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- PDB-7ad3: Class D GPCR Ste2 dimer coupled to two G proteins -

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Basic information

Entry
Database: PDB / ID: 7ad3
TitleClass D GPCR Ste2 dimer coupled to two G proteins
Components
  • (Guanine nucleotide-binding protein ...) x 2
  • Alpha-factor mating pheromone
  • Pheromone alpha factor receptor
  • STE4 isoform 1
KeywordsMEMBRANE PROTEIN / Fungal GPCR / Dimer / Complex / Class D / Active State
Function / homology
Function and homology information


mating projection / : / protein localization to mating projection tip / PLC beta mediated events / G-protein activation / Acetylcholine regulates insulin secretion / G alpha (q) signalling events / ADP signalling through P2Y purinoceptor 1 / Fatty Acids bound to GPR40 (FFAR1) regulate insulin secretion / : ...mating projection / : / protein localization to mating projection tip / PLC beta mediated events / G-protein activation / Acetylcholine regulates insulin secretion / G alpha (q) signalling events / ADP signalling through P2Y purinoceptor 1 / Fatty Acids bound to GPR40 (FFAR1) regulate insulin secretion / : / G-protein beta/gamma-subunit complex / adaptation of signaling pathway by response to pheromone involved in conjugation with cellular fusion / : / G alpha (12/13) signalling events / chemotropism / Cdc24p-Far1p-Gbetagamma complex / mating pheromone activity / mating-type factor pheromone receptor activity / nuclear migration involved in conjugation with cellular fusion / mating / G protein-coupled receptor homodimeric complex / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / karyogamy involved in conjugation with cellular fusion / G-protein gamma-subunit binding / establishment of protein localization to plasma membrane / pheromone-dependent signal transduction involved in conjugation with cellular fusion / invasive growth in response to glucose limitation / pheromone binding / cupric ion binding / G-protein alpha-subunit binding / cell periphery / G protein-coupled receptor binding / G-protein beta/gamma-subunit complex binding / small GTPase binding / G-protein beta-subunit binding / heterotrimeric G-protein complex / scaffold protein binding / endosome membrane / endosome / G protein-coupled receptor signaling pathway / GTPase activity / GTP binding / protein kinase binding / signal transduction / extracellular region / metal ion binding / plasma membrane / cytoplasm / cytosol
Similarity search - Function
Fungal G-protein, alpha subunit / Guanine nucleotide-binding protein subunit gamma, fungal / Mating factor alpha, C-terminal repeat / Mating factor alpha precursor, N-terminal / Yeast mating factor alpha hormone / Mating factor alpha precursor N-terminus / GPCR fungal pheromone mating factor, STE2 / Pheromone alpha factor receptor, double transmembrane domain superfamily / Fungal pheromone mating factor STE2 GPCR / : ...Fungal G-protein, alpha subunit / Guanine nucleotide-binding protein subunit gamma, fungal / Mating factor alpha, C-terminal repeat / Mating factor alpha precursor, N-terminal / Yeast mating factor alpha hormone / Mating factor alpha precursor N-terminus / GPCR fungal pheromone mating factor, STE2 / Pheromone alpha factor receptor, double transmembrane domain superfamily / Fungal pheromone mating factor STE2 GPCR / : / 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-like domain / GGL domain / G protein gamma subunit-like motifs / Guanine nucleotide-binding protein, beta subunit / G-protein, beta subunit / 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
CHOLESTEROL HEMISUCCINATE / Guanine nucleotide-binding protein subunit gamma / BJ4_G0036910.mRNA.1.CDS.1 / Mating factor alpha-1 / Guanine nucleotide-binding protein alpha-1 subunit / Pheromone alpha factor receptor / Guanine nucleotide-binding protein subunit beta / Guanine nucleotide-binding protein subunit gamma
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsVelazhahan, V. / Tate, C.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MRC U105197215 United Kingdom
European Research Council (ERC)EMPSI 339995 United Kingdom
CitationJournal: Nature / Year: 2021
Title: Structure of the class D GPCR Ste2 dimer coupled to two G proteins.
Authors: Vaithish Velazhahan / Ning Ma / Gáspár Pándy-Szekeres / Albert J Kooistra / Yang Lee / David E Gloriam / Nagarajan Vaidehi / Christopher G Tate /
Abstract: G-protein-coupled receptors (GPCRs) are divided phylogenetically into six classes, denoted A to F. More than 370 structures of vertebrate GPCRs (belonging to classes A, B, C and F) have been ...G-protein-coupled receptors (GPCRs) are divided phylogenetically into six classes, denoted A to F. More than 370 structures of vertebrate GPCRs (belonging to classes A, B, C and F) have been determined, leading to a substantial understanding of their function. By contrast, there are no structures of class D GPCRs, which are found exclusively in fungi where they regulate survival and reproduction. Here we determine the structure of a class D GPCR, the Saccharomyces cerevisiae pheromone receptor Ste2, in an active state coupled to the heterotrimeric G protein Gpa1-Ste4-Ste18. Ste2 was purified as a homodimer coupled to two G proteins. The dimer interface of Ste2 is formed by the N terminus, the transmembrane helices H1, H2 and H7, and the first extracellular loop ECL1. We establish a class D1 generic residue numbering system (CD1) to enable comparisons with orthologues and with other GPCR classes. The structure of Ste2 bears similarities in overall topology to class A GPCRs, but the transmembrane helix H4 is shifted by more than 20 Å and the G-protein-binding site is a shallow groove rather than a cleft. The structure provides a template for the design of novel drugs to target fungal GPCRs, which could be used to treat numerous intractable fungal diseases.
History
DepositionSep 14, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 9, 2020Provider: repository / Type: Initial release
Revision 1.1Dec 16, 2020Group: Database references / Category: citation / citation_author / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Jan 13, 2021Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year / _citation_author.identifier_ORCID

