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- PDB-8jkb: Cryo-EM structure of KCTD5 in complex with Gbeta gamma subunits -

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Basic information

Entry
Database: PDB / ID: 8jkb
TitleCryo-EM structure of KCTD5 in complex with Gbeta gamma subunits
Components
  • BTB/POZ domain-containing protein KCTD5
  • Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
  • Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
KeywordsPROTEIN BINDING / ubiquitination / GPCR signaling
Function / homology
Function and homology information


Cul3-RING ubiquitin ligase complex / cullin family protein binding / Olfactory Signaling Pathway / Activation of the phototransduction cascade / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Thromboxane signalling through TP receptor / protein homooligomerization / G-protein activation / G protein-coupled acetylcholine receptor signaling pathway ...Cul3-RING ubiquitin ligase complex / cullin family protein binding / Olfactory Signaling Pathway / Activation of the phototransduction cascade / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Thromboxane signalling through TP receptor / protein homooligomerization / G-protein activation / G protein-coupled acetylcholine receptor signaling pathway / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / Prostacyclin signalling through prostacyclin receptor / Glucagon signaling in metabolic regulation / G beta:gamma signalling through CDC42 / ADP signalling through P2Y purinoceptor 12 / G beta:gamma signalling through BTK / Sensory perception of sweet, bitter, and umami (glutamate) taste / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / photoreceptor disc membrane / Adrenaline,noradrenaline inhibits insulin secretion / Glucagon-type ligand receptors / Vasopressin regulates renal water homeostasis via Aquaporins / G alpha (z) signalling events / cellular response to catecholamine stimulus / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / ADORA2B mediated anti-inflammatory cytokines production / adenylate cyclase-activating dopamine receptor signaling pathway / ADP signalling through P2Y purinoceptor 1 / G beta:gamma signalling through PI3Kgamma / cellular response to prostaglandin E stimulus / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / sensory perception of taste / GPER1 signaling / G-protein beta-subunit binding / heterotrimeric G-protein complex / Inactivation, recovery and regulation of the phototransduction cascade / extracellular vesicle / G alpha (12/13) signalling events / signaling receptor complex adaptor activity / Thrombin signalling through proteinase activated receptors (PARs) / retina development in camera-type eye / GTPase binding / Ca2+ pathway / phospholipase C-activating G protein-coupled receptor signaling pathway / G alpha (i) signalling events / fibroblast proliferation / G alpha (s) signalling events / G alpha (q) signalling events / proteasome-mediated ubiquitin-dependent protein catabolic process / Ras protein signal transduction / cell population proliferation / Extra-nuclear estrogen signaling / G protein-coupled receptor signaling pathway / lysosomal membrane / GTPase activity / synapse / protein-containing complex binding / signal transduction / extracellular exosome / membrane / identical protein binding / nucleus / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Potassium channel tetramerisation-type BTB domain / BTB/POZ domain / Broad-Complex, Tramtrack and Bric a brac / BTB/POZ domain / SKP1/BTB/POZ domain superfamily / G-protein, gamma subunit / G-protein gamma subunit domain profile. / GGL domain / G-protein gamma-like domain superfamily / G-protein gamma-like domain ...Potassium channel tetramerisation-type BTB domain / BTB/POZ domain / Broad-Complex, Tramtrack and Bric a brac / BTB/POZ domain / SKP1/BTB/POZ domain superfamily / G-protein, gamma subunit / G-protein gamma subunit domain profile. / GGL domain / G-protein gamma-like domain superfamily / G-protein gamma-like domain / GGL domain / G protein gamma subunit-like motifs / Guanine nucleotide-binding protein, beta subunit / G-protein, beta subunit / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily
Similarity search - Domain/homology
Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / BTB/POZ domain-containing protein KCTD5
Similarity search - Component
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.27 Å
AuthorsZheng, S. / Jiang, W. / Wang, W. / Kong, Y.
Funding support China, 1items
OrganizationGrant numberCountry
Ministry of Science and Technology (MoST, China) China
CitationJournal: Sci Adv / Year: 2023
Title: Structural basis for the ubiquitination of G protein βγ subunits by KCTD5/Cullin3 E3 ligase.
Authors: Wentong Jiang / Wei Wang / Yinfei Kong / Sanduo Zheng /
Abstract: G protein-coupled receptor (GPCR) signaling is precisely controlled to avoid overstimulation that results in detrimental consequences. Gβγ signaling is negatively regulated by a Cullin3 (Cul3)- ...G protein-coupled receptor (GPCR) signaling is precisely controlled to avoid overstimulation that results in detrimental consequences. Gβγ signaling is negatively regulated by a Cullin3 (Cul3)-dependent E3 ligase, KCTD5, which triggers ubiquitination and degradation of free Gβγ. Here, we report the cryo-electron microscopy structures of the KCTD5-Gβγ fusion complex and the KCTD7-Cul3 complex. KCTD5 in pentameric form engages symmetrically with five copies of Gβγ through its C-terminal domain. The unique pentameric assembly of the KCTD5/Cul3 E3 ligase places the ubiquitin-conjugating enzyme (E2) and the modification sites of Gβγ in close proximity and allows simultaneous transfer of ubiquitin from E2 to five Gβγ subunits. Moreover, we show that ubiquitination of Gβγ by KCTD5 is important for fine-tuning cyclic adenosine 3´,5´-monophosphate signaling of GPCRs. Our studies provide unprecedented insights into mechanisms of substrate recognition by unusual pentameric E3 ligases and highlight the KCTD family as emerging regulators of GPCR signaling.
History
DepositionJun 1, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jul 26, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
B: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
G: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
F: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
L: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
A: BTB/POZ domain-containing protein KCTD5
H: BTB/POZ domain-containing protein KCTD5
C: BTB/POZ domain-containing protein KCTD5
D: BTB/POZ domain-containing protein KCTD5
E: BTB/POZ domain-containing protein KCTD5
I: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
M: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
J: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
N: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
K: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
O: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2


Theoretical massNumber of molelcules
Total (without water)410,49115
Polymers410,49115
Non-polymers00
Water0
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
Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1


Mass: 41088.762 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNB1 / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: P62873
#2: Protein
Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / G gamma-I


Mass: 10228.731 Da / Num. of mol.: 5 / Mutation: C68S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNG2 / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: P59768
#3: Protein
BTB/POZ domain-containing protein KCTD5


Mass: 30780.623 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Kctd5 / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: Q8VC57

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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: Cryo-EM structure of KCTD5 in complex with G protein betagamma subunits
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMHEPESHEPES1
2150 mMSodium ChlorideNaClSodium chloride1
SpecimenConc.: 0.32 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in.
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281.15 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 FIELDBright-field microscopy / Nominal defocus max: 2600 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 49.71 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
11cryoSPARCclassification
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2484234
3D reconstructionResolution: 3.27 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 99265 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingB value: 188.6 / Space: RECIPROCAL
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeSource nameType
13DRY

3dry

13DRYPDBexperimental model
26M8S

6m8s

16M8SPDBexperimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00417464
ELECTRON MICROSCOPYf_angle_d0.64923636
ELECTRON MICROSCOPYf_dihedral_angle_d4.612380
ELECTRON MICROSCOPYf_chiral_restr0.0442677
ELECTRON MICROSCOPYf_plane_restr0.0043060

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