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- PDB-8k8t: Structure of CUL3-RBX1-KLHL22 complex -

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Basic information

Entry
Database: PDB / ID: 8k8t
TitleStructure of CUL3-RBX1-KLHL22 complex
Components
  • Cullin-3
  • Kelch-like protein 22
KeywordsLIGASE / Cullin Ring E3 ubiquitin ligase
Function / homology
Function and homology information


positive regulation of mitotic cell cycle phase transition / trophectodermal cellular morphogenesis / liver morphogenesis / POZ domain binding / nuclear protein quality control by the ubiquitin-proteasome system / polar microtubule / regulation protein catabolic process at postsynapse / COPII vesicle coating / anaphase-promoting complex-dependent catabolic process / cellular response to L-leucine ...positive regulation of mitotic cell cycle phase transition / trophectodermal cellular morphogenesis / liver morphogenesis / POZ domain binding / nuclear protein quality control by the ubiquitin-proteasome system / polar microtubule / regulation protein catabolic process at postsynapse / COPII vesicle coating / anaphase-promoting complex-dependent catabolic process / cellular response to L-leucine / RHOBTB3 ATPase cycle / positive regulation of T cell mediated immune response to tumor cell / positive regulation of mitotic metaphase/anaphase transition / embryonic cleavage / cell projection organization / Notch binding / fibroblast apoptotic process / RHOBTB1 GTPase cycle / negative regulation of type I interferon production / stem cell division / mitotic metaphase chromosome alignment / Cul3-RING ubiquitin ligase complex / mitotic spindle assembly checkpoint signaling / negative regulation of Rho protein signal transduction / stress fiber assembly / positive regulation of cytokinesis / ubiquitin ligase complex scaffold activity / mitotic sister chromatid segregation / protein monoubiquitination / endoplasmic reticulum to Golgi vesicle-mediated transport / sperm flagellum / RHOBTB2 GTPase cycle / ubiquitin-like ligase-substrate adaptor activity / protein K48-linked ubiquitination / protein autoubiquitination / intercellular bridge / gastrulation / 14-3-3 protein binding / positive regulation of TORC1 signaling / regulation of cellular response to insulin stimulus / intrinsic apoptotic signaling pathway / negative regulation of autophagy / cyclin binding / positive regulation of protein ubiquitination / integrin-mediated signaling pathway / cellular response to amino acid stimulus / Degradation of DVL / kidney development / G1/S transition of mitotic cell cycle / Hedgehog 'on' state / protein destabilization / positive regulation of T cell activation / Wnt signaling pathway / protein polyubiquitination / Regulation of RAS by GAPs / spindle pole / mitotic spindle / ubiquitin protein ligase activity / KEAP1-NFE2L2 pathway / cell migration / Antigen processing: Ubiquitination & Proteasome degradation / Neddylation / microtubule cytoskeleton / positive regulation of cell growth / cellular response to oxidative stress / ubiquitin-dependent protein catabolic process / gene expression / Potential therapeutics for SARS / proteasome-mediated ubiquitin-dependent protein catabolic process / lysosome / postsynapse / protein ubiquitination / inflammatory response / cell division / intracellular membrane-bounded organelle / positive regulation of cell population proliferation / ubiquitin protein ligase binding / centrosome / glutamatergic synapse / negative regulation of transcription by RNA polymerase II / Golgi apparatus / extracellular exosome / nucleoplasm / identical protein binding / nucleus / membrane / plasma membrane / cytoplasm / cytosol
Similarity search - Function
Kelch-like protein 22 / KLHDC2/KLHL20/DRC7 Kelch-repeats domain / Cullin protein neddylation domain / BTB-kelch protein / BTB/Kelch-associated / Cullin, conserved site / BTB And C-terminal Kelch / Cullin family signature. / BTB And C-terminal Kelch / Cullin repeat-like-containing domain superfamily ...Kelch-like protein 22 / KLHDC2/KLHL20/DRC7 Kelch-repeats domain / Cullin protein neddylation domain / BTB-kelch protein / BTB/Kelch-associated / Cullin, conserved site / BTB And C-terminal Kelch / Cullin family signature. / BTB And C-terminal Kelch / Cullin repeat-like-containing domain superfamily / Cullin protein, neddylation domain / Cullin / Cullin protein neddylation domain / Kelch motif / Kelch / Kelch repeat type 1 / Cullin / Cullin, N-terminal / Cullin homology domain / Cullin homology domain superfamily / Cullin alpha solenoid domain / Cullin family profile. / Kelch-type beta propeller / BTB/POZ domain / BTB domain profile. / Broad-Complex, Tramtrack and Bric a brac / BTB/POZ domain / SKP1/BTB/POZ domain superfamily / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily
Similarity search - Domain/homology
Cullin-3 / Kelch-like protein 22
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsWang, W. / Ling, L. / Dai, Z. / Zuo, P. / Yin, Y.
Funding support China, 4items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)2021YFA1300601 China
National Natural Science Foundation of China (NSFC)82030081 China
National Natural Science Foundation of China (NSFC)81874235 China
National Natural Science Foundation of China (NSFC)31800626 China
CitationJournal: Nat Commun / Year: 2024
Title: A conserved N-terminal motif of CUL3 contributes to assembly and E3 ligase activity of CRL3.
Authors: Weize Wang / Ling Liang / Zonglin Dai / Peng Zuo / Shang Yu / Yishuo Lu / Dian Ding / Hongyi Chen / Hui Shan / Yan Jin / Youdong Mao / Yuxin Yin /
Abstract: The CUL3-RING E3 ubiquitin ligases (CRL3s) play an essential role in response to extracellular nutrition and stress stimuli. The ubiquitin ligase function of CRL3s is activated through dimerization. ...The CUL3-RING E3 ubiquitin ligases (CRL3s) play an essential role in response to extracellular nutrition and stress stimuli. The ubiquitin ligase function of CRL3s is activated through dimerization. However, how and why such a dimeric assembly is required for its ligase activity remains elusive. Here, we report the cryo-EM structure of the dimeric CRL3 complex and reveal a conserved N-terminal motif in CUL3 that contributes to the dimerization assembly and the E3 ligase activity of CRL3. We show that deletion of the CUL3 N-terminal motif impairs dimeric assembly and the E3 ligase activity of both CRL3 and several other CRL3s. In addition, we found that the dynamics of dimeric assembly of CRL3 generates a variable ubiquitination zone, potentially facilitating substrate recognition and ubiquitination. These findings demonstrate that a CUL3 N-terminal motif participates in the assembly process and provide insights into the assembly and activation of CRL3s.
History
DepositionJul 31, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0May 22, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
C: Cullin-3
D: Cullin-3
K: Kelch-like protein 22
L: Kelch-like protein 22


