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- PDB-4whv: E3 ubiquitin-protein ligase RNF8 in complex with Ubiquitin-conjug... -

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Basic information

Entry
Database: PDB / ID: 4whv
TitleE3 ubiquitin-protein ligase RNF8 in complex with Ubiquitin-conjugating enzyme E2 N and Polyubiquitin-B
Components
  • E3 ubiquitin-protein ligase RNF8
  • Polyubiquitin-B
  • Ubiquitin-conjugating enzyme E2 N
Keywordsligase/protein binding / E3 ligase / E2 conjugating enzyme / ubiquitination / coiled coil / ligase-protein binding complex
Function / homology
Function and homology information


UBC13-MMS2 complex / ubiquitin conjugating enzyme complex / ubiquitin-protein transferase activator activity / sperm DNA condensation / positive regulation of protein K63-linked ubiquitination / DNA double-strand break processing / protein K6-linked ubiquitination / isotype switching / DNA damage tolerance / symbiont entry into host cell via disruption of host cell glycocalyx ...UBC13-MMS2 complex / ubiquitin conjugating enzyme complex / ubiquitin-protein transferase activator activity / sperm DNA condensation / positive regulation of protein K63-linked ubiquitination / DNA double-strand break processing / protein K6-linked ubiquitination / isotype switching / DNA damage tolerance / symbiont entry into host cell via disruption of host cell glycocalyx / E2 ubiquitin-conjugating enzyme / response to ionizing radiation / positive regulation of double-strand break repair / symbiont entry into host cell via disruption of host cell envelope / virus tail / DNA repair-dependent chromatin remodeling / ubiquitin conjugating enzyme activity / positive regulation of intracellular signal transduction / negative regulation of transcription elongation by RNA polymerase II / signal transduction in response to DNA damage / protein K63-linked ubiquitination / protein monoubiquitination / ubiquitin ligase complex / IRAK1 recruits IKK complex / IRAK1 recruits IKK complex upon TLR7/8 or 9 stimulation / interstrand cross-link repair / protein K48-linked ubiquitination / positive regulation of double-strand break repair via homologous recombination / TRAF6 mediated IRF7 activation in TLR7/8 or 9 signaling / regulation of DNA repair / protein autoubiquitination / TICAM1, RIP1-mediated IKK complex recruitment / JNK (c-Jun kinases) phosphorylation and activation mediated by activated human TAK1 / negative regulation of TORC1 signaling / IKK complex recruitment mediated by RIP1 / PINK1-PRKN Mediated Mitophagy / antiviral innate immune response / activated TAK1 mediates p38 MAPK activation / positive regulation of DNA repair / epigenetic regulation of gene expression / ubiquitin binding / NOD1/2 Signaling Pathway / TAK1-dependent IKK and NF-kappa-B activation / double-strand break repair via homologous recombination / Nonhomologous End-Joining (NHEJ) / ISG15 antiviral mechanism / CLEC7A (Dectin-1) signaling / Formation of Incision Complex in GG-NER / positive regulation of NF-kappaB transcription factor activity / G2/M DNA damage checkpoint / FCERI mediated NF-kB activation / RING-type E3 ubiquitin transferase / Interleukin-1 signaling / double-strand break repair via nonhomologous end joining / Aggrephagy / protein polyubiquitination / ubiquitin-protein transferase activity / Downstream TCR signaling / Antigen processing: Ubiquitination & Proteasome degradation / ubiquitin protein ligase activity / 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 / double-strand break repair / T cell receptor signaling pathway / Processing of DNA double-strand break ends / ubiquitin-dependent protein catabolic process / midbody / histone binding / proteasome-mediated ubiquitin-dependent protein catabolic process / chromosome, telomeric region / positive regulation of canonical NF-kappaB signal transduction / protein ubiquitination / cell division / ubiquitin protein ligase binding / DNA damage response / chromatin binding / SARS-CoV-2 activates/modulates innate and adaptive immune responses / protein homodimerization activity / protein-containing complex / RNA binding / extracellular exosome / zinc ion binding / nucleoplasm / ATP binding / identical protein binding / nucleus / cytosol / cytoplasm
Similarity search - Function
