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- PDB-6xjd: Two mouse cGAS catalytic domain binding to human assembled nucleosome -

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Basic information

Entry
Database: PDB / ID: 6xjd
TitleTwo mouse cGAS catalytic domain binding to human assembled nucleosome
Components
  • (DNA (145-MER)) x 2
  • Cyclic GMP-AMP synthase
  • Histone H2A type 1
  • Histone H2B type 1-C/E/F/G/I
  • Histone H3.2
  • Histone H4
KeywordsIMMUNE SYSTEM/DNA / Immunity / IMMUNE SYSTEM / IMMUNE SYSTEM-DNA complex
Function / homology
Function and homology information


2',3'-cyclic GMP-AMP synthase activity / cyclic GMP-AMP synthase / paracrine signaling / poly-ADP-D-ribose modification-dependent protein binding / regulation of type I interferon production / regulation of immunoglobulin production / cGAS/STING signaling pathway / regulation of T cell activation / negative regulation of cGAS/STING signaling pathway / negative regulation of DNA repair ...2',3'-cyclic GMP-AMP synthase activity / cyclic GMP-AMP synthase / paracrine signaling / poly-ADP-D-ribose modification-dependent protein binding / regulation of type I interferon production / regulation of immunoglobulin production / cGAS/STING signaling pathway / regulation of T cell activation / negative regulation of cGAS/STING signaling pathway / negative regulation of DNA repair / cGMP-mediated signaling / cellular response to exogenous dsRNA / positive regulation of type I interferon production / nucleosome binding / regulation of immune response / negative regulation of double-strand break repair via homologous recombination / negative regulation of megakaryocyte differentiation / protein localization to CENP-A containing chromatin / positive regulation of defense response to virus by host / Chromatin modifying enzymes / Replacement of protamines by nucleosomes in the male pronucleus / CENP-A containing nucleosome / Packaging Of Telomere Ends / activation of innate immune response / phosphatidylinositol-4,5-bisphosphate binding / Recognition and association of DNA glycosylase with site containing an affected purine / Cleavage of the damaged purine / Deposition of new CENPA-containing nucleosomes at the centromere / nucleosomal DNA binding / cAMP-mediated signaling / Recognition and association of DNA glycosylase with site containing an affected pyrimidine / Cleavage of the damaged pyrimidine / Inhibition of DNA recombination at telomere / telomere organization / Meiotic synapsis / Interleukin-7 signaling / RNA Polymerase I Promoter Opening / Assembly of the ORC complex at the origin of replication / SUMOylation of chromatin organization proteins / Regulation of endogenous retroelements by the Human Silencing Hub (HUSH) complex / DNA methylation / Condensation of Prophase Chromosomes / SIRT1 negatively regulates rRNA expression / Chromatin modifications during the maternal to zygotic transition (MZT) / ERCC6 (CSB) and EHMT2 (G9a) positively regulate rRNA expression / HCMV Late Events / innate immune response in mucosa / determination of adult lifespan / PRC2 methylates histones and DNA / Regulation of endogenous retroelements by KRAB-ZFP proteins / Defective pyroptosis / Regulation of endogenous retroelements by Piwi-interacting RNAs (piRNAs) / HDACs deacetylate histones / Nonhomologous End-Joining (NHEJ) / RNA Polymerase I Promoter Escape / molecular condensate scaffold activity / Transcriptional regulation by small RNAs / Formation of the beta-catenin:TCF transactivating complex / RUNX1 regulates genes involved in megakaryocyte differentiation and platelet function / NoRC negatively regulates rRNA expression / Activated PKN1 stimulates transcription of AR (androgen receptor) regulated genes KLK2 and KLK3 / G2/M DNA damage checkpoint / HDMs demethylate histones / B-WICH complex positively regulates rRNA expression / DNA Damage/Telomere Stress Induced Senescence / heterochromatin formation / PKMTs methylate histone lysines / Metalloprotease DUBs / Meiotic recombination / Pre-NOTCH Transcription and Translation / RMTs methylate histone arginines / Activation of anterior HOX genes in hindbrain development during early embryogenesis / HCMV Early Events / positive regulation of cellular senescence / Transcriptional regulation of granulopoiesis / structural constituent of chromatin / UCH proteinases / antimicrobial humoral immune response mediated by antimicrobial peptide / nucleosome / nucleosome assembly / E3 ubiquitin ligases ubiquitinate target proteins / antibacterial humoral response / Recruitment and ATM-mediated phosphorylation of repair and signaling proteins at DNA double strand breaks / chromatin organization / site of double-strand break / RUNX1 regulates transcription of genes involved in differentiation of HSCs / Factors involved in megakaryocyte development and platelet production / HATs acetylate histones / Processing of DNA double-strand break ends / double-stranded DNA binding / Senescence-Associated Secretory Phenotype (SASP) / defense response to virus / Oxidative Stress Induced Senescence / Estrogen-dependent gene expression / chromosome, telomeric region / nuclear body / Ub-specific processing proteases / defense response to Gram-positive bacterium / protein heterodimerization activity / Amyloid fiber formation
Similarity search - Function
Mab-21-like, nucleotidyltransferase domain / Mab-21-like / Mab-21-like, HhH/H2TH-like domain / Mab-21 protein HhH/H2TH-like domain / Mab-21 / Mab-21 protein nucleotidyltransferase domain / Histone H2B signature. / Histone H2B / Histone H2B / Histone H2A conserved site ...Mab-21-like, nucleotidyltransferase domain / Mab-21-like / Mab-21-like, HhH/H2TH-like domain / Mab-21 protein HhH/H2TH-like domain / Mab-21 / Mab-21 protein nucleotidyltransferase domain / Histone H2B signature. / Histone H2B / Histone H2B / Histone H2A conserved site / Histone H2A signature. / Histone H2A, C-terminal domain / C-terminus of histone H2A / Histone H4, conserved site / Histone H4 signature. / Histone H4 / Histone H4 / Histone H2A / Histone 2A / CENP-T/Histone H4, histone fold / Centromere kinetochore component CENP-T histone fold / TATA box binding protein associated factor / TATA box binding protein associated factor (TAF), histone-like fold domain / Histone H3 signature 1. / Histone H3 signature 2. / Histone H3 / Histone H3/CENP-A / Histone H2A/H2B/H3 / Core histone H2A/H2B/H3/H4 / Histone-fold
Similarity search - Domain/homology
DNA / DNA (> 10) / DNA (> 100) / Histone H2A type 1 / Histone H4 / Histone H2B type 1-C/E/F/G/I / Histone H3.2 / Cyclic GMP-AMP synthase
Similarity search - Component
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.8 Å
AuthorsXu, P. / Li, P. / Zhao, B.
Funding support United States, 2items
OrganizationGrant numberCountry
Welch FoundationGrant A-1931-20170325 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)Grant R01 AI145287 United States
CitationJournal: Nature / Year: 2020
Title: The molecular basis of tight nuclear tethering and inactivation of cGAS.
Authors: Baoyu Zhao / Pengbiao Xu / Chesley M Rowlett / Tao Jing / Omkar Shinde / Yuanjiu Lei / A Phillip West / Wenshe Ray Liu / Pingwei Li /
Abstract: Nucleic acids derived from pathogens induce potent innate immune responses. Cyclic GMP-AMP synthase (cGAS) is a double-stranded DNA sensor that catalyses the synthesis of the cyclic dinucleotide ...Nucleic acids derived from pathogens induce potent innate immune responses. 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 axis. cGAS was previously thought to not react with self DNA owing to its cytosolic localization; however, recent studies have shown that cGAS is localized mostly in the nucleus and has low activity as a result of tight nuclear tethering. 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 site. 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.
History
DepositionJun 23, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 16, 2020Provider: repository / Type: Initial release
Revision 1.1Sep 23, 2020Group: Database references / Structure summary / Category: audit_author / citation / citation_author
Item: _audit_author.name / _citation.country ..._audit_author.name / _citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.year
Revision 1.2Dec 9, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.title / _citation_author.identifier_ORCID
Revision 1.3Mar 6, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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  • Deposited structure unit
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Assembly

