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- PDB-7b5r: Ubiquitin ligation to F-box protein substrates by SCF-RBR E3-E3 s... -

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Entry
Database: PDB / ID: 7b5r
TitleUbiquitin ligation to F-box protein substrates by SCF-RBR E3-E3 super-assembly: CUL1-RBX1-SKP1-SKP2-CKSHS1-Cyclin A-CDK2-p27
Components
  • (Cyclin-dependent kinase ...) x 2
  • (S-phase kinase-associated protein ...) x 2
  • Cullin-1
  • Cyclin-A2
  • Cyclin-dependent kinases regulatory subunit 1
KeywordsLIGASE / ubiquitin / ubiquitin ligase / E3 ligase / F-box protein / RBR ligase / Cullin-RING-Ligase / CRL / SCF / NEDD8 / Post-translational modification / ubiquitylation
Function / homology
Function and homology information


cyclin-dependent protein kinase regulator activity / regulation of lens fiber cell differentiation / negative regulation of cardiac muscle tissue regeneration / positive regulation of protein polyubiquitination / autophagic cell death / FOXO-mediated transcription of cell cycle genes / Parkin-FBXW7-Cul1 ubiquitin ligase complex / negative regulation of epithelial cell proliferation involved in prostate gland development / cellular response to cell-matrix adhesion / cyclin A2-CDK1 complex ...cyclin-dependent protein kinase regulator activity / regulation of lens fiber cell differentiation / negative regulation of cardiac muscle tissue regeneration / positive regulation of protein polyubiquitination / autophagic cell death / FOXO-mediated transcription of cell cycle genes / Parkin-FBXW7-Cul1 ubiquitin ligase complex / negative regulation of epithelial cell proliferation involved in prostate gland development / cellular response to cell-matrix adhesion / cyclin A2-CDK1 complex / F-box domain binding / Aberrant regulation of mitotic exit in cancer due to RB1 defects / regulation of cell cycle G1/S phase transition / cell cycle G1/S phase transition / cellular response to luteinizing hormone stimulus / epithelial cell proliferation involved in prostate gland development / PcG protein complex / G2/M DNA replication checkpoint / regulation of exit from mitosis / negative regulation of epithelial cell apoptotic process / cyclin-dependent protein serine/threonine kinase inhibitor activity / Transcription of E2F targets under negative control by p107 (RBL1) and p130 (RBL2) in complex with HDAC1 / cellular response to leptin stimulus / cullin-RING ubiquitin ligase complex / maintenance of protein location in nucleus / Cul7-RING ubiquitin ligase complex / RHO GTPases activate CIT / regulation of cyclin-dependent protein serine/threonine kinase activity / male pronucleus / Loss of Function of FBXW7 in Cancer and NOTCH1 Signaling / nuclear export / female pronucleus / cyclin-dependent protein serine/threonine kinase activator activity / negative regulation of mitotic cell cycle / epithelial cell apoptotic process / cellular response to cocaine / AKT phosphorylates targets in the cytosol / response to glucagon / ubiquitin ligase activator activity / cyclin-dependent protein serine/threonine kinase regulator activity / molecular function inhibitor activity / positive regulation of DNA biosynthetic process / cellular response to lithium ion / positive regulation of intracellular estrogen receptor signaling pathway / SCF ubiquitin ligase complex / cyclin A1-CDK2 complex / cyclin E2-CDK2 complex / cyclin E1-CDK2 complex / cellular response to insulin-like growth factor stimulus / cyclin A2-CDK2 complex / positive regulation of DNA-templated DNA replication initiation / G2 Phase / Y chromosome / cyclin-dependent protein kinase activity / regulation of heterochromatin organization / Phosphorylation of proteins involved in G1/S transition by active Cyclin E:Cdk2 complexes / positive regulation of heterochromatin formation / p53-Dependent G1 DNA Damage Response / X chromosome / PTK6 Regulates Cell Cycle / SCF-dependent proteasomal ubiquitin-dependent protein catabolic process / Cul4A-RING E3 ubiquitin ligase complex / Constitutive Signaling by AKT1 E17K in Cancer / Prolactin receptor signaling / regulation of anaphase-promoting complex-dependent catabolic process / cellular response to antibiotic / Defective binding of RB1 mutants to E2F1,(E2F2, E2F3) / inner ear development / ciliary transition zone / centriole replication / Regulation of APC/C activators between G1/S and early anaphase / ubiquitin ligase complex scaffold activity / protein kinase inhibitor activity / telomere maintenance in response to DNA damage / regulation of DNA replication / centrosome duplication / microtubule organizing center / G0 and Early G1 / regulation of G1/S transition of mitotic cell cycle / negative regulation of vascular associated smooth muscle cell proliferation / cochlea development / Telomere Extension By Telomerase / animal organ regeneration / Activation of the pre-replicative complex / Estrogen-dependent nuclear events downstream of ESR-membrane signaling / cullin family protein binding / cyclin-dependent kinase / cyclin-dependent protein serine/threonine kinase activity / TP53 Regulates Transcription of Genes Involved in G1 Cell Cycle Arrest / protein monoubiquitination / protein K63-linked ubiquitination / Activation of ATR in response to replication stress / Regulation of MITF-M-dependent genes involved in cell cycle and proliferation / Cajal body / Cyclin E associated events during G1/S transition / Cyclin A:Cdk2-associated events at S phase entry / positive regulation of microtubule polymerization / positive regulation of double-strand break repair via homologous recombination / cyclin-dependent protein kinase holoenzyme complex / Cyclin A/B1/B2 associated events during G2/M transition
