SASDBR3: Wild type RNF8 complexed with Ubc13 (C87K, K92A mutant): conjugat...

Basic information

• Entry: Database: SASBDB / ID: SASDBR3

Sample

Wild type RNF8 complexed with Ubc13 (C87K, K92A mutant): conjugated to Ubiquitin

• E3 ubiquitin-protein ligase RNF8 (protein), RNF8, Homo sapiens
• Ubiquitin-conjugating enzyme E2 N double mutant (C87K, K92A) (protein), Ubc13 - C87K, K92A, Homo sapiens
• Polyubiquitin-C (protein), UBC, Homo sapiens

Function / homology

Function:

: / : / UBC13-MMS2 complex / ubiquitin conjugating enzyme complex / sperm DNA condensation / ubiquitin-protein transferase activator activity / DNA double-strand break processing / positive regulation of protein K63-linked ubiquitination / isotype switching / postreplication repair / DNA repair-dependent chromatin remodeling / positive regulation of double-strand break repair / E2 ubiquitin-conjugating enzyme / positive regulation of intracellular signal transduction / response to ionizing radiation / negative regulation of transcription elongation by RNA polymerase II / ubiquitin conjugating enzyme activity / protein K63-linked ubiquitination / antiviral innate immune response / protein K48-linked ubiquitination / protein autoubiquitination / regulation of DNA repair / interstrand cross-link repair / ubiquitin ligase complex / epigenetic regulation of gene expression / signal transduction in response to DNA damage / Maturation of protein E / Maturation of protein E / ER Quality Control Compartment (ERQC) / Myoclonic epilepsy of Lafora / negative regulation of TORC1 signaling / 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 / positive regulation of DNA repair / p75NTR recruits signalling complexes / Downregulation of ERBB4 signaling / 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 / Pexophagy / InlA-mediated entry of Listeria monocytogenes into host cells / 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 / NF-kB is activated and signals survival / Regulation of PTEN localization / Regulation of BACH1 activity / Translesion synthesis by REV1 / Synthesis of active ubiquitin: roles of E1 and E2 enzymes / Translesion synthesis by POLK / MAP3K8 (TPL2)-dependent MAPK1/3 activation / TICAM1, RIP1-mediated IKK complex recruitment / Gap-filling DNA repair synthesis and ligation in GG-NER / Downregulation of TGF-beta receptor signaling / Translesion synthesis by POLI / 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 / ubiquitin binding / 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 / NIK-->noncanonical NF-kB signaling / Regulation of NF-kappa B signaling / Ubiquitin-dependent degradation of Cyclin D / AUF1 (hnRNP D0) binds and destabilizes mRNA / NOTCH3 Activation and Transmission of Signal to the Nucleus / TNFR2 non-canonical NF-kB pathway / activated TAK1 mediates p38 MAPK activation / Assembly of the pre-replicative complex / Vpu mediated degradation of CD4 / Negative regulators of DDX58/IFIH1 signaling / Degradation of DVL

Domain/homology:

E3 ubiquitin-protein ligase RNF8 / Forkhead associated domain / Forkhead-associated (FHA) domain profile. / FHA domain / Forkhead-associated (FHA) domain / Zinc finger, C3HC4 type (RING finger) / Ubiquitin-conjugating enzyme, active site / Ubiquitin-conjugating (UBC) active site signature. / SMAD/FHA domain superfamily / Ubiquitin-conjugating enzyme E2, catalytic domain homologues / Ubiquitin-conjugating enzyme E2 / Ubiquitin-conjugating enzyme / Ubiquitin-conjugating (UBC) core domain profile. / Ubiquitin-conjugating enzyme/RWD-like / Zinc finger, RING-type, conserved site / Zinc finger RING-type signature. / Ring finger / Ubiquitin conserved site / Ubiquitin domain / Zinc finger RING-type profile. / Zinc finger, RING-type / Ubiquitin domain signature. / Ubiquitin family / Ubiquitin homologues / Ubiquitin-like domain / Ubiquitin domain profile. / Ubiquitin-like domain superfamily / Zinc finger, RING/FYVE/PHD-type

Component:

E3 ubiquitin-protein ligase RNF8 / Polyubiquitin-C / Ubiquitin-conjugating enzyme E2 N

• Biological species: Homo sapiens (human)
• Citation: Journal: 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-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.

