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- PDB-8uqb: Crystal structure of RNF168 (RING)-UbcH5c fused to H2A-H2B via a ... -

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Basic information

Entry
Database: PDB / ID: 8uqb
TitleCrystal structure of RNF168 (RING)-UbcH5c fused to H2A-H2B via a 20-residue linker (crystallization condition 1)
ComponentsE3 ubiquitin-protein ligase RNF168,Ubiquitin-conjugating enzyme E2 D3,Histone H2B type 2-E,Histone H2A type 1-B/E
KeywordsTRANSFERASE / RNF168 / UbcH5c / Histone H2A / Histone H2B / Chromatin / Ubiquitin ligase / Ubiquitin-conjugating enzyme / DNA damage response / DNA double-strand break repair / PROTEIN BINDING / PROTEIN BINDING-Transferase complex
Function / homology
Function and homology information


histone H2AK15 ubiquitin ligase activity / histone ubiquitin ligase activity / protein K6-linked ubiquitination / Signaling by BMP / (E3-independent) E2 ubiquitin-conjugating enzyme / double-strand break repair via classical nonhomologous end joining / isotype switching / protein K11-linked ubiquitination / positive regulation of protein targeting to mitochondrion / K63-linked polyubiquitin modification-dependent protein binding ...histone H2AK15 ubiquitin ligase activity / histone ubiquitin ligase activity / protein K6-linked ubiquitination / Signaling by BMP / (E3-independent) E2 ubiquitin-conjugating enzyme / double-strand break repair via classical nonhomologous end joining / isotype switching / protein K11-linked ubiquitination / positive regulation of protein targeting to mitochondrion / K63-linked polyubiquitin modification-dependent protein binding / E2 ubiquitin-conjugating enzyme / response to ionizing radiation / DNA repair-dependent chromatin remodeling / ubiquitin conjugating enzyme activity / negative regulation of transcription elongation by RNA polymerase II / protein K63-linked ubiquitination / negative regulation of BMP signaling pathway / protein monoubiquitination / ubiquitin ligase complex / interstrand cross-link repair / protein localization to CENP-A containing chromatin / SUMOylation of DNA damage response and repair proteins / protein K48-linked ubiquitination / Replacement of protamines by nucleosomes in the male pronucleus / nucleosome binding / CENP-A containing nucleosome / protein autoubiquitination / Packaging Of Telomere Ends / Recognition and association of DNA glycosylase with site containing an affected purine / Cleavage of the damaged purine / TICAM1, RIP1-mediated IKK complex recruitment / IKK complex recruitment mediated by RIP1 / Deposition of new CENPA-containing nucleosomes at the centromere / Recognition and association of DNA glycosylase with site containing an affected pyrimidine / Cleavage of the damaged pyrimidine / PINK1-PRKN Mediated Mitophagy / Inhibition of DNA recombination at telomere / RNA Polymerase I Promoter Opening / Meiotic synapsis / Assembly of the ORC complex at the origin of replication / Negative regulators of DDX58/IFIH1 signaling / Regulation of endogenous retroelements by the Human Silencing Hub (HUSH) complex / positive regulation of DNA repair / DNA methylation / Condensation of Prophase Chromosomes / Peroxisomal protein import / Chromatin modifications during the maternal to zygotic transition (MZT) / epigenetic regulation of gene expression / HCMV Late Events / SIRT1 negatively regulates rRNA expression / Downregulation of SMAD2/3:SMAD4 transcriptional activity / ERCC6 (CSB) and EHMT2 (G9a) positively regulate rRNA expression / Regulation of TNFR1 signaling / ubiquitin binding / PRC2 methylates histones and DNA / innate immune response in mucosa / Regulation of endogenous retroelements by KRAB-ZFP proteins / Defective pyroptosis / Regulation of endogenous retroelements by Piwi-interacting RNAs (piRNAs) / HDACs deacetylate histones / Inactivation of CSF3 (G-CSF) signaling / RNA Polymerase I Promoter Escape / Nonhomologous End-Joining (NHEJ) / Transcriptional regulation by small RNAs / Oxygen-dependent proline hydroxylation of Hypoxia-inducible Factor Alpha / Formation of the beta-catenin:TCF transactivating complex / RUNX1 regulates genes involved in megakaryocyte differentiation and platelet function / Activated PKN1 stimulates transcription of AR (androgen receptor) regulated genes KLK2 and KLK3 / G2/M DNA damage checkpoint / Metalloprotease DUBs / NoRC negatively regulates rRNA expression / protein modification process / RING-type E3 ubiquitin transferase / DNA Damage/Telomere Stress Induced Senescence / B-WICH complex positively regulates