E3 ubiquitin-protein ligase RNF8 / Forkhead associated domain / Forkhead-associated (FHA) domain profile. / FHA domain / Forkhead-associated (FHA) domain / Pectate lyase superfamily protein / Rhamnogalacturonase A/epimerase, pectate lyase-like / Zinc finger, C3HC4 type (RING finger) / SMAD/FHA domain superfamily / Ubiquitin-conjugating enzyme, active site ...E3 ubiquitin-protein ligase RNF8 / Forkhead associated domain / Forkhead-associated (FHA) domain profile. / FHA domain / Forkhead-associated (FHA) domain / Pectate lyase superfamily protein / Rhamnogalacturonase A/epimerase, pectate lyase-like / Zinc finger, C3HC4 type (RING finger) / SMAD/FHA domain superfamily / 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 / Pectin lyase fold / Pectin lyase fold/virulence factor / Zinc finger, RING-type, conserved site / Zinc finger RING-type signature. / Ring finger / Zinc finger RING-type profile. / Zinc finger, RING-type / Ubiquitin family / Zinc finger, RING/FYVE/PHD-type
Similarity search - Domain/homology
E3 ubiquitin-protein ligase RNF8 / Tail fiber / Ubiquitin-conjugating enzyme E2 N
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 8.3 Å
AuthorsHodge, C.D. / Edwards, R.A. / Glover, J.N.M.
CitationJournal: J Biol Chem / Year: 2016
Title: RNF8 E3 Ubiquitin Ligase Stimulates Ubc13 E2 Conjugating Activity That Is Essential for DNA Double Strand Break Signaling and BRCA1 Tumor Suppressor Recruitment.
Authors: Curtis D Hodge / Ismail H Ismail / Ross A Edwards / Greg L Hura / Andrew T Xiao / John A Tainer / Michael J Hendzel / J N Mark Glover /
Abstract: DNA double strand break (DSB) responses depend on the sequential actions of the E3 ubiquitin ligases RNF8 and RNF168 plus E2 ubiquitin-conjugating enzyme Ubc13 to specifically generate histone Lys-63- ...DNA double strand break (DSB) responses depend on the sequential actions of the E3 ubiquitin ligases RNF8 and RNF168 plus E2 ubiquitin-conjugating enzyme Ubc13 to specifically generate histone Lys-63-linked ubiquitin chains in DSB signaling. Here, we defined the activated RNF8-Ubc13∼ubiquitin complex by x-ray crystallography and its functional solution conformations by x-ray scattering, as tested by separation-of-function mutations imaged in cells by immunofluorescence. The collective results show that the RING E3 RNF8 targets E2 Ubc13 to DSB sites and plays a critical role in damage signaling by stimulating polyubiquitination through modulating conformations of ubiquitin covalently linked to the Ubc13 active site. Structure-guided separation-of-function mutations show that the RNF8 E2 stimulating activity is essential for DSB signaling in mammalian cells and is necessary for downstream recruitment of 53BP1 and BRCA1. Chromatin-targeted RNF168 rescues 53BP1 recruitment involved in non-homologous end joining but not BRCA1 recruitment for homologous recombination. These findings suggest an allosteric approach to targeting the ubiquitin-docking cleft at the E2-E3 interface for possible interventions in cancer and chronic inflammation, and moreover, they establish an independent RNF8 role in BRCA1 recruitment.
History
DepositionSep 23, 2014Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 30, 2015Provider: repository / Type: Initial release
Revision 1.1Mar 9, 2016Group: Database references
Revision 1.2May 11, 2016Group: Database references / Structure summary
Revision 1.3Nov 22, 2017Group: Derived calculations / Refinement description / Category: pdbx_struct_oper_list / software / Item: _pdbx_struct_oper_list.symmetry_operation
Revision 1.4Sep 27, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: Ubiquitin-conjugating enzyme E2 N
C: E3 ubiquitin-protein ligase RNF8
D: E3 ubiquitin-protein ligase RNF8
E: Ubiquitin-conjugating enzyme E2 N
H: Ubiquitin-conjugating enzyme E2 N
I: E3 ubiquitin-protein ligase RNF8
J: E3 ubiquitin-protein ligase RNF8
K: Ubiquitin-conjugating enzyme E2 N
A: Polyubiquitin-B
F: Polyubiquitin-B
G: Polyubiquitin-B
L: Polyubiquitin-B
hetero molecules


Theoretical massNumber of molelcules
Total (without water)180,15020
Polymers179,62712
Non-polymers5238
Water00
1
B: Ubiquitin-conjugating enzyme E2 N
C: E3 ubiquitin-protein ligase RNF8
D: E3 ubiquitin-protein ligase RNF8
E: Ubiquitin-conjugating enzyme E2 N
A: Polyubiquitin-B
F: Polyubiquitin-B
hetero molecules