Contact author

• Curtis Hodge
• Ross Edwards
• Mark Glover

Models

• Model #461: ; Type: mix / Software: MES-FoXS / Radius of dummy atoms: 1.90 A; Search similar-shape structures of this assembly by Omokage search (details)
• Model #462: ; Type: mix / Software: MES-FoXS / Radius of dummy atoms: 1.90 A; Search similar-shape structures of this assembly by Omokage search (details)
• Model #463: ; Type: mix / Software: MES-FoXS / Radius of dummy atoms: 1.90 A; Search similar-shape structures of this assembly by Omokage search (details)
• Model #464: ; Type: mix / Software: MES-FoXS / Radius of dummy atoms: 1.90 A; Search similar-shape structures of this assembly by Omokage search (details)
• Model #465: ; Type: mix / Software: MES-FoXS / Radius of dummy atoms: 1.90 A; Search similar-shape structures of this assembly by Omokage search (details)

Sample

• Sample: Name: Wild type RNF8 complexed with Ubc13 (C87K, K92A mutant): conjugated to Ubiquitin; Entity id: 304 / 305 / 306
• Buffer: Name: HEPES / Concentration: 20.00 mM / pH: 6.8 / Composition: 200 mM NaCl, 0.01 mM, ZnSO4, and 1 mM DTT

Entity #304

Name: RNF8 / Type: protein / Description: E3 ubiquitin-protein ligase RNF8 / Formula weight: 17.618 / Num. of mol.: 2 / Source: Homo sapiens / References: UniProt: O76064
Sequence:

GPLGSPEFQE HWALMEELNR SKKDFEAIIQ AKNKELEQTK EEKEKMQAQK EEVLSHMNDV LENELQCIIC SEYFIEAVTL NCAHSFCSYC INEWMKRKIE CPICRKDIKS KTYSLVLDNC INKMVNNLSS EVKERRIVLI RERKAKRLF

Entity #305

Name: Ubc13 - C87K, K92A / Type: protein
Description: Ubiquitin-conjugating enzyme E2 N double mutant (C87K, K92A)
Formula weight: 17.89 / Num. of mol.: 2 / Source: Homo sapiens / References: UniProt: P61088
Sequence:

GPLGSPEFMA GLPRRIIKET QRLLAEPVPG IKAEPDESNA RYFHVVIAGP QDSPFEGGTF KLELFLPEEY PMAAPKVRFM TKIYHPNVDK LGRIKLDILA DKWSPALQIR TVLLSIQALL SAPNPDDPLA NDVAEQWKTN EAQAIETARA WTRLYAMNNI

Entity #306

Name: UBC / Type: protein / Description: Polyubiquitin-C / Formula weight: 8.565 / Num. of mol.: 2 / Source: Homo sapiens / References: UniProt: P0CG48
Sequence:

MQIFVKTLTG KTITLEVEPS DTIENVKAKI QDKEGIPPDQ QRLIFAGKQL EDGRTLSDYN IQKESTLHLV LRLRGG

Experimental information

• Beam: Instrument name: Advanced Light Source (ALS) 12.3.1 (SIBYLS); City: Berkeley, CA / 国: USA / Type of source: X-ray synchrotronSynchrotron / Wavelength: 0.103 Å
• Detector: Name: Pilatus 2M

Scan

Title: Wild type RNF8/Ubc13 (C87K K92A)/Ubiquitin complex / Measurement date: Sep 8, 2015 / Unit: 1/nm /

	Min	Max
Q	0.1393	4.0962

Distance distribution function P(R)

Sofotware P(R): GNOM 5.0 / Number of points: 586 /

	Min	Max
Q	0.003482	0.299312
P(R) point	1	586
R	0	188.5

Result

Type of curve: single_conc /

	Experimental	Porod
MW	66 kDa	-
Volume	-	119.05 nm3

	P(R)	Guinier
Forward scattering, I0	665.1	662.4
Radius of gyration, Rg	4.63 nm	4.45 nm

	Min	Max
D	-	18.85
Guinier point	2	14