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- PDB-8t1v: Crystal structure of orphan G protein-coupled receptor 6 with bou... -

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Basic information

Entry
Database: PDB / ID: 8t1v
TitleCrystal structure of orphan G protein-coupled receptor 6 with bound inverse agonist 3h
ComponentsG-protein coupled receptor 6, Soluble cytochrome b562 chimera
KeywordsMEMBRANE PROTEIN / Orphan GPCR / GPR6 / BRIL / inverse agonist / IAG3h / LCP / APS / Parkinson's Disease
Function / homology
Function and homology information


sphingosine-1-phosphate receptor activity / regulation of metabolic process / electron transport chain / G protein-coupled receptor activity / adenylate cyclase-activating G protein-coupled receptor signaling pathway / positive regulation of cytosolic calcium ion concentration / periplasmic space / electron transfer activity / G protein-coupled receptor signaling pathway / iron ion binding ...sphingosine-1-phosphate receptor activity / regulation of metabolic process / electron transport chain / G protein-coupled receptor activity / adenylate cyclase-activating G protein-coupled receptor signaling pathway / positive regulation of cytosolic calcium ion concentration / periplasmic space / electron transfer activity / G protein-coupled receptor signaling pathway / iron ion binding / heme binding / plasma membrane / cytoplasm
Similarity search - Function
G protein-coupled receptor 6 / G protein-coupled receptor 3/6/12 orphan / Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562 / 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)
Similarity search - Domain/homology
: / Soluble cytochrome b562 / G-protein coupled receptor 6
Similarity search - Component
Biological speciesHomo sapiens (human)
Escherichia coli (E. coli)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.6 Å
AuthorsBarekatain, M. / Johansson, L. / Lam, J.H. / Sadybekov, A.V. / Han, G.W. / Popov, P. / Russo, J. / Bliesath, J. / Brice, N. / Beresford, M. ...Barekatain, M. / Johansson, L. / Lam, J.H. / Sadybekov, A.V. / Han, G.W. / Popov, P. / Russo, J. / Bliesath, J. / Brice, N. / Beresford, M. / Carlson, L. / Saikatendu, K.S. / Sun, H. / Murphy, S. / Monenschein, H. / Schiffer, H.H. / Lutomski, C. / Robinson, C.V. / Liu, Z. / Hua, T. / Katritch, V. / Cherezov, V.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM127086 United States
CitationJournal: Sci Signal / Year: 2024
Title: Structural insights into the high basal activity and inverse agonism of the orphan receptor GPR6 implicated in Parkinson's disease.
Authors: Mahta Barekatain / Linda C Johansson / Jordy H Lam / Hao Chang / Anastasiia V Sadybekov / Gye Won Han / Joseph Russo / Joshua Bliesath / Nicola L Brice / Mark B L Carlton / Kumar S ...Authors: Mahta Barekatain / Linda C Johansson / Jordy H Lam / Hao Chang / Anastasiia V Sadybekov / Gye Won Han / Joseph Russo / Joshua Bliesath / Nicola L Brice / Mark B L Carlton / Kumar S Saikatendu / Hukai Sun / Sean T Murphy / Holger Monenschein / Hans H Schiffer / Petr Popov / Corinne A Lutomski / Carol V Robinson / Zhi-Jie Liu / Tian Hua / Vsevolod Katritch / Vadim Cherezov /
Abstract: GPR6 is an orphan G protein-coupled receptor with high constitutive activity found in D2-type dopamine receptor-expressing medium spiny neurons of the striatopallidal pathway, which is aberrantly ...GPR6 is an orphan G protein-coupled receptor with high constitutive activity found in D2-type dopamine receptor-expressing medium spiny neurons of the striatopallidal pathway, which is aberrantly hyperactivated in Parkinson's disease. Here, we solved crystal structures of GPR6 without the addition of a ligand (a pseudo-apo state) and in complex with two inverse agonists, including CVN424, which improved motor symptoms in patients with Parkinson's disease in clinical trials. In addition, we obtained a cryo-electron microscopy structure of the signaling complex between GPR6 and its cognate G heterotrimer. The pseudo-apo structure revealed a strong density in the orthosteric pocket of GPR6 corresponding to a lipid-like endogenous ligand. A combination of site-directed mutagenesis, native mass spectrometry, and computer modeling suggested potential mechanisms for high constitutive activity and inverse agonism in GPR6 and identified a series of lipids and ions bound to the receptor. The structures and results obtained in this study could guide the rational design of drugs that modulate GPR6 signaling.
History
DepositionJun 4, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 4, 2024Provider: repository / Type: Initial release
Revision 1.1Dec 18, 2024Group: Data collection / Database references / Derived calculations
Category: citation / citation_author ...citation / citation_author / pdbx_struct_assembly / pdbx_struct_assembly_gen / pdbx_struct_assembly_prop / pdbx_struct_oper_list / struct_biol
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name / _pdbx_struct_assembly.details / _pdbx_struct_assembly.method_details / _pdbx_struct_assembly.oligomeric_count / _pdbx_struct_assembly.oligomeric_details / _pdbx_struct_assembly_gen.oper_expression

