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- PDB-9bkj: Cholecystokinin 1 receptor (CCK1R) Y140A mutant, Gq chimera (mGsq... -

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Basic information

Entry
Database: PDB / ID: 9bkj
TitleCholecystokinin 1 receptor (CCK1R) Y140A mutant, Gq chimera (mGsqi) complex
Components
  • (Guanine nucleotide-binding protein ...) x 3
  • Cholecystokinin receptor type A
  • Cholecystokinin-8
KeywordsMEMBRANE PROTEIN / cholecystokinin / GPCR / cholesterol / mutant
Function / homology
Function and homology information


cholecystokinin receptor activity / cholecystokinin signaling pathway / regulation of hormone secretion / neuropeptide hormone activity / eating behavior / peptide hormone receptor binding / forebrain development / peptide hormone binding / PKA activation in glucagon signalling / developmental growth ...cholecystokinin receptor activity / cholecystokinin signaling pathway / regulation of hormone secretion / neuropeptide hormone activity / eating behavior / peptide hormone receptor binding / forebrain development / peptide hormone binding / PKA activation in glucagon signalling / developmental growth / hair follicle placode formation / D1 dopamine receptor binding / intracellular transport / vascular endothelial cell response to laminar fluid shear stress / renal water homeostasis / activation of adenylate cyclase activity / adenylate cyclase inhibitor activity / Hedgehog 'off' state / cellular response to hormone stimulus / digestion / positive regulation of protein localization to cell cortex / adenylate cyclase-activating adrenergic receptor signaling pathway / T cell migration / Adenylate cyclase inhibitory pathway / D2 dopamine receptor binding / response to prostaglandin E / adenylate cyclase regulator activity / G protein-coupled serotonin receptor binding / adenylate cyclase-inhibiting serotonin receptor signaling pathway / cellular response to glucagon stimulus / regulation of insulin secretion / cellular response to forskolin / axonogenesis / regulation of mitotic spindle organization / Peptide ligand-binding receptors / adenylate cyclase activator activity / trans-Golgi network membrane / Regulation of insulin secretion / negative regulation of inflammatory response to antigenic stimulus / positive regulation of cholesterol biosynthetic process / negative regulation of insulin secretion / G protein-coupled receptor binding / hormone activity / bone development / response to peptide hormone / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / platelet aggregation / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / centriolar satellite / cognition / G-protein beta/gamma-subunit complex binding / adenylate cyclase-activating G protein-coupled receptor signaling pathway / Olfactory Signaling Pathway / neuron migration / Activation of the phototransduction cascade / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Thromboxane signalling through TP receptor / G protein-coupled acetylcholine receptor signaling pathway / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / G-protein activation / Prostacyclin signalling through prostacyclin receptor / G beta:gamma signalling through CDC42 / Glucagon signaling in metabolic regulation / G beta:gamma signalling through BTK / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / ADP signalling through P2Y purinoceptor 12 / photoreceptor disc membrane / Sensory perception of sweet, bitter, and umami (glutamate) taste / Glucagon-type ligand receptors / Adrenaline,noradrenaline inhibits insulin secretion / GDP binding / Vasopressin regulates renal water homeostasis via Aquaporins / sensory perception of smell / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / G alpha (z) signalling events / cellular response to catecholamine stimulus / ADORA2B mediated anti-inflammatory cytokines production / ADP signalling through P2Y purinoceptor 1 / G beta:gamma signalling through PI3Kgamma / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / adenylate cyclase-activating dopamine receptor signaling pathway / GPER1 signaling / Inactivation, recovery and regulation of the phototransduction cascade / cellular response to prostaglandin E stimulus / G-protein beta-subunit binding / heterotrimeric G-protein complex / G alpha (12/13) signalling events / sensory perception of taste / extracellular vesicle / positive regulation of cold-induced thermogenesis / signaling receptor complex adaptor activity / Thrombin signalling through proteinase activated receptors (PARs) / retina development in camera-type eye / G protein activity / GTPase binding / Ca2+ pathway / fibroblast proliferation / midbody
Similarity search - Function
