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- PDB-6r25: Structure of LSD2/NPAC-linker/nucleosome core particle complex: C... -

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Basic information

Entry
Database: PDB / ID: 6r25
TitleStructure of LSD2/NPAC-linker/nucleosome core particle complex: Class 3
Components
  • (DNA (147-MER)) x 2
  • (Histone H3) x 2
  • H2B
  • H4
  • Histone H2A
  • Lysine-specific histone demethylase 1B
  • NPAC
KeywordsGENE REGULATION / Histone demethylation / chromatin reader / flavoenzyme / epigenetics / evolution of protein function / molecular recognition.
Function / homology
Function and homology information


epigenetic programing of female pronucleus / chromatin-protein adaptor activity / genomic imprinting / [histone H3]-N6,N6-dimethyl-L-lysine4 FAD-dependent demethylase / FAD-dependent H3K4me/H3K4me3 demethylase activity / transcription elongation-coupled chromatin remodeling / histone demethylase activity / nucleosome binding / NR1H3 & NR1H2 regulate gene expression linked to cholesterol transport and efflux / transcription initiation-coupled chromatin remodeling ...epigenetic programing of female pronucleus / chromatin-protein adaptor activity / genomic imprinting / [histone H3]-N6,N6-dimethyl-L-lysine4 FAD-dependent demethylase / FAD-dependent H3K4me/H3K4me3 demethylase activity / transcription elongation-coupled chromatin remodeling / histone demethylase activity / nucleosome binding / NR1H3 & NR1H2 regulate gene expression linked to cholesterol transport and efflux / transcription initiation-coupled chromatin remodeling / methylated histone binding / FAD binding / HDMs demethylate histones / structural constituent of chromatin / UCH proteinases / NAD binding / nucleosome / nucleosome assembly / NADP binding / flavin adenine dinucleotide binding / histone binding / oxidoreductase activity / protein heterodimerization activity / chromatin binding / chromatin / negative regulation of transcription by RNA polymerase II / positive regulation of transcription by RNA polymerase II / DNA binding / zinc ion binding / nucleoplasm / nucleus / cytosol
Similarity search - Function
NP60, PWWP domain / Zinc finger, CW-type / CW-type Zinc Finger / Zinc finger CW-type profile. / 3-hydroxyisobutyrate dehydrogenase-like, NAD-binding domain / NAD-binding of NADP-dependent 3-hydroxyisobutyrate dehydrogenase / 6-phosphogluconate dehydrogenase, NADP-binding / NAD binding domain of 6-phosphogluconate dehydrogenase / SWIRM domain / SWIRM domain ...NP60, PWWP domain / Zinc finger, CW-type / CW-type Zinc Finger / Zinc finger CW-type profile. / 3-hydroxyisobutyrate dehydrogenase-like, NAD-binding domain / NAD-binding of NADP-dependent 3-hydroxyisobutyrate dehydrogenase / 6-phosphogluconate dehydrogenase, NADP-binding / NAD binding domain of 6-phosphogluconate dehydrogenase / SWIRM domain / SWIRM domain / SWIRM domain profile. / 6-phosphogluconate dehydrogenase, domain 2 / Amine oxidase / Flavin containing amine oxidoreductase / 6-phosphogluconate dehydrogenase-like, C-terminal domain superfamily / domain with conserved PWWP motif / PWWP domain / PWWP domain profile. / PWWP 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 / TATA box binding protein associated factor / TATA box binding protein associated factor (TAF), histone-like fold domain / 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 / Homeobox-like domain superfamily / Histone H2A/H2B/H3 / Core histone H2A/H2B/H3/H4 / FAD/NAD(P)-binding domain superfamily / Histone-fold / Winged helix-like DNA-binding domain superfamily / NAD(P)-binding domain superfamily
Similarity search - Domain/homology
FLAVIN-ADENINE DINUCLEOTIDE / DNA / DNA (> 10) / DNA (> 100) / Histone H3 / Histone H2B 1.1 / Histone H2A type 1 / Histone H4 / Histone H3.2 / Cytokine-like nuclear factor N-PAC ...FLAVIN-ADENINE DINUCLEOTIDE / DNA / DNA (> 10) / DNA (> 100) / Histone H3 / Histone H2B 1.1 / Histone H2A type 1 / Histone H4 / Histone H3.2 / Cytokine-like nuclear factor N-PAC / Histone H2A / Lysine-specific histone demethylase 2
Similarity search - Component
Biological speciesHomo sapiens (human)
Xenopus laevis (African clawed frog)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.61 Å
AuthorsMarabelli, C. / Pilotto, S. / Chittori, S. / Subramaniam, S. / Mattevi, A.
Funding support Italy, 1items
OrganizationGrant numberCountry
Italian Association for Cancer ResearchIG-11342 Italy
CitationJournal: Cell Rep / Year: 2019
Title: A Tail-Based Mechanism Drives Nucleosome Demethylation by the LSD2/NPAC Multimeric Complex.
Authors: Chiara Marabelli / Biagina Marrocco / Simona Pilotto / Sagar Chittori / Sarah Picaud / Sara Marchese / Giuseppe Ciossani / Federico Forneris / Panagis Filippakopoulos / Guy Schoehn / Daniela ...Authors: Chiara Marabelli / Biagina Marrocco / Simona Pilotto / Sagar Chittori / Sarah Picaud / Sara Marchese / Giuseppe Ciossani / Federico Forneris / Panagis Filippakopoulos / Guy Schoehn / Daniela Rhodes / Sriram Subramaniam / Andrea Mattevi /
Abstract: LSD1 and LSD2 are homologous histone demethylases with opposite biological outcomes related to chromatin silencing and transcription elongation, respectively. Unlike LSD1, LSD2 nucleosome-demethylase ...LSD1 and LSD2 are homologous histone demethylases with opposite biological outcomes related to chromatin silencing and transcription elongation, respectively. Unlike LSD1, LSD2 nucleosome-demethylase activity relies on a specific linker peptide from the multidomain protein NPAC. We used single-particle cryoelectron microscopy (cryo-EM), in combination with kinetic and mutational analysis, to analyze the mechanisms underlying the function of the human LSD2/NPAC-linker/nucleosome complex. Weak interactions between LSD2 and DNA enable multiple binding modes for the association of the demethylase to the nucleosome. The demethylase thereby captures mono- and dimethyl Lys4 of the H3 tail to afford histone demethylation. Our studies also establish that the dehydrogenase domain of NPAC serves as a catalytically inert oligomerization module. While LSD1/CoREST forms a nucleosome docking platform at silenced gene promoters, LSD2/NPAC is a multifunctional enzyme complex with flexible linkers, tailored for rapid chromatin modification, in conjunction with the advance of the RNA polymerase on actively transcribed genes.
History
DepositionMar 15, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 24, 2019Provider: repository / Type: Initial release

