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- EMDB-36474: cryo-EM structure of NTSR1-GRK2-Galpha(q) complexes 1 -

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Entry
Database: EMDB / ID: EMD-36474
Titlecryo-EM structure of NTSR1-GRK2-Galpha(q) complexes 1
Map data
Sample
  • Complex: NTSR1-GRK2-Galpha(q) complexes 1
    • Protein or peptide: Beta-adrenergic receptor kinase 1G protein-coupled receptor kinase 2
    • Protein or peptide: NTS(8-13)
    • Protein or peptide: Guanine nucleotide-binding protein G(q) subunit alpha
    • Protein or peptide: Neurotensin receptor type 1
  • Ligand: STAUROSPORINE
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: TETRAFLUOROALUMINATE ION
  • Ligand: 2-[{2-(1-fluorocyclopropyl)-4-[4-(2-methoxyphenyl)piperidin-1-yl]quinazolin-6-yl}(methyl)amino]ethan-1-ol
KeywordsBiased signaling / SIGNALING PROTEIN
Function / homology
Function and homology information


Calmodulin induced events / beta-adrenergic-receptor kinase / negative regulation of the force of heart contraction by chemical signal / negative regulation of relaxation of smooth muscle / Activation of SMO / G protein-coupled neurotensin receptor activity / beta-adrenergic receptor kinase activity / inositol phosphate catabolic process / G protein-coupled receptor kinase activity / Edg-2 lysophosphatidic acid receptor binding ...Calmodulin induced events / beta-adrenergic-receptor kinase / negative regulation of the force of heart contraction by chemical signal / negative regulation of relaxation of smooth muscle / Activation of SMO / G protein-coupled neurotensin receptor activity / beta-adrenergic receptor kinase activity / inositol phosphate catabolic process / G protein-coupled receptor kinase activity / Edg-2 lysophosphatidic acid receptor binding / alpha-2A adrenergic receptor binding / positive regulation of inhibitory postsynaptic potential / Fatty Acids bound to GPR40 (FFAR1) regulate insulin secretion / Acetylcholine regulates insulin secretion / tachykinin receptor signaling pathway / symmetric synapse / D-aspartate import across plasma membrane / positive regulation of gamma-aminobutyric acid secretion / negative regulation of striated muscle contraction / desensitization of G protein-coupled receptor signaling pathway / positive regulation of protein localization to cilium / Cargo recognition for clathrin-mediated endocytosis / PLC beta mediated events / phospholipase C-activating dopamine receptor signaling pathway / entrainment of circadian clock / regulation of platelet activation / L-glutamate import across plasma membrane / positive regulation of arachidonic acid secretion / regulation of the force of heart contraction / phototransduction, visible light / regulation of respiratory gaseous exchange / negative regulation of systemic arterial blood pressure / positive regulation of smoothened signaling pathway / G protein-coupled receptor internalization / glutamate receptor signaling pathway / negative regulation of release of sequestered calcium ion into cytosol / positive regulation of glutamate secretion / regulation of canonical Wnt signaling pathway / G alpha (s) signalling events / G alpha (q) signalling events / positive regulation of inositol phosphate biosynthetic process / temperature homeostasis / action potential / response to lipid / regulation of membrane depolarization / detection of temperature stimulus involved in sensory perception of pain / activation of phospholipase C activity / photoreceptor outer segment / neuropeptide signaling pathway / regulation of signal transduction / cardiac muscle contraction / GTPase activator activity / Peptide ligand-binding receptors / viral genome replication / adult locomotory behavior / positive regulation of release of sequestered calcium ion into cytosol / dendritic shaft / learning / cell projection / G protein-coupled receptor binding / G protein-coupled receptor activity / intracellular protein transport / negative regulation of protein kinase activity / G-protein beta/gamma-subunit complex binding / Thromboxane signalling through TP receptor / G-protein activation / G protein-coupled acetylcholine receptor signaling pathway / adenylate cyclase-activating G protein-coupled receptor signaling pathway / terminal bouton / cytoplasmic side of plasma membrane / ADP signalling through P2Y purinoceptor 1 / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / heterotrimeric G-protein complex / blood coagulation / Thrombin signalling through proteinase activated receptors (PARs) / presynapse / heart development / perikaryon / chemical synaptic transmission / postsynapse / G alpha (q) signalling events / peptidyl-serine phosphorylation / nuclear membrane / dendritic spine / protein stabilization / protein kinase activity / symbiont entry into host cell / positive regulation of apoptotic process / membrane raft / G protein-coupled receptor signaling pathway / lysosomal membrane / protein phosphorylation / GTPase activity / synapse / protein-containing complex binding / GTP binding / positive regulation of gene expression / negative regulation of apoptotic process / Golgi apparatus / cell surface
Similarity search - Function
Neurotensin receptor / Neurotensin type 1 receptor / G-protein alpha subunit, group Q / GPCR kinase / Regulator of G protein signaling domain / RGS, subdomain 2 / RGS domain / RGS domain profile. / Regulator of G protein signalling domain / RGS domain superfamily ...Neurotensin receptor / Neurotensin type 1 receptor / G-protein alpha subunit, group Q / GPCR kinase / Regulator of G protein signaling domain / RGS, subdomain 2 / RGS domain / RGS domain profile. / Regulator of G protein signalling domain / RGS domain superfamily / Extension to Ser/Thr-type protein kinases / AGC-kinase, C-terminal / AGC-kinase C-terminal domain profile. / PH domain / PH domain profile. / Pleckstrin homology domain. / Pleckstrin homology domain / 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 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) / PH-like domain superfamily / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Protein kinase domain / Serine/Threonine protein kinases, catalytic domain / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Beta-adrenergic receptor kinase 1 / Neurotensin receptor type 1 / Guanine nucleotide-binding protein G(q) subunit alpha
Similarity search - Component
Biological speciesHomo sapiens (human) / Bos taurus (cattle) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.07 Å
AuthorsDuan J / Liu H / Zhao F / Yuan Q / Ji Y / Xu HE
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: Nature / Year: 2023
Title: GPCR activation and GRK2 assembly by a biased intracellular agonist.
Authors: Jia Duan / Heng Liu / Fenghui Zhao / Qingning Yuan / Yujie Ji / Xiaoqing Cai / Xinheng He / Xinzhu Li / Junrui Li / Kai Wu / Tianyu Gao / Shengnan Zhu / Shi Lin / Ming-Wei Wang / Xi Cheng / ...Authors: Jia Duan / Heng Liu / Fenghui Zhao / Qingning Yuan / Yujie Ji / Xiaoqing Cai / Xinheng He / Xinzhu Li / Junrui Li / Kai Wu / Tianyu Gao / Shengnan Zhu / Shi Lin / Ming-Wei Wang / Xi Cheng / Wanchao Yin / Yi Jiang / Dehua Yang / H Eric Xu /
Abstract: Phosphorylation of G-protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) desensitizes G-protein signalling and promotes arrestin signalling, which is also modulated by biased ligands. The ...Phosphorylation of G-protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) desensitizes G-protein signalling and promotes arrestin signalling, which is also modulated by biased ligands. The molecular assembly of GRKs on GPCRs and the basis of GRK-mediated biased signalling remain largely unknown owing to the weak GPCR-GRK interactions. Here we report the complex structure of neurotensin receptor 1 (NTSR1) bound to GRK2, Gα and the arrestin-biased ligand SBI-553. The density map reveals the arrangement of the intact GRK2 with the receptor, with the N-terminal helix of GRK2 docking into the open cytoplasmic pocket formed by the outward movement of the receptor transmembrane helix 6, analogous to the binding of the G protein to the receptor. SBI-553 binds at the interface between GRK2 and NTSR1 to enhance GRK2 binding. The binding mode of SBI-553 is compatible with arrestin binding but clashes with the binding of Gα protein, thus providing a mechanism for its arrestin-biased signalling capability. In sum, our structure provides a rational model for understanding the details of GPCR-GRK interactions and GRK2-mediated biased signalling.
History
DepositionJun 11, 2023-
Header (metadata) releaseAug 9, 2023-
Map releaseAug 9, 2023-
UpdateAug 30, 2023-
Current statusAug 30, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_36474.png