Cholecystokinin-like / Gastrin/cholecystokinin peptide hormone / Gastrin/cholecystokinin, conserved site / Gastrin/cholecystokinin family / Gastrin / cholecystokinin family signature. / gastrin / cholecystokinin / caerulein family / Cholecystokinin receptor type A / Cholecystokinin A receptor, N-terminal / Cholecystokinin A receptor, N-terminal domain superfamily / Cholecystokinin A receptor, N-terminal ...Cholecystokinin-like / Gastrin/cholecystokinin peptide hormone / Gastrin/cholecystokinin, conserved site / Gastrin/cholecystokinin family / Gastrin / cholecystokinin family signature. / gastrin / cholecystokinin / caerulein family / Cholecystokinin receptor type A / Cholecystokinin A receptor, N-terminal / Cholecystokinin A receptor, N-terminal domain superfamily / Cholecystokinin A receptor, N-terminal / Cholecystokinin receptor / G-protein alpha subunit, group S / G-protein alpha subunit, group I / Serpentine type 7TM GPCR chemoreceptor Srsx / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit / G-alpha domain profile. / G protein alpha subunit / G-protein, gamma subunit / G-protein gamma subunit domain profile. / G-protein gamma-like domain / G-protein gamma-like domain superfamily / GGL domain / G protein gamma subunit-like motifs / GGL domain / G protein beta WD-40 repeat protein / Guanine nucleotide-binding protein, beta subunit / G-protein, beta subunit / G-protein coupled receptors family 1 signature. / G protein-coupled receptor, rhodopsin-like / GPCR, rhodopsin-like, 7TM / G-protein coupled receptors family 1 profile. / 7 transmembrane receptor (rhodopsin family) / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
AMINO GROUP / Cholecystokinin / Cholecystokinin receptor type A / 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 / Guanine nucleotide-binding protein G(s) subunit alpha isoforms short / Guanine nucleotide-binding protein G(i) subunit alpha-1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.59 Å
AuthorsCary, B.P. / Harikumar, K.G. / Zhao, P. / Desai, A.J. / Mobbs, J.M. / Toufaily, C. / Furness, S.G.B. / Christopoulos, A. / Belousoff, M.J. / Wootten, D. ...Cary, B.P. / Harikumar, K.G. / Zhao, P. / Desai, A.J. / Mobbs, J.M. / Toufaily, C. / Furness, S.G.B. / Christopoulos, A. / Belousoff, M.J. / Wootten, D. / Sexton, P.M. / Miller, L.J.
Funding support United States, Australia, 4items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM141003 United States
National Health and Medical Research Council (NHMRC, Australia)1154434 Australia
National Health and Medical Research Council (NHMRC, Australia)1155302 Australia
Australian Research Council (ARC)FT180100543 Australia
CitationJournal: PLoS Biol / Year: 2024
Title: Cholesterol-dependent dynamic changes in the conformation of the type 1 cholecystokinin receptor affect ligand binding and G protein coupling.
Authors: Kaleeckal G Harikumar / Peishen Zhao / Brian P Cary / Xiaomeng Xu / Aditya J Desai / Maoqing Dong / Jesse I Mobbs / Chirine Toufaily / Sebastian G B Furness / Arthur Christopoulos / Matthew ...Authors: Kaleeckal G Harikumar / Peishen Zhao / Brian P Cary / Xiaomeng Xu / Aditya J Desai / Maoqing Dong / Jesse I Mobbs / Chirine Toufaily / Sebastian G B Furness / Arthur Christopoulos / Matthew J Belousoff / Denise Wootten / Patrick M Sexton / Laurence J Miller /
Abstract: Development of optimal therapeutics for disease states that can be associated with increased membrane cholesterol requires better molecular understanding of lipid modulation of the drug target. Type ...Development of optimal therapeutics for disease states that can be associated with increased membrane cholesterol requires better molecular understanding of lipid modulation of the drug target. Type 1 cholecystokinin receptor (CCK1R) agonist actions are affected by increased membrane cholesterol, enhancing ligand binding and reducing calcium signaling, while agonist actions of the closely related CCK2R are not. In this work, we identified a set of chimeric human CCK1R/CCK2R mutations that exchange the cholesterol sensitivity of these 2 receptors, providing powerful tools when expressed in CHO and HEK-293 model cell lines to explore mechanisms. Static, low energy, high-resolution structures of the mutant CCK1R constructs, stabilized in complex with G protein, were not substantially different, suggesting that alterations to receptor dynamics were key to altered function. We reveal that cholesterol-dependent dynamic changes in the conformation of the helical bundle of CCK receptors affects both ligand binding at the extracellular surface and G protein coupling at the cytosolic surface, as well as their interrelationships involved in stimulus-response coupling. This provides an ideal setting for potential allosteric modulators to correct the negative impact of membrane cholesterol on CCK1R.
History
DepositionApr 29, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 22, 2024Provider: repository / Type: Initial release
Revision 1.1Aug 14, 2024Group: Advisory / Data collection ...Advisory / Data collection / Database references / Derived calculations
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_validate_close_contact / struct_conn
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 / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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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
P: Cholecystokinin-8
R: Cholecystokinin receptor type A
A: Guanine nucleotide-binding protein G(i) subunit alpha-1,Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
hetero molecules