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-11720
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
B: Pheromone alpha factor receptor
A: Pheromone alpha factor receptor
K: Alpha-factor mating pheromone
I: Alpha-factor mating pheromone
F: STE4 isoform 1
E: Guanine nucleotide-binding protein alpha-1 subunit,Guanine nucleotide-binding protein alpha-1 subunit
G: Guanine nucleotide-binding protein subunit gamma
H: Guanine nucleotide-binding protein alpha-1 subunit,Guanine nucleotide-binding protein alpha-1 subunit
hetero molecules


Theoretical massNumber of molelcules
Total (without water)214,24216
Polymers210,8798
Non-polymers3,3638
Water00
1


  • Idetical with deposited unit
  • defined by software
  • Evidence: gel filtration, The Ste2 dimer coupled to two heterotrimeric G proteins was purified as a monodisperse complex as analysed by gel filtration and SEC-MALS
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area20620 Å2
ΔGint-141 kcal/mol
Surface area50560 Å2
MethodPISA

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Components

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Protein , 2 types, 3 molecules BAF

#1: Protein Pheromone alpha factor receptor


Mass: 47885.402 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: STE2 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P0CI39
#3: Protein STE4 isoform 1


Mass: 46626.953 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: STE4, GI526_G0005548 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: A0A6A5Q727, UniProt: P18851*PLUS

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Guanine nucleotide-binding protein ... , 2 types, 3 molecules EHG

#4: Protein Guanine nucleotide-binding protein alpha-1 subunit,Guanine nucleotide-binding protein alpha-1 subunit / GP1-alpha


Mass: 26316.305 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: GPA1, CDC70, DAC1, SCG1, YHR005C
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: P08539
#5: Protein Guanine nucleotide-binding protein subunit gamma


Mass: 12477.051 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: STE18, GI526_G0003318, PACBIOSEQ_LOCUS2168, PACBIOSEQ_LOCUS3627, PACBIOSEQ_LOCUS3692, PACBIOSEQ_LOCUS3742, PACBIOSEQ_LOCUS3767
Production host: Trichoplusia ni (cabbage looper) / References: UniProt: A0A6A5PT44, UniProt: P18852*PLUS

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Protein/peptide / Sugars / Non-polymers , 3 types, 10 molecules KI

#2: Protein/peptide Alpha-factor mating pheromone


Mass: 1685.986 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P01149*PLUS
#6: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
#7: Chemical
ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C31H50O4

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Details

Has ligand of interestN

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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