Downloads & links

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Map

FileDownload / File: emd_36474.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.824 Å
Density
Contour LevelBy AUTHOR: 0.3
Minimum - Maximum-2.2118118 - 3.351739
Average (Standard dev.)0.0028308015 (±0.062424373)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 263.68 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_36474_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_36474_half_map_1.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_36474_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : NTSR1-GRK2-Galpha(q) complexes 1

EntireName: NTSR1-GRK2-Galpha(q) complexes 1
Components
  • Complex: NTSR1-GRK2-Galpha(q) complexes 1
    • Protein or peptide: Beta-adrenergic receptor kinase 1G protein-coupled receptor kinase 2
    • Protein or peptide: NTS(8-13)
    • Protein or peptide: Guanine nucleotide-binding protein G(q) subunit alpha
    • Protein or peptide: Neurotensin receptor type 1
  • Ligand: STAUROSPORINE
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: TETRAFLUOROALUMINATE ION
  • Ligand: 2-[{2-(1-fluorocyclopropyl)-4-[4-(2-methoxyphenyl)piperidin-1-yl]quinazolin-6-yl}(methyl)amino]ethan-1-ol

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Supramolecule #1: NTSR1-GRK2-Galpha(q) complexes 1

SupramoleculeName: NTSR1-GRK2-Galpha(q) complexes 1 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Beta-adrenergic receptor kinase 1

MacromoleculeName: Beta-adrenergic receptor kinase 1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: beta-adrenergic-receptor kinase
Source (natural)Organism: Bos taurus (cattle)
Molecular weightTheoretical: 79.644727 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: ADLEAVLADV SYLMAMEKSK ATPAARASKK ILLPEPSIRS VMQKYLEDRG EVTFEKIFSQ KLGYLLFRDF CLKHLEEAKP LVEFYEEIK KYEKLETEEE RLVCSREIFD TYIMKELLAC SHPFSKSAIE HVQGHLVKKQ VPPDLFQPYI EEICQNLRGD V FQKFIESD ...String:
ADLEAVLADV SYLMAMEKSK ATPAARASKK ILLPEPSIRS VMQKYLEDRG EVTFEKIFSQ KLGYLLFRDF CLKHLEEAKP LVEFYEEIK KYEKLETEEE RLVCSREIFD TYIMKELLAC SHPFSKSAIE HVQGHLVKKQ VPPDLFQPYI EEICQNLRGD V FQKFIESD KFTRFCQWKN VELNIHLTMN DFSVHRIIGR GGFGEVYGCR KADTGKMYAM KCLDKKRIKM KQGETLALNE RI MLSLVST GDCPFIVCMS YAFHTPDKLS FILDLMNGGD LHYHLSQHGV FSEPDMIFYA AEIILGLEHM HNRFVVYRDL KPA NILLDE HGHVRISDLG LACDFSKKKP HASVGTHGYM APEVLQKGVA YDSSADWFSL GCMLFKLLRG HSPFRQHKTK DKHE IDRMT LTMAVELPDS FSPELRSLLE GLLQRDVNRR LGCLGRGAQE VKESPFFRDL DWQMVFLQKY PPPLIPPRGE VNAAD AFDI GSFDEEDTKG IKLLDSDQEL YRNFPLTISE RWQQEVAETV FDTINAETDR LEARKKTKNK QLGHEEDYAL GKDCIM HGY MSKMGNPFLT QWQRRYFYLF PNRLEWRGEG EAPQSLLTME EIQSVEETQI KERKCLLLKI RGGKQFVLQC DSDPELV QW KKELRDAYRE AQQLVQRVPK MKNKPRSPVV ELSKVPLIQR GSANGL

UniProtKB: Beta-adrenergic receptor kinase 1

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Macromolecule #2: NTS(8-13)

MacromoleculeName: NTS(8-13) / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 819.007 Da
SequenceString:
RRPYIL

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Macromolecule #3: Guanine nucleotide-binding protein G(q) subunit alpha