Theoretical massNumber of molelcules
Total (without water)121,7576
Polymers121,7415
Non-polymers161
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Guanine nucleotide-binding protein ... , 3 types, 3 molecules BGA

#1: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Transducin beta chain 1


Mass: 37413.863 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNB1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P62873
#2: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / G gamma-I


Mass: 6375.332 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNG2 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P59768
#5: Protein Guanine nucleotide-binding protein G(i) subunit alpha-1,Guanine nucleotide-binding protein G(s) subunit alpha isoforms short / Adenylate cyclase-inhibiting G alpha protein / Adenylate cyclase-stimulating G alpha protein


Mass: 29144.971 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Guanine nucleotide-binding protein G(i) subunit alpha-1,Guanine nucleotide-binding protein G(s) subunit alpha isoforms short,with certain residues mutated to match Guanine nucleotide-binding protein G(q) subunit
Source: (gene. exp.) Homo sapiens (human) / Gene: GNAI1, GNAS, GNAS1, GSP / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P63096, UniProt: P63092

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Protein/peptide / Protein / Non-polymers , 3 types, 3 molecules PR

#3: Protein/peptide Cholecystokinin-8 / CCK8


Mass: 1144.255 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) / References: UniProt: P06307
#4: Protein Cholecystokinin receptor type A / CCK-A receptor / CCK-AR / Cholecystokinin-1 receptor / CCK1-R


Mass: 47662.770 Da / Num. of mol.: 1 / Mutation: Y140A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CCKAR, CCKRA / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P32238
#6: Chemical ChemComp-NH2 / AMINO GROUP


Mass: 16.023 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: NH2 / Feature type: SUBJECT OF INVESTIGATION

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Details

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: Complex of CCK1R (Y140A mutant) bound to miniGs (Gq, Gi chimera), G-beta1, G-gamma-2 and CCK8s.
Type: COMPLEX
Details: Single-chain fragment variable 16 (ScFv16) was included in the complex but left unmodeled.
Entity ID: #1-#5 / Source: RECOMBINANT
Molecular weightValue: 0.150 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 7.4
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 20 mA, negative polarity / Grid material: GOLD / Grid type: UltrAuFoil 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: BRIGHT FIELD / Nominal defocus max: 1200 nm / Nominal defocus min: 600 nm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5884

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Processing

EM software
IDNameVersionCategory
1crYOLO1.7.6particle selection
2EPUimage acquisition
4CTFFIND4.1.4CTF correction
9cryoSPARC4.1.2initial Euler assignment
10cryoSPARC4.1.2final Euler assignment
12cryoSPARC4.1.23D reconstruction
13PHENIX1.19model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3580372
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.59 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 279231 / Symmetry type: POINT
Atomic model buildingPDB-ID: 7MBY

7mby


Accession code: 7MBY / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0066976
ELECTRON MICROSCOPYf_angle_d1.0439477
ELECTRON MICROSCOPYf_dihedral_angle_d7.533954
ELECTRON MICROSCOPYf_chiral_restr0.0561116
ELECTRON MICROSCOPYf_plane_restr0.0071188

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