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- PDB-8byl: Cryo-EM structure of SKP1-SKP2-CKS1 from the SCFSKP2 E3 ligase complex -

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Basic information

Entry
Database: PDB / ID: 8byl
TitleCryo-EM structure of SKP1-SKP2-CKS1 from the SCFSKP2 E3 ligase complex
Components
  • Cyclin-dependent kinase inhibitor 1B
  • Cyclin-dependent kinases regulatory subunit 1
  • S-phase kinase-associated protein 1
  • S-phase kinase-associated protein 2
KeywordsCELL CYCLE / cyclin-dependent kinase / signalling / ubiquitinationCell cycle
Function / homology
Function and homology information


cyclin-dependent protein kinase regulator activity / regulation of lens fiber cell differentiation / negative regulation of cyclin-dependent protein kinase activity / negative regulation of cardiac muscle tissue regeneration / negative regulation of kinase activity / positive regulation of protein polyubiquitination / autophagic cell death / FOXO-mediated transcription of cell cycle genes / F-box domain binding / negative regulation of epithelial cell proliferation involved in prostate gland development ...cyclin-dependent protein kinase regulator activity / regulation of lens fiber cell differentiation / negative regulation of cyclin-dependent protein kinase activity / negative regulation of cardiac muscle tissue regeneration / negative regulation of kinase activity / positive regulation of protein polyubiquitination / autophagic cell death / FOXO-mediated transcription of cell cycle genes / F-box domain binding / negative regulation of epithelial cell proliferation involved in prostate gland development / negative regulation of cyclin-dependent protein serine/threonine kinase activity / cellular response to cell-matrix adhesion / Aberrant regulation of mitotic exit in cancer due to RB1 defects / regulation of cell cycle G1/S phase transition / PcG protein complex / regulation of exit from mitosis / negative regulation of epithelial cell apoptotic process / epithelial cell proliferation involved in prostate gland development / positive regulation of ubiquitin protein ligase activity / cyclin-dependent protein serine/threonine kinase inhibitor activity / Cul7-RING ubiquitin ligase complex / Loss of Function of FBXW7 in Cancer and NOTCH1 Signaling / maintenance of protein location in nucleus / ubiquitin ligase activator activity / regulation of cyclin-dependent protein serine/threonine kinase activity / RHO GTPases activate CIT / cyclin-dependent protein serine/threonine kinase activator activity / nuclear export / negative regulation of mitotic cell cycle / AKT phosphorylates targets in the cytosol / epithelial cell apoptotic process / cellular response to antibiotic / SCF ubiquitin ligase complex / molecular function inhibitor activity / SCF-dependent proteasomal ubiquitin-dependent protein catabolic process / Cul4A-RING E3 ubiquitin ligase complex / cellular response to lithium ion / positive regulation of intracellular estrogen receptor signaling pathway / ubiquitin ligase complex scaffold activity / Prolactin receptor signaling / p53-Dependent G1 DNA Damage Response / Constitutive Signaling by AKT1 E17K in Cancer / PTK6 Regulates Cell Cycle / protein kinase inhibitor activity / Defective binding of RB1 mutants to E2F1,(E2F2, E2F3) / inner ear development / cullin family protein binding / negative regulation of vascular associated smooth muscle cell proliferation / regulation of G1/S transition of mitotic cell cycle / protein K63-linked ubiquitination / Estrogen-dependent nuclear events downstream of ESR-membrane signaling / protein monoubiquitination / TP53 Regulates Transcription of Genes Involved in G1 Cell Cycle Arrest / ubiquitin-like ligase-substrate adaptor activity / protein K48-linked ubiquitination / Cyclin E associated events during G1/S transition / positive regulation of double-strand break repair via homologous recombination / Cyclin A:Cdk2-associated events at S phase entry / Nuclear events stimulated by ALK signaling in cancer / cyclin-dependent protein kinase holoenzyme complex / Notch signaling pathway / positive regulation of microtubule polymerization / FLT3 Signaling / positive regulation of smooth muscle cell proliferation / cyclin binding / regulation of mitotic cell cycle / regulation of cell migration / Regulation of BACH1 activity / molecular function activator activity / MAP3K8 (TPL2)-dependent MAPK1/3 activation / placenta development / positive regulation of DNA replication / ubiquitin binding / SCF-beta-TrCP mediated degradation of Emi1 / NIK-->noncanonical NF-kB signaling / DNA damage response, signal transduction by p53 class mediator / Vpu mediated degradation of CD4 / Dectin-1 mediated noncanonical NF-kB signaling / Activation of NF-kappaB in B cells / Degradation of GLI1 by the proteasome / Iron uptake and transport / GSK3B and BTRC:CUL1-mediated-degradation of NFE2L2 / sensory perception of sound / Negative regulation of NOTCH4 signaling / G1/S transition of mitotic cell cycle / Degradation of GLI2 by the proteasome / GLI3 is processed to GLI3R by the proteasome / FBXL7 down-regulates AURKA during mitotic entry and in early mitosis / APC/C:Cdh1 mediated degradation of Cdc20 and other APC/C:Cdh1 targeted proteins in late mitosis/early G1 / negative regulation of cell growth / Degradation of beta-catenin by the destruction complex / beta-catenin binding / NOTCH1 Intracellular Domain Regulates Transcription / DNA Damage/Telomere Stress Induced Senescence / potassium ion transport / CLEC7A (Dectin-1) signaling / SCF(Skp2)-mediated degradation of p27/p21 / Constitutive Signaling by NOTCH1 PEST Domain Mutants / Constitutive Signaling by NOTCH1 HD+PEST Domain Mutants / FCERI mediated NF-kB activation
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 / A Receptor for Ubiquitination Targets ...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 / A Receptor for Ubiquitination Targets / Leucine-rich repeat, cysteine-containing subtype / Leucine-rich repeat - CC (cysteine-containing) subfamily / F-box domain profile. / 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 / S-phase kinase-associated protein 1-like / SKP1 component, POZ domain / Skp1 family, tetramerisation domain / Found in Skp1 protein family / SKP1/BTB/POZ domain superfamily / Leucine-rich repeat domain superfamily
Similarity search - Domain/homology
Cyclin-dependent kinase inhibitor 1B / Cyclin-dependent kinases regulatory subunit 1 / S-phase kinase-associated protein 1 / S-phase kinase-associated protein 2
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsRowland, R.J. / Salamina, M. / Endicott, J.A. / Noble, M.E.M.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MR/N009738/1 and MR/V029142/1 United Kingdom
CitationJournal: Sci Rep / Year: 2023
Title: Cryo-EM structure of SKP1-SKP2-CKS1 in complex with CDK2-cyclin A-p27KIP1.
Authors: Rhianna J Rowland / Richard Heath / Daniel Maskell / Rebecca F Thompson / Neil A Ranson / James N Blaza / Jane A Endicott / Martin E M Noble / Marco Salamina /
Abstract: p27KIP1 (cyclin-dependent kinase inhibitor 1B, p27) is a member of the CIP/KIP family of CDK (cyclin dependent kinase) regulators that inhibit cell cycle CDKs. p27 phosphorylation by CDK1/2, signals ...p27KIP1 (cyclin-dependent kinase inhibitor 1B, p27) is a member of the CIP/KIP family of CDK (cyclin dependent kinase) regulators that inhibit cell cycle CDKs. p27 phosphorylation by CDK1/2, signals its recruitment to the SCF (S-phase kinase associated protein 1 (SKP1)-cullin-SKP2) E3 ubiquitin ligase complex for proteasomal degradation. The nature of p27 binding to SKP2 and CKS1 was revealed by the SKP1-SKP2-CKS1-p27 phosphopeptide crystal structure. Subsequently, a model for the hexameric CDK2-cyclin A-CKS1-p27-SKP1-SKP2 complex was proposed by overlaying an independently determined CDK2-cyclin A-p27 structure. Here we describe the experimentally determined structure of the isolated CDK2-cyclin A-CKS1-p27-SKP1-SKP2 complex at 3.4 Å global resolution using cryogenic electron microscopy. This structure supports previous analysis in which p27 was found to be structurally dynamic, transitioning from disordered to nascent secondary structure on target binding. We employed 3D variability analysis to further explore the conformational space of the hexameric complex and uncovered a previously unidentified hinge motion centred on CKS1. This flexibility gives rise to open and closed conformations of the hexameric complex that we propose may contribute to p27 regulation by facilitating recognition with SCF. This 3D variability analysis further informed particle subtraction and local refinement approaches to enhance the local resolution of the complex.
History
DepositionDec 13, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 28, 2023Provider: repository / Type: Initial release
Revision 1.1Jul 19, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
Revision 1.2Oct 23, 2024Group: Data collection / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: S-phase kinase-associated protein 1
B: S-phase kinase-associated protein 2
C: Cyclin-dependent kinases regulatory subunit 1
D: Cyclin-dependent kinase inhibitor 1B


