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- PDB-6y5d: Structure of human cGAS (K394E) bound to the nucleosome -

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Basic information

Entry
Database: PDB / ID: 6y5d
TitleStructure of human cGAS (K394E) bound to the nucleosome
Components
  • (DNA (153-MER)) x 2
  • Cyclic GMP-AMP synthase
  • Histone H2A type 2-A
  • Histone H2B type 1-K
  • Histone H3.2
  • Histone H4
KeywordsIMMUNE SYSTEM / cGAS / STING / Nucleosome / Innate Immunity / cGMP-AMP
Function / homology
Function and homology information


2',3'-cyclic GMP-AMP synthase activity / cyclic GMP-AMP synthase / paracrine signaling / STING mediated induction of host immune responses / poly-ADP-D-ribose modification-dependent protein binding / regulation of immunoglobulin production / cGAS/STING signaling pathway / regulation of T cell activation / pattern recognition receptor signaling pathway / : ...2',3'-cyclic GMP-AMP synthase activity / cyclic GMP-AMP synthase / paracrine signaling / STING mediated induction of host immune responses / poly-ADP-D-ribose modification-dependent protein binding / regulation of immunoglobulin production / cGAS/STING signaling pathway / regulation of T cell activation / pattern recognition receptor signaling pathway / : / negative regulation of cGAS/STING signaling pathway / cytoplasmic pattern recognition receptor signaling pathway / cellular response to exogenous dsRNA / positive regulation of type I interferon production / : / negative regulation of double-strand break repair via homologous recombination / nucleosome binding / negative regulation of megakaryocyte differentiation / protein localization to CENP-A containing chromatin / Chromatin modifying enzymes / Replacement of protamines by nucleosomes in the male pronucleus / CENP-A containing nucleosome / positive regulation of defense response to virus by host / phosphatidylinositol-4,5-bisphosphate binding / Packaging Of Telomere Ends / 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 / telomere organization / activation of innate immune response / Recognition and association of DNA glycosylase with site containing an affected pyrimidine / Cleavage of the damaged pyrimidine / Interleukin-7 signaling / RNA Polymerase I Promoter Opening / Inhibition of DNA recombination at telomere / Assembly of the ORC complex at the origin of replication / Meiotic synapsis / SUMOylation of chromatin organization proteins / Regulation of endogenous retroelements by the Human Silencing Hub (HUSH) complex / DNA methylation / Condensation of Prophase Chromosomes / Chromatin modifications during the maternal to zygotic transition (MZT) / SIRT1 negatively regulates rRNA expression / HCMV Late Events / ERCC6 (CSB) and EHMT2 (G9a) positively regulate rRNA expression / determination of adult lifespan / PRC2 methylates histones and DNA / Regulation of endogenous retroelements by KRAB-ZFP proteins / innate immune response in mucosa / Defective pyroptosis / HDACs deacetylate histones / Regulation of endogenous retroelements by Piwi-interacting RNAs (piRNAs) / RNA Polymerase I Promoter Escape / Nonhomologous End-Joining (NHEJ) / molecular condensate scaffold activity / Transcriptional regulation by small RNAs / Formation of the beta-catenin:TCF transactivating complex / Activated PKN1 stimulates transcription of AR (androgen receptor) regulated genes KLK2 and KLK3 / RUNX1 regulates genes involved in megakaryocyte differentiation and platelet function / HDMs demethylate histones / G2/M DNA damage checkpoint / NoRC negatively regulates rRNA expression / B-WICH complex positively regulates rRNA expression / PKMTs methylate histone lysines / DNA Damage/Telomere Stress Induced Senescence / Pre-NOTCH Transcription and Translation / Meiotic recombination / Activation of anterior HOX genes in hindbrain development during early embryogenesis / Metalloprotease DUBs / RMTs methylate histone arginines / Transcriptional regulation of granulopoiesis / HCMV Early Events / antimicrobial humoral immune response mediated by antimicrobial peptide / structural constituent of chromatin / positive regulation of cellular senescence / UCH proteinases / antibacterial humoral response / heterochromatin formation / nucleosome / nucleosome assembly / E3 ubiquitin ligases ubiquitinate target proteins / Recruitment and ATM-mediated phosphorylation of repair and signaling proteins at DNA double strand breaks / site of double-strand break / HATs acetylate histones / RUNX1 regulates transcription of genes involved in differentiation of HSCs / Factors involved in megakaryocyte development and platelet production / MLL4 and MLL3 complexes regulate expression of PPARG target genes in adipogenesis and hepatic steatosis / chromatin organization / Processing of DNA double-strand break ends / Senescence-Associated Secretory Phenotype (SASP) / double-stranded DNA binding / Oxidative Stress Induced Senescence / defense response to Gram-negative bacterium / gene expression / defense response to virus / Estrogen-dependent gene expression / killing of cells of another organism / chromosome, telomeric region / defense response to Gram-positive bacterium / Ub-specific processing proteases
