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- PDB-8h37: Cryo-EM Structure of the KBTBD2-CUL3-Rbx1-p85a tetrameric complex -

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Basic information

Entry
Database: PDB / ID: 8h37
TitleCryo-EM Structure of the KBTBD2-CUL3-Rbx1-p85a tetrameric complex
Components
  • Cullin-3
  • E3 ubiquitin-protein ligase RBX1
  • Kelch repeat and BTB domain-containing protein 2
  • Phosphatidylinositol 3-kinase regulatory subunit alpha
KeywordsLIGASE / complex
Function / homology
Function and homology information


positive regulation of mitotic cell cycle phase transition / trophectodermal cellular morphogenesis / liver morphogenesis / POZ domain binding / perinuclear endoplasmic reticulum membrane / regulation protein catabolic process at postsynapse / regulation of toll-like receptor 4 signaling pathway / polar microtubule / nuclear protein quality control by the ubiquitin-proteasome system / anaphase-promoting complex-dependent catabolic process ...positive regulation of mitotic cell cycle phase transition / trophectodermal cellular morphogenesis / liver morphogenesis / POZ domain binding / perinuclear endoplasmic reticulum membrane / regulation protein catabolic process at postsynapse / regulation of toll-like receptor 4 signaling pathway / polar microtubule / nuclear protein quality control by the ubiquitin-proteasome system / anaphase-promoting complex-dependent catabolic process / phosphatidylinositol kinase activity / COPII vesicle coating / stem cell division / phosphatidylinositol 3-kinase regulator activity / IRS-mediated signalling / positive regulation of focal adhesion disassembly / phosphatidylinositol 3-kinase activator activity / cullin-RING-type E3 NEDD8 transferase / interleukin-18-mediated signaling pathway / PI3K events in ERBB4 signaling / RHOBTB3 ATPase cycle / myeloid leukocyte migration / NEDD8 transferase activity / embryonic cleavage / cullin-RING ubiquitin ligase complex / 1-phosphatidylinositol-3-kinase regulator activity / cell projection organization / positive regulation of mitotic metaphase/anaphase transition / phosphatidylinositol 3-kinase regulatory subunit binding / neurotrophin TRKA receptor binding / Activated NTRK2 signals through PI3K / cellular response to chemical stress / Cul7-RING ubiquitin ligase complex / ubiquitin-dependent protein catabolic process via the C-end degron rule pathway / Activated NTRK3 signals through PI3K / cis-Golgi network / ErbB-3 class receptor binding / Loss of Function of FBXW7 in Cancer and NOTCH1 Signaling / kinase activator activity / transmembrane receptor protein tyrosine kinase adaptor activity / RHOF GTPase cycle / Signaling by cytosolic FGFR1 fusion mutants / positive regulation of endoplasmic reticulum unfolded protein response / positive regulation of protein autoubiquitination / RHOD GTPase cycle / protein neddylation / phosphatidylinositol 3-kinase complex, class IA / phosphatidylinositol 3-kinase complex / enzyme-substrate adaptor activity / Nephrin family interactions / Notch binding / Signaling by LTK in cancer / NEDD8 ligase activity / Costimulation by the CD28 family / RND1 GTPase cycle / Signaling by LTK / RHOBTB1 GTPase cycle / fibroblast apoptotic process / MET activates PI3K/AKT signaling / positive regulation of leukocyte migration / PI3K/AKT activation / RND2 GTPase cycle / Cul5-RING ubiquitin ligase complex / positive regulation of filopodium assembly / negative regulation of response to oxidative stress / RND3 GTPase cycle / growth hormone receptor signaling pathway / negative regulation of stress fiber assembly / negative regulation of Rho protein signal transduction / insulin binding / ubiquitin-ubiquitin ligase activity / SCF ubiquitin ligase complex / Cul2-RING ubiquitin ligase complex / Cul4A-RING E3 ubiquitin ligase complex / natural killer cell mediated cytotoxicity / mitotic metaphase chromosome alignment / RHOV GTPase cycle / negative regulation of cell-matrix adhesion / Signaling by ALK / negative regulation of type I interferon production / Cul4B-RING E3 ubiquitin ligase complex / SCF-dependent proteasomal ubiquitin-dependent protein catabolic process / RHOB GTPase cycle / GP1b-IX-V activation signalling / Cul3-RING ubiquitin ligase complex / ubiquitin ligase complex scaffold activity / PI-3K cascade:FGFR3 / Erythropoietin activates Phosphoinositide-3-kinase (PI3K) / stress fiber assembly / PI-3K cascade:FGFR2 / PI-3K cascade:FGFR4 / PI-3K cascade:FGFR1 / Prolactin receptor signaling / RHOC GTPase cycle / RHOJ GTPase cycle / intracellular glucose homeostasis / negative regulation of osteoclast differentiation / positive regulation of cytokinesis / protein monoubiquitination / Synthesis of PIPs at the plasma membrane
Similarity search - Function