rRNA expression / Meiotic recombination / Pre-NOTCH Transcription and Translation / double-strand break repair via nonhomologous end joining / RMTs methylate histone arginines / Activation of anterior HOX genes in hindbrain development during early embryogenesis / Transcriptional regulation of granulopoiesis / UCH proteinases / HCMV Early Events / protein polyubiquitination / ubiquitin-protein transferase activity / antimicrobial humoral immune response mediated by antimicrobial peptide / Antigen processing: Ubiquitination & Proteasome degradation / structural constituent of chromatin / ubiquitin protein ligase activity / antibacterial humoral response / E3 ubiquitin ligases ubiquitinate target proteins / Recruitment and ATM-mediated phosphorylation of repair and signaling proteins at DNA double strand breaks / nucleosome / heterochromatin formation / site of double-strand break / double-strand break repair / RUNX1 regulates transcription of genes involved in differentiation of HSCs / Neddylation / nucleosome assembly / Processing of DNA double-strand break ends
Similarity search - Function
E3 ubiquitin-protein ligase RNF168 / : / Prokaryotic RING finger family 4 / Ubiquitin-conjugating enzyme, active site / Ubiquitin-conjugating (UBC) active site signature. / Ubiquitin-conjugating enzyme E2 / Ubiquitin-conjugating enzyme / Ubiquitin-conjugating (UBC) core domain profile. / Ubiquitin-conjugating enzyme E2, catalytic domain homologues / Ubiquitin-conjugating enzyme/RWD-like ...E3 ubiquitin-protein ligase RNF168 / : / Prokaryotic RING finger family 4 / Ubiquitin-conjugating enzyme, active site / Ubiquitin-conjugating (UBC) active site signature. / Ubiquitin-conjugating enzyme E2 / Ubiquitin-conjugating enzyme / Ubiquitin-conjugating (UBC) core domain profile. / Ubiquitin-conjugating enzyme E2, catalytic domain homologues / Ubiquitin-conjugating enzyme/RWD-like / Ring finger / : / 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 / Zinc finger RING-type profile. / Zinc finger, RING-type / Histone H2A/H2B/H3 / Core histone H2A/H2B/H3/H4 / Histone-fold / Zinc finger, RING/FYVE/PHD-type
Similarity search - Domain/homology
Histone H2A type 1-B/E / Ubiquitin-conjugating enzyme E2 D3 / Histone H2B type 2-E / E3 ubiquitin-protein ligase RNF168
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.484 Å
AuthorsHu, Q. / Botuyan, M.V. / Mer, G.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM136262 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)R01 CA132878 United States
CitationJournal: Mol Cell / Year: 2024
Title: Mechanisms of RNF168 nucleosome recognition and ubiquitylation.
Authors: Qi Hu / Debiao Zhao / Gaofeng Cui / Janarjan Bhandari / James R Thompson / Maria Victoria Botuyan / Georges Mer /
Abstract: RNF168 plays a central role in the DNA damage response (DDR) by ubiquitylating histone H2A at K13 and K15. These modifications direct BRCA1-BARD1 and 53BP1 foci formation in chromatin, essential for ...RNF168 plays a central role in the DNA damage response (DDR) by ubiquitylating histone H2A at K13 and K15. These modifications direct BRCA1-BARD1 and 53BP1 foci formation in chromatin, essential for cell-cycle-dependent DNA double-strand break (DSB) repair pathway selection. The mechanism by which RNF168 catalyzes the targeted accumulation of H2A ubiquitin conjugates to form repair foci around DSBs remains unclear. Here, using cryoelectron microscopy (cryo-EM), nuclear magnetic resonance (NMR) spectroscopy, and functional assays, we provide a molecular description of the reaction cycle and dynamics of RNF168 as it modifies the nucleosome and recognizes its ubiquitylation products. We demonstrate an interaction of a canonical ubiquitin-binding domain within full-length RNF168, which not only engages ubiquitin but also the nucleosome surface, clarifying how such site-specific ubiquitin recognition propels a signal amplification loop. Beyond offering mechanistic insights into a key DDR protein, our study aids in understanding site specificity in both generating and interpreting chromatin ubiquitylation.
History
DepositionOct 23, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 17, 2024Provider: repository / Type: Initial release
Revision 1.1Jan 31, 2024Group: 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_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Mar 20, 2024Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: E3 ubiquitin-protein ligase RNF168,Ubiquitin-conjugating enzyme E2 D3,Histone H2B type 2-E,Histone H2A type 1-B/E
hetero molecules