Theoretical massNumber of molelcules
Total (without water)90,07510
Polymers89,8136
Non-polymers2624
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
H: Ubiquitin-conjugating enzyme E2 N
I: E3 ubiquitin-protein ligase RNF8
J: E3 ubiquitin-protein ligase RNF8
K: Ubiquitin-conjugating enzyme E2 N
G: Polyubiquitin-B
L: Polyubiquitin-B
hetero molecules


Theoretical massNumber of molelcules
Total (without water)90,07510
Polymers89,8136
Non-polymers2624
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)341.379, 341.379, 113.447
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number181
Space group name H-MP6422
DetailsThe biological unit is a dimer set (2RNF8 protomers, 2Ubc13~Ub). There are 2 biological units in the asymmetric unit (chains A, B, C, D, E, F and chains G, H, I, J, K, L

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Components

#1: Protein
Ubiquitin-conjugating enzyme E2 N / Bendless-like ubiquitin-conjugating enzyme / Ubc13 / UbcH13 / Ubiquitin carrier protein N / ...Bendless-like ubiquitin-conjugating enzyme / Ubc13 / UbcH13 / Ubiquitin carrier protein N / Ubiquitin-protein ligase N


Mass: 17968.668 Da / Num. of mol.: 4 / Fragment: unp residues 1-152 / Mutation: C87K
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: UBE2N, BLU / Plasmid: pGEX6p1 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: P61088, ubiquitin-protein ligase
#2: Protein
E3 ubiquitin-protein ligase RNF8 / hRNF8 / RING finger protein 8


Mass: 17646.484 Da / Num. of mol.: 4 / Fragment: unp residues 345-485
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RNF8, KIAA0646 / Plasmid: pGEX6p1 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)
References: UniProt: O76064, Ligases; Forming carbon-nitrogen bonds; Acid-amino-acid ligases (peptide synthases)
#3: Protein
Polyubiquitin-B


Mass: 9291.554 Da / Num. of mol.: 4 / Fragment: unp residues 1-76
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: UBB / Plasmid: pET47b+ / Production host: Escherichia coli (E. coli) / References: UniProt: P0CG47
#4: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Zn

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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: 5.31 Å3/Da / Density % sol: 76.84 %
Crystal growTemperature: 298 K / Method: vapor diffusion, sitting drop / pH: 8 / Details: 1.04 M (NH4)2HPO4

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Data collection

DiffractionMean temperature: 105 K
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 12.3.1 / Wavelength: 1.03321 Å
DetectorType: ADSC QUANTUM 315 / Detector: CCD / Date: Jul 19, 2013
RadiationMonochromator: Unk / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.03321 Å / Relative weight: 1
ReflectionResolution: 8.3→50 Å / Num. obs: 4094 / % possible obs: 100 % / Redundancy: 21.8 % / Biso Wilson estimate: 468.44 Å2 / Rmerge(I) obs: 0.191 / Χ2: 1.042 / Net I/av σ(I): 24.273 / Net I/σ(I): 6.7 / Num. measured all: 89114
Reflection shell

Diffraction-ID: 1 / Rejects: _

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique allΧ2% possible all
8.3-8.624.20.9013801.075100
8.6-8.9423.80.7464001.067100
8.94-9.3423.90.4824041.116100
9.34-9.8323.50.3513991.068100
9.83-10.4423.20.2854051.082100
10.44-11.2424.10.2073990.965100
11.24-12.3523.40.1314051.012100
12.35-14.1122.70.1124120.989100
14.11-17.6417.20.0984240.98499.8
17.64-5013.30.0554661.049100

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Processing

Software
NameVersionClassification
HKL-2000data reduction
PHENIX(phenix.refine: 1.9_1692)refinement
PDB_EXTRACT3.15data extraction
HKL-2000data scaling
DENZOdata reduction
SCALEPACKdata scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4ORH