Kelch repeat and BTB domain-containing protein 2 / : / Phosphatidylinositol 3-kinase regulatory subunit alpha, SH3 domain / PIK3R1, inter-SH2 domain / PI3K p85 subunit, C-terminal SH2 domain / PI3K regulatory subunit p85-related , inter-SH2 domain / PI3K p85 subunit, N-terminal SH2 domain / Phosphatidylinositol 3-kinase regulatory subunit P85 inter-SH2 domain / Zinc finger, RING-H2-type / RING-H2 zinc finger domain ...Kelch repeat and BTB domain-containing protein 2 / : / Phosphatidylinositol 3-kinase regulatory subunit alpha, SH3 domain / PIK3R1, inter-SH2 domain / PI3K p85 subunit, C-terminal SH2 domain / PI3K regulatory subunit p85-related , inter-SH2 domain / PI3K p85 subunit, N-terminal SH2 domain / Phosphatidylinositol 3-kinase regulatory subunit P85 inter-SH2 domain / Zinc finger, RING-H2-type / RING-H2 zinc finger domain / BTB-kelch protein / BTB/Kelch-associated / BTB And C-terminal Kelch / BTB And C-terminal Kelch / Cullin protein neddylation domain / : / Cullin, conserved site / Cullin family signature. / Cullin repeat-like-containing domain superfamily / Cullin protein, neddylation domain / Cullin / Kelch / Cullin protein neddylation domain / Rho GTPase-activating protein domain / RhoGAP domain / Rho GTPase-activating proteins domain profile. / GTPase-activator protein for Rho-like GTPases / Kelch motif / Kelch repeat type 1 / Cullin / Cullin, N-terminal / Cullin homology domain / Cullin homology domain superfamily / Cullin family / Cullin family profile. / Kelch-type beta propeller / Rho GTPase activation protein / BTB/POZ domain / BTB domain profile. / Broad-Complex, Tramtrack and Bric a brac / BTB/POZ domain / SKP1/BTB/POZ domain superfamily / SH2 domain / Zinc finger RING-type profile. / Zinc finger, RING-type / Src homology 2 (SH2) domain profile. / Src homology 2 domains / SH2 domain / Src homology 3 domains / SH2 domain superfamily / SH3-like domain superfamily / Src homology 3 (SH3) domain profile. / SH3 domain / Zinc finger, RING/FYVE/PHD-type / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily
Similarity search - Domain/homology
Phosphatidylinositol 3-kinase regulatory subunit alpha / E3 ubiquitin-protein ligase RBX1 / Cullin-3 / Kelch repeat and BTB domain-containing protein 2
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 7.52 Å
AuthorsHu, Y. / Mao, Q. / Chen, Z. / Sun, L.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)81900729 China
CitationJournal: Nat Struct Mol Biol / Year: 2024
Title: Dynamic molecular architecture and substrate recruitment of cullin3-RING E3 ligase CRL3.
Authors: Yuxia Hu / Zhao Zhang / Qiyu Mao / Xiang Zhang / Aihua Hao / Yu Xun / Yeda Wang / Lin Han / Wuqiang Zhan / Qianying Liu / Yue Yin / Chao Peng / Eva Marie Y Moresco / Zhenguo Chen / Bruce Beutler / Lei Sun /
Abstract: Phosphatidylinositol 3-kinase α, a heterodimer of catalytic p110α and one of five regulatory subunits, mediates insulin- and insulin like growth factor-signaling and, frequently, oncogenesis. ...Phosphatidylinositol 3-kinase α, a heterodimer of catalytic p110α and one of five regulatory subunits, mediates insulin- and insulin like growth factor-signaling and, frequently, oncogenesis. Cellular levels of the regulatory p85α subunit are tightly controlled by regulated proteasomal degradation. In adipose tissue and growth plates, failure of K48-linked p85α ubiquitination causes diabetes, lipodystrophy and dwarfism in mice, as in humans with SHORT syndrome. Here we elucidated the structures of the key ubiquitin ligase complexes regulating p85α availability. Specificity is provided by the substrate receptor KBTBD2, which recruits p85α to the cullin3-RING E3 ubiquitin ligase (CRL3). CRL3 forms multimers, which disassemble into dimers upon substrate binding (CRL3-p85α) and/or neddylation by the activator NEDD8 (CRL3~N8), leading to p85α ubiquitination and degradation. Deactivation involves dissociation of NEDD8 mediated by the COP9 signalosome and displacement of KBTBD2 by the inhibitor CAND1. The hereby identified structural basis of p85α regulation opens the way to better understanding disturbances of glucose regulation, growth and cancer.
History
DepositionOct 8, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Oct 11, 2023Provider: repository / Type: Initial release
Revision 1.1Mar 20, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
C: Cullin-3
F: Cullin-3
M: Cullin-3
O: Cullin-3
E: E3 ubiquitin-protein ligase RBX1
D: E3 ubiquitin-protein ligase RBX1
R: E3 ubiquitin-protein ligase RBX1
Q: E3 ubiquitin-protein ligase RBX1
A: Kelch repeat and BTB domain-containing protein 2
G: Phosphatidylinositol 3-kinase regulatory subunit alpha
B: Kelch repeat and BTB domain-containing protein 2
H: Phosphatidylinositol 3-kinase regulatory subunit alpha
I: Phosphatidylinositol 3-kinase regulatory subunit alpha
N: Kelch repeat and BTB domain-containing protein 2
J: Phosphatidylinositol 3-kinase regulatory subunit alpha
P: Kelch repeat and BTB domain-containing protein 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,026,76528
Polymers1,025,98016
Non-polymers78512
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Cullin-3 / CUL-3