Similarity search - Function
Cyclin-dependent kinase inhibitor domain / Cyclin-dependent kinase inhibitor domain superfamily / Cyclin-dependent kinase inhibitor / Cyclin-dependent kinase, regulatory subunit / Cyclin-dependent kinase, regulatory subunit superfamily / Cyclin-dependent kinase regulatory subunit / Cyclin-dependent kinases regulatory subunits signature 1. / Cyclin-dependent kinases regulatory subunits signature 2. / Cyclin-dependent kinase regulatory subunit / Leucine-rich repeat, cysteine-containing subtype ...Cyclin-dependent kinase inhibitor domain / Cyclin-dependent kinase inhibitor domain superfamily / Cyclin-dependent kinase inhibitor / Cyclin-dependent kinase, regulatory subunit / Cyclin-dependent kinase, regulatory subunit superfamily / Cyclin-dependent kinase regulatory subunit / Cyclin-dependent kinases regulatory subunits signature 1. / Cyclin-dependent kinases regulatory subunits signature 2. / Cyclin-dependent kinase regulatory subunit / Leucine-rich repeat, cysteine-containing subtype / Leucine-rich repeat - CC (cysteine-containing) subfamily / A Receptor for Ubiquitination Targets / F-box domain profile. / Cyclin-A, N-terminal APC/C binding region / Cyclin-A N-terminal APC/C binding region / F-box-like / F-box-like domain superfamily / SKP1 component, dimerisation / S-phase kinase-associated protein 1 / SKP1-like, dimerisation domain superfamily / Skp1 family, dimerisation domain / : / F-box domain / Cyclin, C-terminal domain / : / Cyclins signature. / Cyclin / Cyclin, C-terminal domain / Cyclin_C / Cullin, N-terminal / Cullin protein neddylation domain / Cullin, conserved site / Cullin alpha solenoid domain / Cullin family signature. / Cullin repeat-like-containing domain superfamily / Cullin protein, neddylation domain / Cullin / Cullin protein neddylation domain / Cullin / : / Cullin alpha+beta domain / Cullin homology domain / Cullin homology domain superfamily / Cullin family profile. / Cyclin, N-terminal / Cyclin, N-terminal domain / S-phase kinase-associated protein 1-like / SKP1 component, POZ domain / Skp1 family, tetramerisation domain / Found in Skp1 protein family / Cyclin-like / domain present in cyclins, TFIIB and Retinoblastoma / Cyclin-like superfamily / : / SKP1/BTB/POZ domain superfamily / Leucine-rich repeat domain superfamily / Winged helix DNA-binding domain superfamily / Transferase(Phosphotransferase) domain 1 / Transferase(Phosphotransferase); domain 1 / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Protein kinase domain / Serine/Threonine protein kinases, catalytic domain / Winged helix-like DNA-binding domain superfamily / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily / Orthogonal Bundle / Mainly Alpha
Similarity search - Domain/homology
Cyclin-A2 / Cyclin-dependent kinase 2 / Cyclin-dependent kinase inhibitor 1B / Cyclin-dependent kinases regulatory subunit 1 / S-phase kinase-associated protein 1 / S-phase kinase-associated protein 2 / Cullin-1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsHorn-Ghetko, D. / Prabu, J.R. / Schulman, B.A.
Funding support Germany, 2items
OrganizationGrant numberCountry
European Research Council (ERC)H2020 789016-NEDD8Activate Germany
German Research Foundation (DFG)SCHU 3196/1-1 Germany
CitationJournal: Nature / Year: 2021
Title: Ubiquitin ligation to F-box protein targets by SCF-RBR E3-E3 super-assembly.
Authors: Daniel Horn-Ghetko / David T Krist / J Rajan Prabu / Kheewoong Baek / Monique P C Mulder / Maren Klügel / Daniel C Scott / Huib Ovaa / Gary Kleiger / Brenda A Schulman /
Abstract: E3 ligases are typically classified by hallmark domains such as RING and RBR, which are thought to specify unique catalytic mechanisms of ubiquitin transfer to recruited substrates. However, rather ...E3 ligases are typically classified by hallmark domains such as RING and RBR, which are thought to specify unique catalytic mechanisms of ubiquitin transfer to recruited substrates. However, rather than functioning individually, many neddylated cullin-RING E3 ligases (CRLs) and RBR-type E3 ligases in the ARIH family-which together account for nearly half of all ubiquitin ligases in humans-form E3-E3 super-assemblies. Here, by studying CRLs in the SKP1-CUL1-F-box (SCF) family, we show how neddylated SCF ligases and ARIH1 (an RBR-type E3 ligase) co-evolved to ubiquitylate diverse substrates presented on various F-box proteins. We developed activity-based chemical probes that enabled cryo-electron microscopy visualization of steps in E3-E3 ubiquitylation, initiating with ubiquitin linked to the E2 enzyme UBE2L3, then transferred to the catalytic cysteine of ARIH1, and culminating in ubiquitin linkage to a substrate bound to the SCF E3 ligase. The E3-E3 mechanism places the ubiquitin-linked active site of ARIH1 adjacent to substrates bound to F-box proteins (for example, substrates with folded structures or limited length) that are incompatible with previously described conventional RING E3-only mechanisms. The versatile E3-E3 super-assembly may therefore underlie widespread ubiquitylation.
History
DepositionDec 7, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 10, 2021Provider: repository / Type: Initial release
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Structure visualization