4orh


Resolution: 8.3→49.274 Å / SU ML: 0.58 / Cross valid method: FREE R-VALUE / σ(F): 1.36 / Phase error: 37.74 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.3374 189 4.64 %
Rwork0.3293 3883 -
obs0.3298 4072 99.41 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 607.75 Å2 / Biso mean: 522.6599 Å2 / Biso min: 429.98 Å2
Refinement stepCycle: final / Resolution: 8.3→49.274 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms9612 0 8 0 9620
Biso mean--498.95 --
Num. residues----1349
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0079764
X-RAY DIFFRACTIONf_angle_d1.08913345
X-RAY DIFFRACTIONf_chiral_restr0.0571640
X-RAY DIFFRACTIONf_plane_restr0.0041733
X-RAY DIFFRACTIONf_dihedral_angle_d12.9763377
LS refinement shellResolution: 8.2056→49.2747 Å / Total num. of bins used: 1
RfactorNum. reflection% reflection
Rfree0.3374 189 -
Rwork0.3293 3883 -
all-4072 -
obs--99 %
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
10.08320.0448-0.03950.2806-0.0490.0054-1.7433-0.3495-1.02450.50460.64520.15350.9222.55140.27125.85920.3197-1.40566.14-0.08715.7536-55.6348-116.09610.2947
2-0.04660.1198-0.00270.22740.68611.43911.2304-0.4749-1.44771.4608-0.55410.5705-1.3437-0.89170.45625.7974-0.26620.07624.34960.36015.1482-90.3906-109.34931.6418
31.4811-1.465-0.03241.2880.45690.5387-0.62740.323-4.7742-0.17180.45761.49850.1697-0.4897-1.91865.44270.5045-0.1455.8910.06867.5364-99.2962-104.8491-7.9739
40.6958-0.278-0.23690.14660.03430.04080.24080.6909-0.02741.61380.1172-0.5093-0.2063-0.2152.82755.7150.4943-0.17585.37921.1637.1025-115.0228-72.9423-9.4638
50.0258-0.0296-0.03940.19640.40540.83590.8326-1.4725-0.27790.8728-0.47910.3103-0.32820.2211.1476.67950.0196-0.5926.84240.46535.3549-62.525-73.188131.5667
60.5465-0.83620.64011.2764-0.9551.2715-0.66-0.5532-0.6091-0.0551-0.6709-2.4614-0.59070.632-0.43125.17590.95320.53426.1314-1.04894.7387-59.1374-70.89191.0045
70.3258-0.36340.38440.4206-0.41780.9658-0.1378-0.43360.699-0.3076-0.28010.3944-0.0959-1.03250.46575.40120.92671.54445.14160.20635.3009-71.3089-63.0116-10.9716
80.2622-0.21410.24470.156-0.17730.24080.6618-0.0875-0.2368-0.3948-0.6739-0.198-0.7362-0.3072-0.42577.06140.1107-0.71675.4310.04385.7511-71.5807-66.3725-42.2015
9-0.0024-0.0139-0.00980.0069-0.00510.0087-0.08320.596-0.10940.01290.11420.0432-0.05590.6498-0.12377.3310.78520.13235.51860.11867.0914-69.1139-110.6676-19.3505
100.05840.03630.02410.0080.02450.0084-0.6557-0.26050.44250.6171-0.0196-0.2317-0.65150.2918-0.39976.52570.09390.79556.0166-0.83855.9082-104.4062-83.06318.8897
110.2365-0.0565-0.00410.07960.05140.01360.3522-0.5761-0.0566-0.00720.2481-0.0716-0.1674-0.70380.49467.28640.29-0.46995.9258-0.94495.8237-77.9775-59.026417.6071
120.07020.0429-0.0940.0611-0.08110.09170.01370.1793-0.1948-0.44310.26250.0821-0.01840.17961.51216.68470.691.16796.9134-0.12157.2149-55.2023-75.5734-28.2982
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1(chain 'B' and resid 4 through 150)B0
2X-RAY DIFFRACTION2(chain 'C' and resid 345 through 480)C0
3X-RAY DIFFRACTION3(chain 'D' and resid 342 through 480)D0
4X-RAY DIFFRACTION4(chain 'E' and resid 4 through 150)E0
5X-RAY DIFFRACTION5(chain 'H' and resid 4 through 150)H0
6X-RAY DIFFRACTION6(chain 'I' and resid 380 through 480)I0
7X-RAY DIFFRACTION7(chain 'J' and resid 380 through 480)J0
8X-RAY DIFFRACTION8(chain 'K' and resid 4 through 150)K0
9X-RAY DIFFRACTION9(chain 'A' and resid 1 through 71)A0
10X-RAY DIFFRACTION10(chain 'F' and resid 1 through 71)F0
11X-RAY DIFFRACTION11(chain 'G' and resid 1 through 71)G0
12X-RAY DIFFRACTION12(chain 'L' and resid 1 through 71)L0

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