Similarity search - Function
Mab-21-like, nucleotidyltransferase domain / Mab-21 protein nucleotidyltransferase domain / Mab-21-like / Mab-21-like, HhH/H2TH-like domain / Mab-21 protein HhH/H2TH-like domain / Mab-21 / : / Histone H2B signature. / Histone H2A conserved site / Histone H2A signature. ...Mab-21-like, nucleotidyltransferase domain / Mab-21 protein nucleotidyltransferase domain / Mab-21-like / Mab-21-like, HhH/H2TH-like domain / Mab-21 protein HhH/H2TH-like domain / Mab-21 / : / Histone H2B signature. / Histone H2A conserved site / Histone H2A signature. / Histone H2B / Histone H2B / Histone H2A, C-terminal domain / C-terminus of histone H2A / Histone 2A / Histone H2A / TATA box binding protein associated factor / TATA box binding protein associated factor (TAF), histone-like fold domain / Histone H4, conserved site / Histone H4 signature. / Histone H4 / Histone H4 / CENP-T/Histone H4, histone fold / Centromere kinetochore component CENP-T histone fold / Histone H3 signature 1. / Histone H3 signature 2. / Histone H3 / Histone H3/CENP-A / Histone H2A/H2B/H3 / Core histone H2A/H2B/H3/H4 domain / Histone-fold
Similarity search - Domain/homology
PENTANEDIAL / DNA / DNA (> 10) / DNA (> 100) / Histone H2B type 1-K / Histone H4 / Histone H2A type 2-A / Histone H3.2 / Cyclic GMP-AMP synthase
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsPathare, G.R. / Cavadini, S. / Kempf, G. / Thoma, N.H.
Funding support Switzerland, 4items
OrganizationGrant numberCountry
European Research Council (ERC)666068 Switzerland
European Research Council (ERC)724022 Switzerland
European Research Council (ERC)804933 Switzerland
Swiss National Science FoundationBSSGI0-155984 Switzerland
CitationJournal: Nature / Year: 2020
Title: Structural mechanism of cGAS inhibition by the nucleosome.
Authors: Ganesh R Pathare / Alexiane Decout / Selene Glück / Simone Cavadini / Kristina Makasheva / Ruud Hovius / Georg Kempf / Joscha Weiss / Zuzanna Kozicka / Baptiste Guey / Pauline Melenec / ...Authors: Ganesh R Pathare / Alexiane Decout / Selene Glück / Simone Cavadini / Kristina Makasheva / Ruud Hovius / Georg Kempf / Joscha Weiss / Zuzanna Kozicka / Baptiste Guey / Pauline Melenec / Beat Fierz / Nicolas H Thomä / Andrea Ablasser /
Abstract: The DNA sensor cyclic GMP-AMP synthase (cGAS) initiates innate immune responses following microbial infection, cellular stress and cancer. Upon activation by double-stranded DNA, cytosolic cGAS ...The DNA sensor cyclic GMP-AMP synthase (cGAS) initiates innate immune responses following microbial infection, cellular stress and cancer. Upon activation by double-stranded DNA, cytosolic cGAS produces 2'3' cGMP-AMP, which triggers the induction of inflammatory cytokines and type I interferons . cGAS is also present inside the cell nucleus, which is replete with genomic DNA, where chromatin has been implicated in restricting its enzymatic activity. However, the structural basis for inhibition of cGAS by chromatin remains unknown. Here we present the cryo-electron microscopy structure of human cGAS bound to nucleosomes. cGAS makes extensive contacts with both the acidic patch of the histone H2A-H2B heterodimer and nucleosomal DNA. The structural and complementary biochemical analysis also find cGAS engaged to a second nucleosome in trans. Mechanistically, binding of the nucleosome locks cGAS into a monomeric state, in which steric hindrance suppresses spurious activation by genomic DNA. We find that mutations to the cGAS-acidic patch interface are sufficient to abolish the inhibitory effect of nucleosomes in vitro and to unleash the activity of cGAS on genomic DNA in living cells. Our work uncovers the structural basis of the interaction between cGAS and chromatin and details a mechanism that permits self-non-self discrimination of genomic DNA by cGAS.
History
DepositionFeb 25, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 23, 2020Provider: repository / Type: Initial release
Revision 1.1Dec 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_author.identifier_ORCID
Revision 1.2Oct 23, 2024Group: Data collection / Database references / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update