Mass: 89063.328 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CUL3, KIAA0617 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q13618
#2: Protein
E3 ubiquitin-protein ligase RBX1 / E3 ubiquitin-protein transferase RBX1 / Protein ZYP / RING finger protein 75 / RING-box protein 1 / ...E3 ubiquitin-protein transferase RBX1 / Protein ZYP / RING finger protein 75 / RING-box protein 1 / Rbx1 / Regulator of cullins 1 / ROC1


Mass: 12289.977 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RBX1, RNF75, ROC1 / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: P62877, RING-type E3 ubiquitin transferase, cullin-RING-type E3 NEDD8 transferase
#3: Protein
Kelch repeat and BTB domain-containing protein 2 / BTB and kelch domain-containing protein 1


Mass: 71431.375 Da / Num. of mol.: 4 / Mutation: S252D
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: KBTBD2, BKLHD1, KIAA1489, CGI-73 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q8IY47
#4: Protein
Phosphatidylinositol 3-kinase regulatory subunit alpha / PI3-kinase regulatory subunit alpha / PI3K regulatory subunit alpha / PtdIns-3-kinase regulatory ...PI3-kinase regulatory subunit alpha / PI3K regulatory subunit alpha / PtdIns-3-kinase regulatory subunit alpha / Phosphatidylinositol 3-kinase 85 kDa regulatory subunit alpha / PI3-kinase subunit p85-alpha / PtdIns-3-kinase regulatory subunit p85-alpha


Mass: 83710.281 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PIK3R1, GRB1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P27986
#5: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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

ComponentName: KBTBD2-CUL3-Rbx1-p85a tetrameric complex / Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 8.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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 defocus max: 2200 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 45 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameVersionCategory
2SerialEMimage acquisition
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 7.52 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 239068 / Symmetry type: POINT

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