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
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Structure viewerMolecule:
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Assembly

Deposited unit
K: Lysine-specific histone demethylase 1B
L: NPAC
M: Histone H3
A: Histone H3
B: H4
C: Histone H2A
D: H2B
E: Histone H3
F: H4
G: Histone H2A
H: H2B
I: DNA (147-MER)
J: DNA (147-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)304,53917
Polymers303,55713
Non-polymers9824
Water5,657314
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: SAXS
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area63320 Å2
ΔGint-451 kcal/mol
Surface area108150 Å2
MethodPISA

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Components

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Protein , 6 types, 10 molecules KMAEBFCGDH

#1: Protein Lysine-specific histone demethylase 1B / Flavin-containing amine oxidase domain-containing protein 1 / Lysine-specific histone demethylase 2


Mass: 87051.547 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: KDM1B, AOF1, C6orf193, LSD2 / Production host: Escherichia coli (E. coli) / References: UniProt: Q8NB78, Oxidoreductases
#3: Protein Histone H3


Mass: 15305.945 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Gene: XELAEV_18002543mg / Production host: Escherichia coli (E. coli) / References: UniProt: A0A310TTQ1, UniProt: P84233*PLUS
#4: Protein Histone H3


Mass: 15303.930 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Gene: XELAEV_18002543mg / Production host: Escherichia coli (E. coli) / References: UniProt: A0A310TTQ1, UniProt: P84233*PLUS
#5: Protein 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) / Production host: Escherichia coli (E. coli) / References: UniProt: P62799*PLUS
#6: Protein Histone H2A


Mass: 13978.241 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Gene: hist1h2aj, LOC494591, XELAEV_18003602mg / Production host: Escherichia coli (E. coli) / References: UniProt: Q6AZJ8, UniProt: P06897*PLUS
#7: Protein H2B


Mass: 13939.228 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Production host: Escherichia coli (E. coli) / References: UniProt: P02281*PLUS

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Protein/peptide , 1 types, 1 molecules L

#2: Protein/peptide NPAC


Mass: 1481.632 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) / References: UniProt: Q49A26*PLUS

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DNA chain , 2 types, 2 molecules IJ

#8: DNA chain DNA (147-MER)


Mass: 45610.043 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#9: DNA chain DNA (147-MER)


Mass: 45138.770 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 3 types, 318 molecules

#10: Chemical ChemComp-FAD / FLAVIN-ADENINE DINUCLEOTIDE


Mass: 785.550 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C27H33N9O15P2 / Comment: FAD*YM
#11: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Zn
#12: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 314 / Source method: isolated from a natural source / Formula: H2O

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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
1LSD2/NPAC(214-225)/nucleosomeCOMPLEXXenopus laevis histones recombinantly expressed. Alkylated K4C-C110A H3. 601 Widom DNA sequence. Human LSD2 Human NPAC#1-#90MULTIPLE SOURCES
2histonesCOMPLEX#3-#71RECOMBINANT
3NPACCOMPLEX#21RECOMBINANT
4DNACOMPLEX#8-#91RECOMBINANT
5Lysine-specific histone demethylase 1BCOMPLEX#11RECOMBINANT
Molecular weightValue: 0.29 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Xenopus laevis (African clawed frog)8355
33Homo sapiens (human)9606
44synthetic construct (others)32630
55Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli (E. coli)562
33synthetic construct (others)32630
44synthetic construct (others)32630
55Escherichia coli (E. coli)562
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTrisC4H11NO31
210 mMPotassium ChlorideKCl1
SpecimenConc.: 0.87 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: LSD2/NPAC(214-225)/nucleosome was monodisperse (gel filtration peak isolation and concentration in buffer described.
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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 3.05 nm / Nominal defocus min: 0.7 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 8 sec. / Electron dose: 1.25 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2078
Image scansMovie frames/image: 40

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Processing

EM software
IDNameVersionCategoryFitting-ID
1RELION2.1particle selection
8UCSF Chimeramodel fitting1
12UCSF Chimeramodel fitting2
17RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 490558
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.61 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 34607 / Symmetry type: POINT
Atomic model building
IDProtocolSpace
1RIGID BODY FITREAL
2RIGID BODY FITREAL
Atomic model building
IDPDB-ID 3D fitting-ID
16ESF

6esf

1
24HSU

4hsu

2

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