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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
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / 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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