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: G-protein coupled receptor 6, Soluble cytochrome b562 chimera
hetero molecules


Theoretical massNumber of molelcules
Total (without water)50,1513
Polymers49,7151
Non-polymers4352
Water905
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)63.136, 63.136, 249.670
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number154
Space group name H-MP3221

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Components

#1: Protein G-protein coupled receptor 6, Soluble cytochrome b562 chimera / Sphingosine 1-phosphate receptor GPR6 / Cytochrome b-562


Mass: 49715.480 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human), (gene. exp.) Escherichia coli (E. coli)
Gene: GPR6, cybC / Production host: Homo sapiens (human) / References: UniProt: P46095, UniProt: P0ABE7
#2: Chemical ChemComp-XY8 / 3-{4-[(2,4-difluorophenyl)methyl]piperazin-1-yl}-7-methyl-N-(propan-2-yl)pyrido[3,4-b]pyrazin-2-amine


Mass: 412.479 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C22H26F2N6 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: Na
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 5 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.89 Å3/Da / Density % sol: 57.43 %
Crystal growTemperature: 293 K / Method: lipidic cubic phase / Details: NaCH3COO, PEG 400, NaCl, PPG P40

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Data collection

DiffractionMean temperature: 123 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 23-ID-B / Wavelength: 1.0332 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Aug 10, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.0332 Å / Relative weight: 1
ReflectionResolution: 2.6→50 Å / Num. obs: 14570 / % possible obs: 78.2 % / Redundancy: 6.6 % / CC1/2: 0.986 / Rmerge(I) obs: 0.147 / Net I/σ(I): 9.1
Reflection shellResolution: 2.6→2.69 Å / Rmerge(I) obs: 0.693 / Mean I/σ(I) obs: 1.4 / Num. unique obs: 467 / CC1/2: 0.819

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Processing

Software
NameVersionClassification
PHENIX(1.20.1_4487: ???)refinement
PHASERphasing
HKL-2000data reduction
HKL-2000data scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.6→41.13 Å / SU ML: 0 / Cross valid method: FREE R-VALUE / σ(F): 49.85 / Phase error: 33.88 / Stereochemistry target values: TWIN_LSQ_F
RfactorNum. reflection% reflection
Rfree0.2576 676 5.09 %
Rwork0.2296 --
obs0.2327 13293 71.51 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.6→41.13 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2635 0 31 5 2671
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0022727
X-RAY DIFFRACTIONf_angle_d0.4423738
X-RAY DIFFRACTIONf_dihedral_angle_d11.174919
X-RAY DIFFRACTIONf_chiral_restr0.031464
X-RAY DIFFRACTIONf_plane_restr0.004458
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.6-2.80.3181480.325833X-RAY DIFFRACTION23
2.81-3.090.2585700.28651478X-RAY DIFFRACTION41
3.09-3.530.27291370.25263135X-RAY DIFFRACTION85
3.53-4.450.23242060.21513488X-RAY DIFFRACTION94
4.45-41.130.27142020.2233696X-RAY DIFFRACTION94
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
11.18680.24490.26051.87081.04092.6263-0.13760.1027-0.19340.20310.2533-0.34620.74420.79530.09580.14570.18970.07540.4177-0.01830.28216.348-3.902-1.875
21.31-0.6314-1.25710.37720.50041.3554-0.6236-0.20290.132-0.3910.2487-0.0192-0.21961.0293-0.09570.6362-0.3486-0.10410.81750.01690.53829.7110.547-48.707
31.90450.26780.29212.4947-0.1641.1264-0.07190.5318-0.5464-0.08570.0573-0.40571.2460.4647-0.1210.34770.17760.13250.2445-0.12170.27057.684-9.701-6.594
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1( CHAIN A AND RESID 68:256 )A68 - 256
2X-RAY DIFFRACTION2( CHAIN A AND RESID 1001:1106 )A1001 - 1106
3X-RAY DIFFRACTION3( CHAIN A AND RESID 270:900 )A270 - 900

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