Theoretical massNumber of molelcules
Total (without water)98,3654
Polymers98,3654
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area5870 Å2
ΔGint-42 kcal/mol
Surface area28310 Å2

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Components

#1: 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 BL21(DE3) (bacteria) / Variant (production host): pLYSs / References: UniProt: P63208
#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 BL21(DE3) (bacteria) / Variant (production host): pLYSs / References: UniProt: Q13309
#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 BL21(DE3) (bacteria) / Variant (production host): pLYSs / References: UniProt: P61024
#4: 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
Details: DGSPNAGSVEQ(TPO)PKK / Source: (gene. exp.) Homo sapiens (human) / Gene: CDKN1B, KIP1 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): pLYSs / References: UniProt: P46527
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: Locally refined complex of SKP1-SKP2-CKS1-p27 from the hexametric SCFSKP2 E3 Ligase complex
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.066 MDa / Experimental value: NO
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
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 278.15 K

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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 magnification: 130000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 9 sec. / Electron dose: 65 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameCategoryDetails
1cryoSPARCparticle selection
2EPUimage acquisition
4cryoSPARCCTF correction
7UCSF ChimeraXmodel fitting
9PHENIXmodel refinement
10cryoSPARCinitial Euler assignmenthomogenous refinement
11cryoSPARCfinal Euler assignmentlocal refinement
12cryoSPARCclassification
13cryoSPARC3D reconstructionParticle subtraction followed by local refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1110356
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 136325 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 122 / Protocol: RIGID BODY FIT / Space: REAL
Details: Initial fitting was performed in chimera followed by real space refinement in Phenix
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0034480
ELECTRON MICROSCOPYf_angle_d0.6016107
ELECTRON MICROSCOPYf_dihedral_angle_d7.49602
ELECTRON MICROSCOPYf_chiral_restr0.045712
ELECTRON MICROSCOPYf_plane_restr0.005790

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