Theoretical massNumber of molelcules
Total (without water)330,2254
Polymers330,2254
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Cullin-3


Mass: 90134.555 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CUL3 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q13618
#2: Protein Kelch-like protein 22


Mass: 74978.117 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: his-tev cleave site-klhl22 / Source: (gene. exp.) Homo sapiens (human) / Gene: KLHL22 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q53GT1

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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: CUL3-RBX1-KLHL22 complex without CUL3 NA motif / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaCl1
220 mMtris(hydroxymethyl)aminomethaneTris1
31 mM2-Amino-2-(hydroxymethyl)propane-1,3-diolTCEP1
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 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: DIFFRACTION / Nominal defocus max: 2000 nm / Nominal defocus min: 1500 nm
Image recordingElectron dose: 50 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameVersionCategory
4cryoSPARC4.2CTF correction
10cryoSPARC4.2initial Euler assignment
11cryoSPARC4.2final Euler assignment
12cryoSPARC4.2classification
13cryoSPARC4.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 142416 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 98.32 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.014310067
ELECTRON MICROSCOPYf_angle_d0.968513507
ELECTRON MICROSCOPYf_chiral_restr0.05491515
ELECTRON MICROSCOPYf_plane_restr0.00691726
ELECTRON MICROSCOPYf_dihedral_angle_d18.58543847

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