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Assembly

Deposited unit
C: Cullin-1
T: S-phase kinase-associated protein 2
K: Cyclin-dependent kinases regulatory subunit 1
S: S-phase kinase-associated protein 1
L: Cyclin-dependent kinase 2
Y: Cyclin-A2
P: Cyclin-dependent kinase inhibitor 1B


Theoretical massNumber of molelcules
Total (without water)270,8327
Polymers270,8327
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area16260 Å2
ΔGint-86 kcal/mol
Surface area65820 Å2
MethodPISA

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Components

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Protein , 3 types, 3 molecules CKY

#1: Protein Cullin-1 / CUL-1


Mass: 89800.367 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CUL1 / Production host: Escherichia coli (E. coli) / References: UniProt: Q13616
#3: Protein Cyclin-dependent kinases regulatory subunit 1 / CKS-1


Mass: 9679.211 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CKS1B, CKS1, PNAS-143, PNAS-16 / Production host: Escherichia coli (E. coli) / References: UniProt: P61024
#6: Protein Cyclin-A2 / Cyclin-A / Cyclin A


Mass: 48609.574 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CCNA2, CCN1, CCNA / Production host: Escherichia coli (E. coli) / References: UniProt: P20248

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S-phase kinase-associated protein ... , 2 types, 2 molecules TS

#2: Protein S-phase kinase-associated protein 2 / Cyclin-A/CDK2-associated protein p45 / F-box protein Skp2 / F-box/LRR-repeat protein 1 / p45skp2


Mass: 47817.785 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SKP2, FBXL1 / Production host: Escherichia coli (E. coli) / References: UniProt: Q13309
#4: Protein S-phase kinase-associated protein 1 / Cyclin-A/CDK2-associated protein p19 / p19A / Organ of Corti protein 2 / OCP-2 / Organ of Corti ...Cyclin-A/CDK2-associated protein p19 / p19A / Organ of Corti protein 2 / OCP-2 / Organ of Corti protein II / OCP-II / RNA polymerase II elongation factor-like protein / SIII / Transcription elongation factor B polypeptide 1-like / p19skp1


Mass: 18679.965 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SKP1, EMC19, OCP2, SKP1A, TCEB1L / Production host: Escherichia coli (E. coli) / References: UniProt: P63208

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Cyclin-dependent kinase ... , 2 types, 2 molecules LP

#5: Protein Cyclin-dependent kinase 2 / Cell division protein kinase 2 / p33 protein kinase


Mass: 34056.469 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CDK2, CDKN2 / Production host: Escherichia coli (E. coli) / References: UniProt: P24941, cyclin-dependent kinase
#7: Protein Cyclin-dependent kinase inhibitor 1B / Cyclin-dependent kinase inhibitor p27 / p27Kip1


Mass: 22188.303 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CDKN1B, KIP1 / Production host: Escherichia coli (E. coli) / References: UniProt: P46527

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Details

Has ligand of interestN
Has protein modificationY

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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: NEDD8-CUL1-RBX1-SKP1-SKP2-CKSHS1-Cyclin A-CDK2-p27-UBE2L3~Ub~ARIH1. Transition State 1 composite map.
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.30 MDa
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.8
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
Image recordingElectron dose: 70 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 213213 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00511857
ELECTRON MICROSCOPYf_angle_d0.72316056
ELECTRON MICROSCOPYf_dihedral_angle_d6.6041549
ELECTRON MICROSCOPYf_chiral_restr0.0451807
ELECTRON MICROSCOPYf_plane_restr0.0052034

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