Deposited unit
A: Histone H3.2
B: Histone H4
C: Histone H2A type 1
D: Histone H2B type 1-C/E/F/G/I
E: Histone H3.2
F: Histone H4
G: Histone H2A type 1
H: Histone H2B type 1-C/E/F/G/I
I: DNA (145-MER)
J: DNA (145-MER)
K: Cyclic GMP-AMP synthase
L: Cyclic GMP-AMP synthase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)286,79014
Polymers286,65912
Non-polymers1312
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 5 types, 10 molecules AEBFCGDHKL

#1: Protein Histone H3.2 / H3-clustered histone 13 / H3-clustered histone 14 / H3-clustered histone 15 / Histone H3/m / Histone H3/o


Mass: 15257.838 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human)
Gene: H3C15, HIST2H3A, H3C14, H3F2, H3FM, HIST2H3C, H3C13, HIST2H3D
Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q71DI3
#2: Protein Histone H4


Mass: 11263.231 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human)
Gene: HIST1H4A, H4/A, H4FA, HIST1H4B, H4/I, H4FI, HIST1H4C, H4/G, H4FG, HIST1H4D, H4/B, H4FB, HIST1H4E, H4/J, H4FJ, HIST1H4F, H4/C, H4FC, HIST1H4H, H4/H, H4FH, HIST1H4I, H4/M, H4FM, HIST1H4J, H4/E, ...Gene: HIST1H4A, H4/A, H4FA, HIST1H4B, H4/I, H4FI, HIST1H4C, H4/G, H4FG, HIST1H4D, H4/B, H4FB, HIST1H4E, H4/J, H4FJ, HIST1H4F, H4/C, H4FC, HIST1H4H, H4/H, H4FH, HIST1H4I, H4/M, H4FM, HIST1H4J, H4/E, H4FE, HIST1H4K, H4/D, H4FD, HIST1H4L, H4/K, H4FK, HIST2H4A, H4/N, H4F2, H4FN, HIST2H4, HIST2H4B, H4/O, H4FO, HIST4H4
Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P62805
#3: Protein Histone H2A type 1 / H2A.1 / Histone H2A/ptl


Mass: 13990.342 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human)
Gene: H2AC11, H2AFP, HIST1H2AG, H2AC13, H2AFC, HIST1H2AI, H2AC15, H2AFD, HIST1H2AK, H2AC16, H2AFI, HIST1H2AL, H2AC17, H2AFN, HIST1H2AM
Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0C0S8
#4: Protein Histone H2B type 1-C/E/F/G/I / Histone H2B.1 A / Histone H2B.a / H2B/a / Histone H2B.g / H2B/g / Histone H2B.h / H2B/h / Histone ...Histone H2B.1 A / Histone H2B.a / H2B/a / Histone H2B.g / H2B/g / Histone H2B.h / H2B/h / Histone H2B.k / H2B/k / Histone H2B.l / H2B/l


Mass: 13795.980 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human)
Gene: H2BC4, H2BFL, HIST1H2BC, H2BC6, H2BFH, HIST1H2BE, H2BC7, H2BFG, HIST1H2BF, H2BC8, H2BFA, HIST1H2BG, H2BC10, H2BFK, HIST1H2BI
Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P62807
#7: Protein Cyclic GMP-AMP synthase / m-cGAS / 2'3'-cGAMP synthase / Mab-21 domain-containing protein 1


Mass: 43647.352 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Cgas, Mb21d1 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q8C6L5, cyclic GMP-AMP synthase

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DNA chain , 2 types, 2 molecules IJ

#5: DNA chain DNA (145-MER)


Mass: 45145.754 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#6: DNA chain DNA (145-MER)


Mass: 45604.047 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)

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Non-polymers , 1 types, 2 molecules

#8: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn

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Details

Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeDetailsEntity IDParent-IDSource
1cGAS-nucleosome complexCOMPLEXTwo mouse cGAS catalytic domain binding to assembled human nucleosome#1-#70MULTIPLE SOURCES
2HistoneCOMPLEX#1-#41RECOMBINANT
3DNACOMPLEX#5-#61RECOMBINANT
4Cyclic GMP-AMP synthaseCOMPLEX#71RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Homo sapiens (human)9606
34Mus musculus (house mouse)10090
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli BL21(DE3) (bacteria)469008
24Escherichia coli BL21(DE3) (bacteria)469008
Buffer solutionpH: 7.4
SpecimenConc.: 0.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1800 nm / Nominal defocus min: 700 nm / Cs: 2.7 mm
Image recordingElectron dose: 48 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.16_3549: / Classification: refinement
EM software
IDNameVersionCategory
4RELION3.0bCTF correction
10RELION3.0binitial Euler assignment
11RELION3.0bfinal Euler assignment
12RELION3.0bclassification
13RELION3.0b3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 6.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 9454 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00818887
ELECTRON MICROSCOPYf_angle_d1.53126697
ELECTRON MICROSCOPYf_dihedral_angle_d24.48110460
ELECTRON MICROSCOPYf_chiral_restr0.0752980
ELECTRON MICROSCOPYf_plane_restr0.0122380

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