Component
IDNameTypeEntity IDParent-IDSource
1Ste2 dimer coupled to two G proteinsCOMPLEX#1-#50MULTIPLE SOURCES
2Ste2 dimer coupled to two G proteinsCOMPLEX#1, #3, #51RECOMBINANT
3Alpha-factor mating pheromoneCOMPLEX#21RECOMBINANT
4Guanine nucleotide-binding protein alpha-1 subunit,Guanine nucleotide-binding protein alpha-1 subunitCOMPLEX#41RECOMBINANT
Molecular weightValue: 0.2667 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Saccharomyces cerevisiae (brewer's yeast)4932
23Saccharomyces cerevisiae (brewer's yeast)4932
34Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)559292
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Trichoplusia ni (cabbage looper)7111
23synthetic construct (others)32630
34Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)866768
Buffer solutionpH: 7.5 / Details: Solutions were made fresh and filtered
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESHEPES1
2100 mMSodium ChlorideNaCl1
32 mMMagnesium ChlorideMgCl21
40.001 %Lauryl Maltose Neopentyl GlycolLMNG1
50.001 mMAlpha-factorWHWLQLKPGQPMY1
SpecimenConc.: 0.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: The sample was purified as a monodisperse complex
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
EM imaging

Accelerating voltage: 300 kV / Calibrated defocus max: 2700 nm / Calibrated defocus min: 900 nm / Cryogen: NITROGEN / Electron source: FIELD EMISSION GUN / Illumination mode: FLOOD BEAM / Model: FEI TITAN KRIOS / Mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2700 nm / Nominal defocus min: 900 nm / Temperature (max): 70 K / Temperature (min): 70 K / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Specimen-ID: 1

IDC2 aperture diameter (µm)DetailsNominal magnification (X)
170eBIC Krios1130000
250LMB Krios1105000
350LMB Krios 2105000
Image recording
IDImaging-IDElectron dose (e/Å2)Detector modeFilm or detector model
1145COUNTINGGATAN K2 QUANTUM (4k x 4k)
2248GATAN K2 QUANTUM (4k x 4k)
3340GATAN K2 QUANTUM (4k x 4k)
EM imaging optics
Energyfilter nameIDImaging-IDEnergyfilter slit width (eV)
GIF Quantum SE1120
GIF Quantum SE2220
GIF Quantum SE3320

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Processing

SoftwareName: PHENIX / Version: 1.17.1_3660: / Classification: refinement
EM software
IDNameVersionCategoryImage processing-ID
7Coot0.9 pre-ELmodel fitting
11RELION3.1initial Euler assignment1
12RELION3.1final Euler assignment1
14RELION3.13D reconstruction1
17RELION3.1initial Euler assignment2
18RELION3.1final Euler assignment2
20RELION3.13D reconstruction2
23RELION3.1initial Euler assignment3
24RELION3.1final Euler assignment3
26RELION3D reconstruction3
29RELION3.1initial Euler assignment4
30RELION3.1final Euler assignment4
33PHENIX1.17.1-3660model refinement
Image processing
IDImage recording-ID
11
22
33
41
CTF correction
IDEM image processing-IDType
11NONE
22NONE
33NONE
44NONE
Particle selection
IDImage processing-IDNum. of particles selected
112193519
22485568
33781447
442193519
Symmetry
IDImage processing-IDEntry-IDPoint symmetry
117AD3C1 (asymmetric)
227AD3C1 (asymmetric)
337AD3C1 (asymmetric)
447AD3
3D reconstruction

Entry-ID: 7AD3 / Resolution method: FSC 0.143 CUT-OFF / Symmetry type: POINT

IDResolution (Å)Num. of particlesImage processing-IDDetails
13.5966111
23.316418216418 particles from this dataset contributed to the final 131274 particles that provided map 2 at 3.3 angstrom global resolution
33.3455634556 particles from this dataset contributed to the final 131274 particles that provided map 2 at 3.3 angstrom global resolution
43.31103004110300 particles from this dataset contributed to the final 131274 particles that provided map 2 at 3.3 angstrom global resolution
Atomic model buildingB value: 112 / Space: REAL / Target criteria: Correlation coefficient
Details: Manual building was performed in Coot iterated with real space refinement in PHENIX.
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-ID
16G79

6g79

A1
26G79

6g79

B1
36G79

6g79

G1
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0059614
ELECTRON MICROSCOPYf_angle_d0.89413109
ELECTRON MICROSCOPYf_dihedral_angle_d8.5581457
ELECTRON MICROSCOPYf_chiral_restr0.0531604
ELECTRON MICROSCOPYf_plane_restr0.0061598

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