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- PDB-6nqa: Active state Dot1L bound to the H2B-Ubiquitinated nucleosome, 1-t... -

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Basic information

Entry
Database: PDB / ID: 6nqa
TitleActive state Dot1L bound to the H2B-Ubiquitinated nucleosome, 1-to-1 complex
Components
  • (601 DNA Strand ...) x 2
  • Histone H2A type 1
  • Histone H2B 1.1
  • Histone H3.2
  • Histone H4
  • Histone-lysine N-methyltransferase, H3 lysine-79 specificHistone methyltransferase
  • Ubiquitin
KeywordsSTRUCTURAL PROTEIN/TRANSFERASE/DNA / Ubiquitin / Nucleosome / Methyltransferase / STRUCTURAL PROTEIN-TRANSFERASE-DNA complex
Function / homology
Function and homology information


: / histone H3K79 trimethyltransferase activity / [histone H3]-lysine79 N-trimethyltransferase / histone H3K79 methyltransferase activity / regulation of transcription regulatory region DNA binding / regulation of receptor signaling pathway via JAK-STAT / histone H3 methyltransferase activity / histone methyltransferase activity / heterochromatin formation / Maturation of protein E ...: / histone H3K79 trimethyltransferase activity / [histone H3]-lysine79 N-trimethyltransferase / histone H3K79 methyltransferase activity / regulation of transcription regulatory region DNA binding / regulation of receptor signaling pathway via JAK-STAT / histone H3 methyltransferase activity / histone methyltransferase activity / heterochromatin formation / Maturation of protein E / Maturation of protein E / ER Quality Control Compartment (ERQC) / Myoclonic epilepsy of Lafora / FLT3 signaling by CBL mutants / Prevention of phagosomal-lysosomal fusion / IRAK2 mediated activation of TAK1 complex / Alpha-protein kinase 1 signaling pathway / Glycogen synthesis / IRAK1 recruits IKK complex / IRAK1 recruits IKK complex upon TLR7/8 or 9 stimulation / Membrane binding and targetting of GAG proteins / Constitutive Signaling by NOTCH1 HD Domain Mutants / Endosomal Sorting Complex Required For Transport (ESCRT) / NOTCH2 Activation and Transmission of Signal to the Nucleus / IRAK2 mediated activation of TAK1 complex upon TLR7/8 or 9 stimulation / PTK6 Regulates RTKs and Their Effectors AKT1 and DOK1 / Negative regulation of FLT3 / Regulation of FZD by ubiquitination / TICAM1,TRAF6-dependent induction of TAK1 complex / TICAM1-dependent activation of IRF3/IRF7 / APC/C:Cdc20 mediated degradation of Cyclin B / Downregulation of ERBB4 signaling / p75NTR recruits signalling complexes / TRAF6 mediated IRF7 activation in TLR7/8 or 9 signaling / APC-Cdc20 mediated degradation of Nek2A / PINK1-PRKN Mediated Mitophagy / TRAF6-mediated induction of TAK1 complex within TLR4 complex / InlA-mediated entry of Listeria monocytogenes into host cells / Pexophagy / telomere organization / Regulation of innate immune responses to cytosolic DNA / VLDLR internalisation and degradation / Downregulation of ERBB2:ERBB3 signaling / NRIF signals cell death from the nucleus / Activated NOTCH1 Transmits Signal to the Nucleus / Translesion synthesis by REV1 / NF-kB is activated and signals survival / Regulation of PTEN localization / Translesion synthesis by POLK / Regulation of BACH1 activity / Synthesis of active ubiquitin: roles of E1 and E2 enzymes / Translesion synthesis by POLI / Gap-filling DNA repair synthesis and ligation in GG-NER / MAP3K8 (TPL2)-dependent MAPK1/3 activation / TICAM1, RIP1-mediated IKK complex recruitment / Downregulation of TGF-beta receptor signaling / Josephin domain DUBs / Activation of IRF3, IRF7 mediated by TBK1, IKKε (IKBKE) / Regulation of activated PAK-2p34 by proteasome mediated degradation / InlB-mediated entry of Listeria monocytogenes into host cell / IKK complex recruitment mediated by RIP1 / JNK (c-Jun kinases) phosphorylation and activation mediated by activated human TAK1 / TGF-beta receptor signaling in EMT (epithelial to mesenchymal transition) / N-glycan trimming in the ER and Calnexin/Calreticulin cycle / Autodegradation of Cdh1 by Cdh1:APC/C / TNFR1-induced NF-kappa-B signaling pathway / DNA damage checkpoint signaling / APC/C:Cdc20 mediated degradation of Securin / Asymmetric localization of PCP proteins / TCF dependent signaling in response to WNT / SCF-beta-TrCP mediated degradation of Emi1 / Regulation of NF-kappa B signaling / NIK-->noncanonical NF-kB signaling / Ubiquitin-dependent degradation of Cyclin D / AUF1 (hnRNP D0) binds and destabilizes mRNA / Negative regulators of DDX58/IFIH1 signaling / TNFR2 non-canonical NF-kB pathway / NOTCH3 Activation and Transmission of Signal to the Nucleus / activated TAK1 mediates p38 MAPK activation / Assembly of the pre-replicative complex / Vpu mediated degradation of CD4 / Deactivation of the beta-catenin transactivating