structure. Li et al., 2013Li X. Shu C. Yi G. Chaton C.T. Shelton C.L. Diao J. Zuo X. Kao C.C. Herr A.B. Li P. Immunity. 2013; 39 (this issue): 1019-1031Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar further confirm the importance of conserved amino acids within the secondary DNA binding site by demonstrating that mutations to this region abolish interferon-beta reporter signaling in a cell-based assay, suggesting that DNA-induced dimerization is critical for full enzyme activation. Oligomerization appears to be a general engineering principle in pathogen-sensor proteins. TLRs and PKR are well-characterized examples of dimerized ligand-bound complexes, and recent reports also implicate oligomerization in activation of signaling by RIG-I, MDA-5, AIM2, and inflammasome components (reviewed in Wu, 2013Wu H. Cell. 2013; 153: 287-292Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar) (Figure 1). The addition of cGAS to the growing list of innate sensors requiring oligomerization for full activation suggests that innate immune signaling receptors have evolved high-order complex formation to both safeguard the cell from aberrant activation and to augment signal transduction upon accurate ligand identification. Additionally, Li et al., 2013Li X. Shu C. Yi G. Chaton C.T. Shelton C.L. Diao J. Zuo X. Kao C.C. Herr A.B. Li P. Immunity. 2013; 39 (this issue): 1019-1031Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar’s discovery of cGAS dimerization may foretell a yet undiscovered role of oligomerization in dsRNA sensing by OAS1 and additional members of the evolutionarily related OLR enzyme superfamily. In contrast to the structure of dimerized TLRs bound to nucleic acid duplexes, the nearly parallel DNA binding sites presented in the mouse cGAS-DNA structure by Li et al., 2013Li X. Shu C. Yi G. Chaton C.T. Shelton C.L. Diao J. Zuo X. Kao C.C. Herr A.B. Li P. Immunity. 2013; 39 (this issue): 1019-1031Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar indicate that a cGAS dimer cannot assemble along a single short dsDNA duplex (Figure 1). cGAS dimers must therefore form around two separate DNA duplexes or possibly on a single DNA duplex that is long enough to bend and occupy each cGAS-binding site. Further confounding the situation, in the structure presented by Li et al., 2013Li X. Shu C. Yi G. Chaton C.T. Shelton C.L. Diao J. Zuo X. Kao C.C. Herr A.B. Li P. Immunity. 2013; 39 (this issue): 1019-1031Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar, extension of the two 18 base-pair DNA duplexes would result in a steric clash in one direction. Perhaps these limitations prevent inappropriate cGAS signaling by requiring multiple DNA ligands for activation or specific binding of cGAS at the ends of a longer piece of dsDNA, as would be present in some viral and bacterial genomes. All structures of cGAS to date contain the enzyme’s catalytic and dsDNA recognition domains but lack its 150 amino acid N-terminal tail. This tail is required for high-affinity human cGAS-DNA interactions (Kranzusch et al., 2013Kranzusch P.J. Lee A.S. Berger J.M. Doudna J.A. Cell Rep. 2013; 3: 1362-1368Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar), and regions of the tail are capable of interacting independently with dsDNA in cells (Sun et al., 2013Sun L. Wu J. Du F. Chen X. Chen Z.J. Science. 2013; 339: 786-791Crossref PubMed Scopus (2508) Google Scholar). Perhaps the N-terminal tail becomes ordered upon DNA binding or plays a role in facilitating the dimerized conformation or limiting further oligomerization. The functional or regulatory role of the cGAS N-terminal tail will require future cellular and biochemical studies. The discovery of cGAS represents a major advance in our understanding of the immune response to foreign DNA. Identification of the unique 2′,5′ cGAMP nucleotide produced by activated cGAS (Ablasser et al., 2013Ablasser A. Goldeck M. Cavlar T. Deimling T. Witte G. Röhl I. Hopfner K.P. Ludwig J. Hornung V. Nature. 2013; 498: 380-384Crossref PubMed Scopus (923) Google Scholar, Diner et al., 2013Diner E.J. Burdette D.L. Wilson S.C. Monroe K.M. Kellenberger C.A. Hyodo M. Hayakawa Y. Hammond M.C. Vance R.E. Cell Rep. 2013; 3: 1355-1361Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar, Gao et al., 2013Gao P. Ascano M. Wu Y. Barchet W. Gaffney B.L. Zillinger T. Serganov A.A. Liu Y. Jones R.A. Hartmann G. et al.Cell. 2013; 153: 1094-1107Abstract Full Text Full Text PDF PubMed Scopus (614) Google Scholar, Zhang et al., 2013Zhang X. Shi H. Wu J. Zhang X. Sun L. Chen C. Chen Z.J. Mol. Cell. 2013; 51: 226-235Abstract Full Text Full Text PDF PubMed Scopus (627) Google Scholar) suggests strategies for therapeutic intervention in this important innate immune signaling pathway, and cGAS enzymatic activity is also an attractive target for pharmacological inhibition in systemic lupus erythematosus and other DNA-specific autoimmune diseases. Identification of DNA-induced dimerization adds an important new twist to our understanding of cGAS, with many more surprises likely to emerge in this fast-moving field. Cyclic GMP-AMP Synthase Is Activated by Double-Stranded DNA-Induced OligomerizationLi et al.ImmunityDecember 12, 2013In BriefCyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor mediating innate antimicrobial immunity. It catalyzes the synthesis of a noncanonical cyclic dinucleotide, 2′,5′ cGAMP, that binds to STING and mediates the activation of TBK1 and IRF-3. Activated IRF-3 translocates to the nucleus and initiates the transcription of the IFN-β gene. The structure of mouse cGAS bound to an 18 bp dsDNA revealed that cGAS interacts with dsDNA through two binding sites, forming a 2:2 complex. Enzyme assays and IFN-β reporter assays of cGAS mutants demonstrated that interactions at both DNA binding sites are essential for cGAS activation. Full-Text PDF Open Archive
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