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Structure visualization

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Assembly

Deposited unit
A: Histone H3.2
B: Histone H4
C: Histone H2A type 2-A
D: Histone H2B type 1-K
E: Histone H3.2
F: Histone H4
G: Histone H2A type 2-A
H: Histone H2B type 1-K
I: DNA (153-MER)
J: DNA (153-MER)
K: Cyclic GMP-AMP synthase
L: Cyclic GMP-AMP synthase
M: Histone H3.2
N: Histone H4
O: Histone H2A type 2-A
P: Histone H2B type 1-K
Q: Histone H3.2
R: Histone H4
S: Histone H2A type 2-A
T: Histone H2B type 1-K
U: DNA (153-MER)
V: DNA (153-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)478,34433
Polymers477,31222
Non-polymers1,03211
Water00
1


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

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Components

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Protein , 5 types, 18 molecules AEMQBFNRCGOSDHPTKL

#1: Protein
Histone H3.2 / Histone H3/m / Histone H3/o


Mass: 11456.345 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: HIST2H3A, HIST2H3C, H3F2, H3FM, HIST2H3D / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: Q71DI3
#2: Protein
Histone H4


Mass: 11394.426 Da / Num. of mol.: 4
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 K-12 (bacteria) / References: UniProt: P62805
#3: Protein
Histone H2A type 2-A / Histone H2A.2 / Histone H2A/o


Mass: 14125.549 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: HIST2H2AA3, H2AFO, HIST2H2AA, HIST2H2AA4 / Production host: Escherichia coli (E. coli) / References: UniProt: Q6FI13
#4: Protein
Histone H2B type 1-K / H2B K / HIRA-interacting protein 1


Mass: 13921.213 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: HIST1H2BK, H2BFT, HIRIP1 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: O60814
#7: Protein Cyclic GMP-AMP synthase / h-cGAS / 2'3'-cGAMP synthase / Mab-21 domain-containing protein 1


Mass: 42404.840 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CGAS, C6orf150, MB21D1 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: Q8N884, cyclic GMP-AMP synthase

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DNA chain , 2 types, 4 molecules IUJV

#5: DNA chain DNA (153-MER)


Mass: 46998.945 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#6: DNA chain DNA (153-MER)


Mass: 47457.234 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)

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Non-polymers , 2 types, 11 molecules

#8: Chemical
ChemComp-PTD / PENTANEDIAL


Mass: 100.116 Da / Num. of mol.: 9 / Source method: obtained synthetically / Formula: C5H8O2
#9: 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 protein modificationY

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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
IDNameTypeEntity IDParent-IDSource
1Cryo-EM structure of cGAS-NCP complexCOMPLEX#1-#70MULTIPLE SOURCES
2ProteinCOMPLEX#1-#2, #4, #71RECOMBINANT
3Histone H2A type 2-ACOMPLEX#31RECOMBINANT
4DNACOMPLEX#5-#61RECOMBINANT
Molecular weightValue: 0.24 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
24Homo sapiens (human)9606
33Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli K-12 (bacteria)83333
23Escherichia coli (E. coli)562
34synthetic construct (others)32630
Buffer solutionpH: 7.4
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Cs: 0.01 mm / C2 aperture diameter: 100 µm
Image recordingElectron dose: 45 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 13943 / Symmetry type: POINT

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