complex / Degradation of DVL / Ubiquitin Mediated Degradation of Phosphorylated Cdc25A / Recognition of DNA damage by PCNA-containing replication complex / Regulation of signaling by CBL / Dectin-1 mediated noncanonical NF-kB signaling / Hh mutants are degraded by ERAD / Cdc20:Phospho-APC/C mediated degradation of Cyclin A / Fanconi Anemia Pathway / Negative regulation of FGFR3 signaling / Termination of translesion DNA synthesis / Peroxisomal protein import / Degradation of AXIN / Downregulation of SMAD2/3:SMAD4 transcriptional activity / Regulation of TNFR1 signaling / Defective CFTR causes cystic fibrosis / Degradation of GLI1 by the proteasome / Activation of NF-kappaB in B cells / Hedgehog ligand biogenesis
Similarity search - Function
434 Repressor (Amino-terminal Domain) - #60 / Histone H3-K79 methyltransferase, metazoa / Histone-lysine N-methyltransferase DOT1 domain / Histone H3-K79 methyltransferase / Histone methylation protein DOT1 / Histone-lysine N-methyltransferase DOT1 (EC 2.1.1.43) domain profile. / Histone, subunit A / Histone, subunit A / 434 Repressor (Amino-terminal Domain) / Histone H2B signature. ...434 Repressor (Amino-terminal Domain) - #60 / Histone H3-K79 methyltransferase, metazoa / Histone-lysine N-methyltransferase DOT1 domain / Histone H3-K79 methyltransferase / Histone methylation protein DOT1 / Histone-lysine N-methyltransferase DOT1 (EC 2.1.1.43) domain profile. / Histone, subunit A / Histone, subunit A / 434 Repressor (Amino-terminal 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 H2A / Histone 2A / Histone H4, conserved site / Histone H4 signature. / Histone H4 / Histone H4 / 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 / Vaccinia Virus protein VP39 / Histone H3 signature 1. / Ubiquitin conserved site / Ubiquitin domain / Histone H3 signature 2. / Histone H3 / Histone H3/CENP-A / Ubiquitin domain signature. / Histone H2A/H2B/H3 / Core histone H2A/H2B/H3/H4 / Histone-fold / Ubiquitin family / Ubiquitin homologues / Ubiquitin-like domain / Ubiquitin domain profile. / Ubiquitin-like domain superfamily / S-adenosyl-L-methionine-dependent methyltransferase superfamily / Rossmann fold / Orthogonal Bundle / 3-Layer(aba) Sandwich / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
S-ADENOSYLMETHIONINE / DNA / DNA (> 10) / DNA (> 100) / Histone H2B 1.1 / Histone H2A type 1 / Polyubiquitin-C / Histone H4 / Histone H3.2 / Histone-lysine N-methyltransferase, H3 lysine-79 specific
Similarity search - Component
Biological speciessynthetic construct (others)
Xenopus laevis (African clawed frog)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.54 Å
AuthorsWorden, E.J. / Hoffmann, N.A. / Wolberger, C.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM095822 United States
CitationJournal: Cell / Year: 2019
Title: Mechanism of Cross-talk between H2B Ubiquitination and H3 Methylation by Dot1L.
Authors: Evan J Worden / Niklas A Hoffmann / Chad W Hicks / Cynthia Wolberger /
Abstract: Methylation of histone H3 K79 by Dot1L is a hallmark of actively transcribed genes that depends on monoubiquitination of H2B K120 (H2B-Ub) and is an example of histone modification cross-talk that is ...Methylation of histone H3 K79 by Dot1L is a hallmark of actively transcribed genes that depends on monoubiquitination of H2B K120 (H2B-Ub) and is an example of histone modification cross-talk that is conserved from yeast to humans. We report here cryo-EM structures of Dot1L bound to ubiquitinated nucleosome that show how H2B-Ub stimulates Dot1L activity and reveal a role for the histone H4 tail in positioning Dot1L. We find that contacts mediated by Dot1L and the H4 tail induce a conformational change in the globular core of histone H3 that reorients K79 from an inaccessible position, thus enabling this side chain to insert into the active site in a position primed for catalysis. Our study provides a comprehensive mechanism of cross-talk between histone ubiquitination and methylation and reveals structural plasticity in histones that makes it possible for histone-modifying enzymes to access residues within the nucleosome core.
History
DepositionJan 19, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 20, 2019Provider: repository / Type: Initial release
Revision 1.1Feb 27, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Mar 20, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Jan 8, 2020Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization

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Structure visualization

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  • Deposited structure unit
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Structure viewerMolecule:
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Assembly