Theoretical massNumber of molelcules
Total (without water)50,3137
Polymers50,0401
Non-polymers2736
Water73941
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)101.948, 101.948, 86.992
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number154
Space group name H-MP3221

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Components

#1: Protein E3 ubiquitin-protein ligase RNF168,Ubiquitin-conjugating enzyme E2 D3,Histone H2B type 2-E,Histone H2A type 1-B/E


Mass: 50040.320 Da / Num. of mol.: 1 / Mutation: C85K in UbcH5c
Source method: isolated from a genetically manipulated source
Details: residues 1-94 of RNF168, followed by residues 2-147 of UbcH5c, followed by residues 33-123 of H2B, followed by residues 12-105 of H2A
Source: (gene. exp.) Homo sapiens (human)
Gene: RNF168, UBE2D3, UBC5C, UBCH5C, H2BC21, H2BFQ, HIST2H2BE, H2AC4, H2AFM, HIST1H2AB, H2AC8, H2AFA, HIST1H2AE
Production host: Escherichia coli (E. coli)
References: UniProt: Q8IYW5, UniProt: P61077, UniProt: Q16778, UniProt: P04908, RING-type E3 ubiquitin transferase, E2 ubiquitin-conjugating enzyme, (E3-independent) E2 ubiquitin-conjugating enzyme
#2: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical
ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Cl
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 41 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.75 Å3/Da / Density % sol: 55.27 %
Crystal growTemperature: 288 K / Method: vapor diffusion, hanging drop / pH: 7
Details: Crystals were obtained by mixing 2 microliters of the protein fusion (12 mg/mL) in 10 mM HEPES, pH 7.5, 600 mM NaCl, 1 mM TCEP and 2 microliters of the reservoir solution containing 50 mM ...Details: Crystals were obtained by mixing 2 microliters of the protein fusion (12 mg/mL) in 10 mM HEPES, pH 7.5, 600 mM NaCl, 1 mM TCEP and 2 microliters of the reservoir solution containing 50 mM BIS-TRIS propane, pH 7.0, 1 M NaCl

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 19-BM / Wavelength: 0.9792 Å
DetectorType: ADSC QUANTUM 315r / Detector: CCD / Date: Feb 2, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9792 Å / Relative weight: 1
ReflectionResolution: 2.484→50 Å / Num. obs: 18662 / % possible obs: 99.36 % / Redundancy: 5.3 % / Biso Wilson estimate: 44.24 Å2 / CC1/2: 0.996 / CC star: 0.999 / Rmerge(I) obs: 0.113 / Rpim(I) all: 0.05322 / Rrim(I) all: 0.1255 / Net I/σ(I): 16.39
Reflection shellResolution: 2.484→2.573 Å / Redundancy: 4.6 % / Rmerge(I) obs: 0.7775 / Mean I/σ(I) obs: 2.66 / Num. unique obs: 1718 / CC1/2: 0.731 / CC star: 0.919 / Rpim(I) all: 0.3962 / Rrim(I) all: 0.8774 / % possible all: 94.24

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Processing

Software
NameVersionClassification
PHENIX1.19.2_4158refinement
HKL-2000data reduction
HKL-2000data scaling
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4GB0, 5EGG

4gb0

5egg


Resolution: 2.484→39.37 Å / SU ML: 0.34 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 26.4 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2596 1869 10.02 %
Rwork0.2182 --
obs0.2223 18659 99.39 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.484→39.37 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3158 0 6 41 3205
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0043225
X-RAY DIFFRACTIONf_angle_d0.6564375
X-RAY DIFFRACTIONf_dihedral_angle_d4.442447
X-RAY DIFFRACTIONf_chiral_restr0.04498
X-RAY DIFFRACTIONf_plane_restr0.007560
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.484-2.550.36331270.29721178X-RAY DIFFRACTION93
2.55-2.630.33721420.28691285X-RAY DIFFRACTION100
2.63-2.710.30681420.28231259X-RAY DIFFRACTION100
2.71-2.810.30961450.26761294X-RAY DIFFRACTION100
2.81-2.920.30671440.27761296X-RAY DIFFRACTION100
2.92-3.050.3051410.30111264X-RAY DIFFRACTION100
3.05-3.210.33791460.25061310X-RAY DIFFRACTION100
3.21-3.420.32841450.23431288X-RAY DIFFRACTION100
3.42-3.680.27071440.20751284X-RAY DIFFRACTION100
3.68-4.050.23751440.18851305X-RAY DIFFRACTION100
4.05-4.630.1871480.17241318X-RAY DIFFRACTION100
4.64-5.840.21681500.18691325X-RAY DIFFRACTION100
5.84-39.370.22671510.19631384X-RAY DIFFRACTION100

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