EMDB-44642: Cholecystokinin 1 receptor (CCK1R) Y140A mutant, Gq chimera (mGsq...

Basic information

Entry

Database: EMDB / ID: EMD-44642

• Title: Cholecystokinin 1 receptor (CCK1R) Y140A mutant, Gq chimera (mGsqi) complex
• Map data: best class, local refinement (micelle and ScFv16 masked out), sharpened

Sample

• Complex: Complex of CCK1R (Y140A mutant) bound to miniGs (Gq, Gi chimera), G-beta1, G-gamma-2 and CCK8s.
  • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
  • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
  • Protein or peptide: Cholecystokinin-8
  • Protein or peptide: Cholecystokinin receptor type A
  • Protein or peptide: Guanine nucleotide-binding protein G(i) subunit alpha-1,Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
• Ligand: AMINO GROUP

• Keywords: cholecystokinin / GPCR / cholesterol / mutant / MEMBRANE PROTEIN

Function / homology

Function:

cholecystokinin receptor activity / cholecystokinin signaling pathway / regulation of hormone secretion / neuropeptide hormone activity / peptide hormone receptor binding / eating behavior / peptide hormone binding / PKA activation in glucagon signalling / hair follicle placode formation / developmental growth / D1 dopamine receptor binding / intracellular transport / vascular endothelial cell response to laminar fluid shear stress / renal water homeostasis / cellular response to hormone stimulus / Hedgehog 'off' state / positive regulation of protein localization to cell cortex / Adenylate cyclase inhibitory pathway / T cell migration / forebrain development / adenylate cyclase-activating adrenergic receptor signaling pathway / activation of adenylate cyclase activity / D2 dopamine receptor binding / digestion / response to prostaglandin E / G protein-coupled serotonin receptor binding / adenylate cyclase regulator activity / adenylate cyclase-inhibiting serotonin receptor signaling pathway / cellular response to forskolin / regulation of insulin secretion / cellular response to glucagon stimulus / regulation of mitotic spindle organization / axonogenesis / adenylate cyclase activator activity / Peptide ligand-binding receptors / trans-Golgi network membrane / Regulation of insulin secretion / negative regulation of inflammatory response to antigenic stimulus / positive regulation of cholesterol biosynthetic process / G protein-coupled receptor binding / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / hormone activity / bone development / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / response to peptide hormone / G-protein beta/gamma-subunit complex binding / centriolar satellite / platelet aggregation / Olfactory Signaling Pathway / cognition / 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 / adenylate cyclase-activating G protein-coupled receptor signaling pathway / G-protein activation / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / Prostacyclin signalling through prostacyclin receptor / G beta:gamma signalling through CDC42 / Glucagon signaling in metabolic regulation / G beta:gamma signalling through BTK / neuron migration / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / ADP signalling through P2Y purinoceptor 12 / Sensory perception of sweet, bitter, and umami (glutamate) taste / photoreceptor disc membrane / Glucagon-type ligand receptors / Adrenaline,noradrenaline inhibits insulin secretion / Vasopressin regulates renal water homeostasis via Aquaporins / GDP binding / G alpha (z) signalling events / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / 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 / sensory perception of smell / 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 / signaling receptor complex adaptor activity / Thrombin signalling through proteinase activated receptors (PARs) / positive regulation of cold-induced thermogenesis / G protein activity / GTPase binding / Ca2+ pathway / retina development in camera-type eye / midbody / cell cortex / High laminar flow shear stress activates signaling by PIEZO1 and PECAM1:CDH5:KDR in endothelial cells / fibroblast proliferation

Domain/homology:

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 / 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. / WD domain, G-beta repeat / 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

Component:

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

• Biological species: Homo sapiens (human)
• Method: single particle reconstruction / cryo EM / Resolution: 2.59 Å
• Authors: Cary BP / Harikumar KG / Zhao P / Desai AJ / Mobbs JM / Toufaily C / Furness SGB / Christopoulos A / Belousoff MJ / Wootten D / Sexton PM / Miller LJ

Funding support

United States, Australia, 4 items

Organization	Grant number	Country
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

• Citation: Journal: 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 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 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