Deposited unit
I: 601 DNA Strand 1
J: 601 DNA Strand 2
F: Histone H4
G: Histone H2A type 1
H: Histone H2B 1.1
K: Histone-lysine N-methyltransferase, H3 lysine-79 specific
L: Ubiquitin
E: Histone H3.2
A: Histone H3.2
D: Histone H2B 1.1
C: Histone H2A type 1
B: Histone H4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)254,98213
Polymers254,58412
Non-polymers3981
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area61910 Å2
ΔGint-413 kcal/mol
Surface area97870 Å2

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Components

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601 DNA Strand ... , 2 types, 2 molecules IJ

#1: DNA chain 601 DNA Strand 1


Mass: 44825.559 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Plasmid: pST55-16x601 / Production host: Escherichia coli (E. coli)
#2: DNA chain 601 DNA Strand 2


Mass: 45305.852 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Plasmid: pST55-16x601 / Production host: Escherichia coli (E. coli)

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Protein , 6 types, 10 molecules FBGCHDKLEA

#3: Protein Histone H4 /


Mass: 11263.231 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Plasmid: pET3a / Production host: Escherichia coli (E. coli) / References: UniProt: P62799
#4: Protein Histone H2A type 1


Mass: 13978.241 Da / Num. of mol.: 2 / Mutation: G99R, A123S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Plasmid: pET3a / Production host: Escherichia coli (E. coli) / References: UniProt: P06897
#5: Protein Histone H2B 1.1 / H2B1.1


Mass: 13498.715 Da / Num. of mol.: 2 / Mutation: S32T, K120C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Plasmid: pET3a / Production host: Escherichia coli (E. coli) / References: UniProt: P02281
#6: Protein Histone-lysine N-methyltransferase, H3 lysine-79 specific / Histone methyltransferase / DOT1-like protein / Histone H3-K79 methyltransferase / H3-K79-HMTase / Lysine N-methyltransferase 4


Mass: 47432.012 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: DOT1L, KIAA1814, KMT4 / Plasmid: pET32a / Production host: Escherichia coli (E. coli)
References: UniProt: Q8TEK3, histone-lysine N-methyltransferase
#7: Protein Ubiquitin /


Mass: 9036.393 Da / Num. of mol.: 1 / Mutation: G76C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: UBC / Production host: Escherichia coli (E. coli) / References: UniProt: P0CG48
#8: Protein Histone H3.2


Mass: 15251.830 Da / Num. of mol.: 2 / Mutation: G102A, K79Nle, M90Nle, M120Nle
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Plasmid: pQE-81L / Production host: Escherichia coli (E. coli) / References: UniProt: P84233

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Non-polymers , 1 types, 1 molecules

#9: Chemical ChemComp-SAM / S-ADENOSYLMETHIONINE / S-Adenosyl methionine


Mass: 398.437 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C15H22N6O5S

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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
1Active state Dot1L in complex with the H2B-Ub nucleosomeCOMPLEX1:1 complex of Dot1L bound to the nucleosome#1-#80MULTIPLE SOURCES
2Dot1LCOMPLEX#61RECOMBINANT
3H2B-Ub nucleosomeCOMPLEX#1-#5, #7-#81MULTIPLE SOURCES
4histone coreCOMPLEX#3-#5, #83RECOMBINANT
5ubiquitinCOMPLEX#73RECOMBINANT
6DNACOMPLEX#1-#23RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Xenopus laevis (African clawed frog)8355
24Xenopus laevis (African clawed frog)8355
35Homo sapiens (human)9606
46synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDPlasmid
12Escherichia coli (E. coli)562pET32a
24Escherichia coli (E. coli)562pET3a,pQE-81L
35Escherichia coli (E. coli)562
46Escherichia coli (E. coli)562pST55-16x601
Buffer solutionpH: 7.5
Details: Solutions were prepared on the day of freezing and filtered though a 0.2 um filter prior to use.
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMsodium chlorideNaClSodium chloride1
22 mMdithiothreitolC4H10O2S21
320 mMHEPESC8H18N2O4S1
SpecimenConc.: 0.75 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Crosslinked with glutaraldehyde
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: Blot once for 3.5 seconds before freezing.

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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 FIELDBright-field microscopy / Nominal magnification: 130000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 8.2 sec. / Electron dose: 50 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 2284 / Details: 3 exposures per hole
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

EM software
IDNameVersionCategory
1RELION3particle selection
2Latitudeimage acquisition
4CTFFIND4.1CTF correction
9PHENIXmodel refinement
10RELION3initial Euler assignment
11RELION3final Euler assignment
12RELION3classification
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 654532
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.54 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 220681 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-ID
11KX3

1kx3

A1
51KX3

1kx3

B1
61KX3

1kx3

C1
71KX3

1kx3

D1
81KX3

1kx3

E1
91KX3

1kx3

F1
101KX3

1kx3

G1
111KX3

1kx3

H1
21NW3

1nw3

K1
31UBQ

1ubq

L1
43MVD

3mvd

I1
123MVD

3mvd

J1

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