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- EMDB-41766: CryoEM structure of D2 dopamine receptor in complex with GoA KE m... -

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Entry
Database: EMDB / ID: EMD-41766
TitleCryoEM structure of D2 dopamine receptor in complex with GoA KE mutant, scFv16, and dopamine
Map data
Sample
  • Complex: Human DRD2 in complex with heterotrimeric G protein GoA (K46E), scFv16, and dopamine
    • Protein or peptide: scFv16
    • 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: D(2) dopamine receptor
    • Protein or peptide: Guanine nucleotide-binding protein G(o) subunit alpha
  • Ligand: L-DOPAMINE
KeywordsGPCR / Dopamine / DRD2 / Dominant Negative / MEMBRANE PROTEIN
Function / homology
Function and homology information


regulation of locomotion involved in locomotory behavior / negative regulation of dopamine receptor signaling pathway / positive regulation of dopamine uptake involved in synaptic transmission / negative regulation of circadian sleep/wake cycle, sleep / positive regulation of glial cell-derived neurotrophic factor production / acid secretion / auditory behavior / nervous system process involved in regulation of systemic arterial blood pressure / dopamine neurotransmitter receptor activity, coupled via Gi/Go / regulation of synapse structural plasticity ...regulation of locomotion involved in locomotory behavior / negative regulation of dopamine receptor signaling pathway / positive regulation of dopamine uptake involved in synaptic transmission / negative regulation of circadian sleep/wake cycle, sleep / positive regulation of glial cell-derived neurotrophic factor production / acid secretion / auditory behavior / nervous system process involved in regulation of systemic arterial blood pressure / dopamine neurotransmitter receptor activity, coupled via Gi/Go / regulation of synapse structural plasticity / response to histamine / positive regulation of renal sodium excretion / neuron-neuron synaptic transmission / adenohypophysis development / cerebral cortex GABAergic interneuron migration / negative regulation of cellular response to hypoxia / hyaloid vascular plexus regression / negative regulation of neuron migration / orbitofrontal cortex development / regulation of potassium ion transport / adenylate cyclase-inhibiting dopamine receptor signaling pathway / response to inactivity / Dopamine receptors / branching morphogenesis of a nerve / negative regulation of voltage-gated calcium channel activity / regulation of dopamine uptake involved in synaptic transmission / dopamine binding / negative regulation of dopamine secretion / positive regulation of growth hormone secretion / heterotrimeric G-protein binding / behavioral response to ethanol / drinking behavior / peristalsis / G protein-coupled receptor complex / grooming behavior / phospholipase C-activating dopamine receptor signaling pathway / dopaminergic synapse / positive regulation of G protein-coupled receptor signaling pathway / mu-type opioid receptor binding / positive regulation of urine volume / corticotropin-releasing hormone receptor 1 binding / striatum development / negative regulation of adenylate cyclase activity / negative regulation of synaptic transmission, glutamatergic / positive regulation of multicellular organism growth / G protein-coupled receptor internalization / non-motile cilium / vesicle docking involved in exocytosis / response to iron ion / adult walking behavior / response to morphine / ciliary membrane / pigmentation / arachidonate secretion / G protein-coupled dopamine receptor signaling pathway / regulation of synaptic transmission, GABAergic / temperature homeostasis / regulation of heart contraction / postsynaptic modulation of chemical synaptic transmission / positive regulation of neuroblast proliferation / heterocyclic compound binding / dopamine uptake involved in synaptic transmission / negative regulation of cytosolic calcium ion concentration / regulation of dopamine secretion / positive regulation of cytokinesis / dopamine metabolic process / associative learning / behavioral response to cocaine / positive regulation of receptor internalization / endocytic vesicle / negative regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / lateral plasma membrane / response to light stimulus / neuroblast proliferation / G-protein alpha-subunit binding / response to axon injury / negative regulation of protein secretion / sperm flagellum / negative regulation of insulin secretion / potassium channel regulator activity / long-term memory / prepulse inhibition / adenylate cyclase-activating adrenergic receptor signaling pathway / G protein-coupled serotonin receptor binding / GABA-ergic synapse / regulation of sodium ion transport / axon terminus / release of sequestered calcium ion into cytosol / muscle contraction / synapse assembly / negative regulation of blood pressure / presynaptic modulation of chemical synaptic transmission / ionotropic glutamate receptor binding / negative regulation of innate immune response / response to amphetamine / phosphatidylinositol 3-kinase/protein kinase B signal transduction / regulation of heart rate / axonogenesis / acrosomal vesicle / excitatory postsynaptic potential
Similarity search - Function
Dopamine D2 receptor / Dopamine receptor family / 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 ...Dopamine D2 receptor / Dopamine receptor family / 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. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD domain, G-beta repeat / 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
Guanine nucleotide-binding protein G(o) subunit alpha / D(2) dopamine receptor / 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
Similarity search - Component
Biological speciesHomo sapiens (human) / Escherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsKrumm BE / Kapolka NJ / Fay JF / Roth BL
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Mental Health (NIH/NIMH)MH112205 United States
CitationJournal: Nat Commun / Year: 2024
Title: A neurodevelopmental disorder mutation locks G proteins in the transitory pre-activated state.
Authors: Kevin M Knight / Brian E Krumm / Nicholas J Kapolka / W Grant Ludlam / Meng Cui / Sepehr Mani / Iya Prytkova / Elizabeth G Obarow / Tyler J Lefevre / Wenyuan Wei / Ning Ma / Xi-Ping Huang / ...Authors: Kevin M Knight / Brian E Krumm / Nicholas J Kapolka / W Grant Ludlam / Meng Cui / Sepehr Mani / Iya Prytkova / Elizabeth G Obarow / Tyler J Lefevre / Wenyuan Wei / Ning Ma / Xi-Ping Huang / Jonathan F Fay / Nagarajan Vaidehi / Alan V Smrcka / Paul A Slesinger / Diomedes E Logothetis / Kirill A Martemyanov / Bryan L Roth / Henrik G Dohlman /
Abstract: Many neurotransmitter receptors activate G proteins through exchange of GDP for GTP. The intermediate nucleotide-free state has eluded characterization, due largely to its inherent instability. Here ...Many neurotransmitter receptors activate G proteins through exchange of GDP for GTP. The intermediate nucleotide-free state has eluded characterization, due largely to its inherent instability. Here we characterize a G protein variant associated with a rare neurological disorder in humans. Gα has a charge reversal that clashes with the phosphate groups of GDP and GTP. As anticipated, the purified protein binds poorly to guanine nucleotides yet retains wild-type affinity for G protein βγ subunits. In cells with physiological concentrations of nucleotide, Gα forms a stable complex with receptors and Gβγ, impeding effector activation. Further, we demonstrate that the mutant can be easily purified in complex with dopamine-bound D2 receptors, and use cryo-electron microscopy to determine the structure, including both domains of Gα, without nucleotide or stabilizing nanobodies. These findings reveal the molecular basis for the first committed step of G protein activation, establish a mechanistic basis for a neurological disorder, provide a simplified strategy to determine receptor-G protein structures, and a method to detect high affinity agonist binding in cells.
History
DepositionAug 27, 2023-
Header (metadata) releaseAug 21, 2024-
Map releaseAug 21, 2024-
UpdateNov 6, 2024-
Current statusNov 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41766.png