Deposition	Apr 29, 2024	-
Header (metadata) release	May 22, 2024	-
Map release	May 22, 2024	-
Update	Aug 14, 2024	-
Current status	Aug 14, 2024	Processing site: RCSB / Status: Released

Map

• File: Download / File: emd_44642.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: best class, local refinement (micelle and ScFv16 masked out), sharpened
• Voxel size: X=Y=Z: 0.85 Å

Density

Contour Level	By AUTHOR: 0.66
Minimum - Maximum	-3.2525418 - 4.681007
Average (Standard dev.)	0.0045112907 (±0.10297948)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 256 256 256 Spacing 256 256 256
Cell	A=B=C: 217.6 Å α=β=γ: 90.0 °

Supplemental data

Additional map: best class, local refinement (micelle, G protein, and...

• File: emd_44642_additional_1.map
• Annotation: best class, local refinement (micelle, G protein, and ScFv16 masked out), unsharpened

Additional map: best class, local refinement (micelle, G protein, and...

• File: emd_44642_additional_2.map
• Annotation: best class, local refinement (micelle, G protein, and ScFv16 masked out), sharpened

Additional map: Consensus map, unsharpened

• File: emd_44642_additional_3.map
• Annotation: Consensus map, unsharpened

Additional map: Consensus refinement, half map A

• File: emd_44642_additional_4.map
• Annotation: Consensus refinement, half map A

Additional map: Consensus refinement, half map B

• File: emd_44642_additional_5.map
• Annotation: Consensus refinement, half map B

Half map: Best class, half-map B

• File: emd_44642_half_map_1.map
• Annotation: Best class, half-map B

Half map: Best class, half-map A

• File: emd_44642_half_map_2.map
• Annotation: Best class, half-map A

Sample components

Entire : Complex of CCK1R (Y140A mutant) bound to miniGs (Gq, Gi chimera),...

• Entire: Name: Complex of CCK1R (Y140A mutant) bound to miniGs (Gq, Gi chimera), G-beta1, G-gamma-2 and CCK8s.

Components

• Complex: Complex of CCK1R (Y140A mutant) bound to miniGs (Gq, Gi chimera), G-beta1, G-gamma-2 and CCK8s.
  • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
  • Protein or peptide: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
  • Protein or peptide: Cholecystokinin-8
  • Protein or peptide: Cholecystokinin receptor type A
  • Protein or peptide: Guanine nucleotide-binding protein G(i) subunit alpha-1,Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
• Ligand: AMINO GROUP

Supramolecule #1: Complex of CCK1R (Y140A mutant) bound to miniGs (Gq, Gi chimera),...

• Supramolecule: Name: Complex of CCK1R (Y140A mutant) bound to miniGs (Gq, Gi chimera), G-beta1, G-gamma-2 and CCK8s.; type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5; Details: Single-chain fragment variable 16 (ScFv16) was included in the complex but left unmodeled.
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 150 KDa

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

• Macromolecule: Name: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1; type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 37.413863 KDa
• Recombinant expression: Organism: Trichoplusia ni (cabbage looper)

Sequence

String:

QSELDQLRQE AEQLKNQIRD ARKACADATL SQITNNIDPV GRIQMRTRRT LRGHLAKIYA MHWGTDSRLL VSASQDGKLI IWDSYTTNK VHAIPLRSSW VMTCAYAPSG NYVACGGLDN ICSIYNLKTR EGNVRVSREL AGHTGYLSCC RFLDDNQIVT S SGDTTCAL WDIETGQQTT TFTGHTGDVM SLSLAPDTRL FVSGACDASA KLWDVREGMC RQTFTGHESD INAICFFPNG NA FATGSDD ATCRLFDLRA DQELMTYSHD NIICGITSVS FSKSGRLLLA GYDDFNCNVW DALKADRAGV LAGHDNRVSC LGV TDDGMA VATGSWDSFL KIWN

UniProtKB: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1

Macromolecule #2: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2

• Macromolecule: Name: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2; type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 6.375332 KDa
• Recombinant expression: Organism: Trichoplusia ni (cabbage looper)

Sequence

String:

