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- PDB-8u02: CryoEM structure of D2 dopamine receptor in complex with GoA KE m... -

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Basic information

Entry
Database: PDB / ID: 8u02
TitleCryoEM structure of D2 dopamine receptor in complex with GoA KE mutant and dopamine
Components
  • 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
  • Guanine nucleotide-binding protein G(o) subunit alpha
KeywordsMEMBRANE PROTEIN / GPCR / Dopamine / DRD2 / Dominant Negative
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 / acid secretion / positive regulation of glial cell-derived neurotrophic factor production / dopamine neurotransmitter receptor activity, coupled via Gi/Go / auditory behavior / nervous system process involved in regulation of systemic arterial blood pressure / 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 / acid secretion / positive regulation of glial cell-derived neurotrophic factor production / dopamine neurotransmitter receptor activity, coupled via Gi/Go / auditory behavior / nervous system process involved in regulation of systemic arterial blood pressure / 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 / regulation of potassium ion transport / hyaloid vascular plexus regression / negative regulation of neuron migration / Dopamine receptors / adenylate cyclase-inhibiting dopamine receptor signaling pathway / negative regulation of cellular response to hypoxia / orbitofrontal cortex development / response to inactivity / regulation of dopamine uptake involved in synaptic transmission / branching morphogenesis of a nerve / negative regulation of voltage-gated calcium channel activity / 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 / phospholipase C-activating dopamine receptor signaling pathway / dopaminergic synapse / grooming behavior / positive regulation of urine volume / positive regulation of G protein-coupled receptor signaling pathway / 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 morphine / adult walking behavior / response to iron ion / ciliary membrane / regulation of synaptic transmission, GABAergic / G protein-coupled dopamine receptor signaling pathway / regulation of heart contraction / arachidonate secretion / temperature homeostasis / pigmentation / dopamine uptake involved in synaptic transmission / postsynaptic modulation of chemical synaptic transmission / dopamine metabolic process / regulation of dopamine secretion / positive regulation of neuroblast proliferation / heterocyclic compound binding / negative regulation of cytosolic calcium ion concentration / positive regulation of cytokinesis / associative learning / positive regulation of receptor internalization / behavioral response to cocaine / endocytic vesicle / negative regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / mu-type opioid receptor binding / lateral plasma membrane / corticotropin-releasing hormone receptor 1 binding / G-protein alpha-subunit binding / neuroblast proliferation / response to light stimulus / sperm flagellum / response to axon injury / potassium channel regulator activity / GABA-ergic synapse / negative regulation of protein secretion / negative regulation of insulin secretion / long-term memory / G protein-coupled serotonin receptor binding / prepulse inhibition / adenylate cyclase-activating adrenergic receptor signaling pathway / muscle contraction / regulation of sodium ion transport / axon terminus / release of sequestered calcium ion into cytosol / synapse assembly / response to amphetamine / presynaptic modulation of chemical synaptic transmission / negative regulation of blood pressure / negative regulation of innate immune response / phosphatidylinositol 3-kinase/protein kinase B signal transduction / regulation of heart rate / axonogenesis / acrosomal vesicle / excitatory postsynaptic potential / negative regulation of protein phosphorylation
Similarity search - Function
Dopamine D2 receptor / Dopamine receptor family / G-protein alpha subunit, group I / Serpentine type 7TM GPCR chemoreceptor Srsx / G-alpha domain profile. / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit / 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 / G-alpha domain profile. / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit / G protein alpha subunit / 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 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
L-DOPAMINE / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.28 Å
AuthorsKrumm, B.E. / Kapolka, N.J. / Fay, J.F. / Roth, B.L.
Funding support United States, 1items
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 28, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 21, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
R: D(2) dopamine receptor
B: Guanine nucleotide-binding protein G(o) subunit alpha
A: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
C: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)138,2185
Polymers138,0654
Non-polymers1531
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 D(2) dopamine receptor


Mass: 50685.355 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: DRD2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P14416
#2: Protein Guanine nucleotide-binding protein G(o) subunit alpha


Mass: 40100.434 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNAO1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P09471
#3: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Transducin beta chain 1


Mass: 39418.086 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNB1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P62873
#4: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / G gamma-I


Mass: 7861.143 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNG2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P59768
#5: Chemical ChemComp-LDP / L-DOPAMINE / DOPAMINE


Mass: 153.178 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C8H11NO2 / Feature type: SUBJECT OF INVESTIGATION / Comment: medication*YM
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: Human DRD2 in complex with heterotrimeric G protein GoA (K46E) and dopamine
Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightValue: 0.12 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
SpecimenConc.: 3.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1500 nm / Nominal defocus min: 700 nm
Image recordingElectron dose: 55 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.28 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 153270 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0048150
ELECTRON MICROSCOPYf_angle_d0.88211041
ELECTRON MICROSCOPYf_dihedral_angle_d9.5432958
ELECTRON MICROSCOPYf_chiral_restr0.0491276
ELECTRON MICROSCOPYf_plane_restr0.0141397

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