Downloads & links

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Map

FileDownload / File: emd_41766.map.gz / Format: CCP4 / Size: 172.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.87 Å/pix.
x 356 pix.
= 311.144 Å		0.87 Å/pix.
x 356 pix.
= 311.144 Å		0.87 Å/pix.
x 356 pix.
= 311.144 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.874 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum-0.29627788 - 0.5969459
Average (Standard dev.)-0.0006310877 (±0.011523307)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions356356356
Spacing356356356
CellA=B=C: 311.144 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_41766_half_map_1.map
Projections & Slices
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Half map: #1

Fileemd_41766_half_map_2.map
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Sample components

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Entire : Human DRD2 in complex with heterotrimeric G protein GoA (K46E), s...

EntireName: Human DRD2 in complex with heterotrimeric G protein GoA (K46E), scFv16, and dopamine
Components
  • Complex: Human DRD2 in complex with heterotrimeric G protein GoA (K46E), scFv16, and dopamine
    • Protein or peptide: scFv16
    • 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: D(2) dopamine receptor
    • Protein or peptide: Guanine nucleotide-binding protein G(o) subunit alpha
  • Ligand: L-DOPAMINE

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Supramolecule #1: Human DRD2 in complex with heterotrimeric G protein GoA (K46E), s...

SupramoleculeName: Human DRD2 in complex with heterotrimeric G protein GoA (K46E), scFv16, and dopamine
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 120 KDa

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Macromolecule #1: scFv16

MacromoleculeName: scFv16 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 28.668922 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: DVQLVESGGG LVQPGGSRKL SCSASGFAFS SFGMHWVRQA PEKGLEWVAY ISSGSGTIYY ADTVKGRFTI SRDDPKNTLF LQMTSLRSE DTAMYYCVRS IYYYGSSPFD FWGQGTTLTV SSGGGGSGGG GSGGGGSDIV MTQATSSVPV TPGESVSISC R SSKSLLHS ...String:
DVQLVESGGG LVQPGGSRKL SCSASGFAFS SFGMHWVRQA PEKGLEWVAY ISSGSGTIYY ADTVKGRFTI SRDDPKNTLF LQMTSLRSE DTAMYYCVRS IYYYGSSPFD FWGQGTTLTV SSGGGGSGGG GSGGGGSDIV MTQATSSVPV TPGESVSISC R SSKSLLHS NGNTYLYWFL QRPGQSPQLL IYRMSNLASG VPDRFSGSGS GTAFTLTISR LEAEDVGVYY CMQHLEYPLT FG AGTKLEL KAAALEVLFQ GPHHHHHHHH

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Macromolecule #2: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1