NTASIAQARK LVEQLKMEAN IDRIKVSKAA ADLMAYCEAH AKEDPLLTPV PASENPFR

UniProtKB: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2

Macromolecule #3: Cholecystokinin-8

• Macromolecule: Name: Cholecystokinin-8 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 1.144255 KDa

Sequence

String:

D(TYS)MGWMDF

UniProtKB: Cholecystokinin

Macromolecule #4: Cholecystokinin receptor type A

• Macromolecule: Name: Cholecystokinin receptor type A / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 47.66277 KDa
• Recombinant expression: Organism: Trichoplusia ni (cabbage looper)

Sequence

String:

DVVDSLLVNG SNITPPCELG LENETLFCLD QPRPSKEWQP AVQILLYSLI FLLSVLGNTL VITVLIRNKR MRTVTNIFLL SLAVSDLML CLFCMPFNLI PNLLKDFIFG SAVCKTTTYF MGTSVSVSTF NLVAISLERA GAICKPLQSR VWQTKSHALK V IAATWCLS FTIMTPYPIY SNLVPFTKNN NQTANMCRFL LPNDVMQQSW HTFLLLILFL IPGIVMMVAY GLISLELYQG IK FEASQKK SAKERKPSTT SSGKYEDSDG CYLQKTRPPR KLELRQLSTG SSSRANRIRS NSSAANLMAK KRVIRMLIVI VVL FFLCWM PIFSANAWRA YDTASAERRL SGTPISFILL LSYTSSCVNP IIYCFMNKRF RLGFMATFPC CPNPGPPGAR GEVG EEEEG GTTGASLSRF SYSHMSASVP PQ

UniProtKB: Cholecystokinin receptor type A

Macromolecule #5: Guanine nucleotide-binding protein G(i) subunit alpha-1,Guanine n...

• Macromolecule: Name: Guanine nucleotide-binding protein G(i) subunit alpha-1,Guanine nucleotide-binding protein G(s) subunit alpha isoforms short; type: protein_or_peptide / ID: 5 ; 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; Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 29.144971 KDa
• Recombinant expression: Organism: Trichoplusia ni (cabbage looper)

Sequence

String:

HHHHHHHHGC TLSAEDKAAV ERSKMIDRNL REDGEKARRT LRLLLLGADN SGKSTIVKQM RILHGGSGGS GGTSGIFETK FQVDKVNFH MFDVGGQRDE RRKWIQCFND VTAIIFVVDS SDYNRLQEAL NDFKSIWNNR WLRTISVILF LNKQDLLAEK V LAGKSKIE DYFPEFARYT TPEDATPEPG EDPRVTRAKY FIRKEFVDIS TASGDGRHIC YPHFTCAVDT ENARRIFNDC KD IILQMNL REYNLV

UniProtKB: Guanine nucleotide-binding protein G(i) subunit alpha-1, Guanine nucleotide-binding protein G(s) subunit alpha isoforms short

Macromolecule #6: AMINO GROUP

• Macromolecule: Name: AMINO GROUP / type: ligand / ID: 6 / Number of copies: 1 / Formula: NH2
• Molecular weight: Theoretical: 16.023 Da

Chemical component information

ChemComp-NH2:
AMINO GROUP

Experimental details

Structure determination

• Method: cryo EM
• Processing: single particle reconstruction
• Aggregation state: particle

Sample preparation

• Concentration: 5 mg/mL
• Buffer: pH: 7.4
• Grid: Model: UltrAuFoil R1.2/1.3 / Material: GOLD / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR / Details: 20 mA, negative polarity
• Vitrification: Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

Electron microscopy

• Microscope: FEI TITAN KRIOS
• Image recording: Film or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 5884 / Average electron dose: 60.0 e/Ų
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.2 µm / Nominal defocus min: 0.6 µm
• Sample stage: Cooling holder cryogen: NITROGEN
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing

• Particle selection: Number selected: 3580372
• Startup model: Type of model: NONE
• Final reconstruction: Applied symmetry - Point group: C₁ (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.59 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.1.2) / Number images used: 279231
• Initial angle assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.1.2)
• Final angle assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.1.2)

Atomic model buiding 1

Initial model

PDB ID:

7mby

Chain - Source name: PDB / Chain - Initial model type: experimental model

Output model

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