MacromoleculeName: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 39.418086 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MHHHHHHLEV LFQGPGSSGS ELDQLRQEAE QLKNQIRDAR KACADATLSQ ITNNIDPVGR IQMRTRRTLR GHLAKIYAMH WGTDSRLLV SASQDGKLII WDSYTTNKVH AIPLRSSWVM TCAYAPSGNY VACGGLDNIC SIYNLKTREG NVRVSRELAG H TGYLSCCR ...String:
MHHHHHHLEV LFQGPGSSGS ELDQLRQEAE QLKNQIRDAR KACADATLSQ ITNNIDPVGR IQMRTRRTLR GHLAKIYAMH WGTDSRLLV SASQDGKLII WDSYTTNKVH AIPLRSSWVM TCAYAPSGNY VACGGLDNIC SIYNLKTREG NVRVSRELAG H TGYLSCCR FLDDNQIVTS SGDTTCALWD IETGQQTTTF TGHTGDVMSL SLAPDTRLFV SGACDASAKL WDVREGMCRQ TF TGHESDI NAICFFPNGN AFATGSDDAT CRLFDLRADQ ELMTYSHDNI ICGITSVSFS KSGRLLLAGY DDFNCNVWDA LKA DRAGVL AGHDNRVSCL GVTDDGMAVA TGSWDSFLKI WN

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

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Macromolecule #3: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2

MacromoleculeName: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 7.861143 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString:
MASNNTASIA QARKLVEQLK MEANIDRIKV SKAAADLMAY CEAHAKEDPL LTPVPASENP FREKKFFCAI L

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

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Macromolecule #4: D(2) dopamine receptor

MacromoleculeName: D(2) dopamine receptor / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 50.685355 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MDPLNLSWYD DDLERQNWSR PFNGSDGKAD RPHYNYYATL LTLLIAVIVF GNVLVCMAVS REKALQTTTN YLIVSLAVAD LLVATLVMP WVVYLEVVGE WKFSRIHCDI FVTLDVMMCT ASILNLCAIS IDRYTAVAMP MLYNTRYSSK RRVTVMISIV W VLSFTISC ...String:
MDPLNLSWYD DDLERQNWSR PFNGSDGKAD RPHYNYYATL LTLLIAVIVF GNVLVCMAVS REKALQTTTN YLIVSLAVAD LLVATLVMP WVVYLEVVGE WKFSRIHCDI FVTLDVMMCT ASILNLCAIS IDRYTAVAMP MLYNTRYSSK RRVTVMISIV W VLSFTISC PLLFGLNNAD QNECIIANPA FVVYSSIVSF YVPFIVTLLV YIKIYIVLRR RRKRVNTKRS SRAFRAHLRA PL KGNCTHP EDMKLCTVIM KSNGSFPVNR RRVEAARRAQ ELEMEMLSST SPPERTRYSP IPPSHHQLTL PDPSHHGLHS TPD SPAKPE KNGHAKDHPK IAKIFEIQTM PNGKTRTSLK TMSRRKLSQQ KEKKATQMLA IVLGVFIICW LPFFITHILN IHCD CNIPP VLYSAFTWLG YVNSAVNPII YTTFNIEFRK AFLKILHC

UniProtKB: D(2) dopamine receptor

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Macromolecule #5: Guanine nucleotide-binding protein G(o) subunit alpha

MacromoleculeName: Guanine nucleotide-binding protein G(o) subunit alpha / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 40.100434 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MGCTLSAEER AALERSKAIE KNLKEDGISA AKDVKLLLLG AGESGESTIV KQMKIIHEDG FSGEDVKQYK PVVYSNTIQS LAAIVRAMD TLGIEYGDKE RKADAKMVCD VVSRMEDTEP FSAELLSAMM RLWGDSGIQE CFNRSREYQL NDSAKYYLDS L DRIGAADY ...String:
MGCTLSAEER AALERSKAIE KNLKEDGISA AKDVKLLLLG AGESGESTIV KQMKIIHEDG FSGEDVKQYK PVVYSNTIQS LAAIVRAMD TLGIEYGDKE RKADAKMVCD VVSRMEDTEP FSAELLSAMM RLWGDSGIQE CFNRSREYQL NDSAKYYLDS L DRIGAADY QPTEQDILRT RVKTTGIVET HFTFKNLHFR LFDVGGQRSE RKKWIHCFED VTAIIFCVAL SGYDQVLHED ET TNRMHES LMLFDSICNN KFFIDTSIIL FLNKKDLFGE KIKKSPLTIC FPEYTGPNTY EDAAAYIQAQ FESKNRSPNK EIY CHMTCA TDTNNIQVVF DAVTDIIIAN NLRGCGLY

UniProtKB: Guanine nucleotide-binding protein G(o) subunit alpha

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Macromolecule #6: L-DOPAMINE

MacromoleculeName: L-DOPAMINE / type: ligand / ID: 6 / Number of copies: 1 / Formula: LDP
Molecular weightTheoretical: 153.178 Da
Chemical component information

ChemComp-LDP:
L-DOPAMINE / medication*YM

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration3.5 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 55.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.8 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

8irs

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 216188
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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