MacromoleculeName: Guanine nucleotide-binding protein G(q) subunit alpha / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 41.282895 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MGCTLSAEDK AAVERSKMID RNLREDGERS RRELKLLLLG TGESGKSTFI KQMRIIHGSG YSDEDKRGFT KLVYQNIFTA MQAMIRAMD TLKIPYKYEH NKAHAQLVRE VDVEKVSAFE NPYVDAIKSL WNDPGIQECY DRRREYQLSD STKYYLNDLD R VADPAYLP ...String:
MGCTLSAEDK AAVERSKMID RNLREDGERS RRELKLLLLG TGESGKSTFI KQMRIIHGSG YSDEDKRGFT KLVYQNIFTA MQAMIRAMD TLKIPYKYEH NKAHAQLVRE VDVEKVSAFE NPYVDAIKSL WNDPGIQECY DRRREYQLSD STKYYLNDLD R VADPAYLP TQQDVLRVRV PTTGIIEYPF DLQSVIFRMV DVGGQRSERR KWIHCFENVT SIMFLVALSE YDQVLVESDN EN RMEESKA LFRTIITYPW FQNSSVILFL NKKDLLEEKI MYSHLVDYFP EYDGPQRDAQ AAREFILKMF VDLNPDSDKI IYS HFTCAT DTENIRFVFA AVKDTILQLN LKEYNLV

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

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Macromolecule #4: Neurotensin receptor type 1

MacromoleculeName: Neurotensin receptor type 1 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 46.307594 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MRLNSSAPGT PGTPAADPFQ RAQAGLEEAL LAPGFGNASG NASERVLAAP SSELDVNTDI YSKVLVTAVY LALFVVGTVG NTVTAFTLA RKKSLQSLQS TVHYHLGSLA LSDLLTLLLA MPVELYNFIW VHHPWAFGDA GCRGYYFLRD ACTYATALNV A SLSVERYL ...String:
MRLNSSAPGT PGTPAADPFQ RAQAGLEEAL LAPGFGNASG NASERVLAAP SSELDVNTDI YSKVLVTAVY LALFVVGTVG NTVTAFTLA RKKSLQSLQS TVHYHLGSLA LSDLLTLLLA MPVELYNFIW VHHPWAFGDA GCRGYYFLRD ACTYATALNV A SLSVERYL AICHPFKAKT LMSRSRTKKF ISAIWLASAL LAVPMLFTMG EQNRSADGQH AGGLVCTPTI HTATVKVVIQ VN TFMSFIF PMVVISVLNT IIANKLTVMV RQAAEQGQVC TVGGEHSTFS MAIEPGRVQA LRHGVRVLRA VVIAFVVCWL PYH VRRLMF CYISDEQWTP FLYDFYHYFY MVTNALFYVS STINPILYNL VSANFRHIFL ATLACLCPVW RRRRKRPAFS RKAD SVSSN HTLSSNATRE TLY

UniProtKB: Neurotensin receptor type 1

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Macromolecule #5: STAUROSPORINE

MacromoleculeName: STAUROSPORINE / type: ligand / ID: 5 / Number of copies: 1 / Formula: STU
Molecular weightTheoretical: 466.531 Da
Chemical component information

ChemComp-STO:
STAUROSPORINE / anticancer, antifungal, antibiotic, alkaloid*YM / Staurosporine

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Macromolecule #6: GUANOSINE-5'-DIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-DIPHOSPHATE / type: ligand / ID: 6 / Number of copies: 1 / Formula: GDP
Molecular weightTheoretical: 443.201 Da
Chemical component information

ChemComp-GDP:
GUANOSINE-5'-DIPHOSPHATE / GDP, energy-carrying molecule*YM / Guanosine diphosphate

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Macromolecule #7: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 7 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #8: TETRAFLUOROALUMINATE ION

MacromoleculeName: TETRAFLUOROALUMINATE ION / type: ligand / ID: 8 / Number of copies: 1 / Formula: ALF
Molecular weightTheoretical: 102.975 Da
Chemical component information

ChemComp-ALF:
TETRAFLUOROALUMINATE ION

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Macromolecule #9: 2-[{2-(1-fluorocyclopropyl)-4-[4-(2-methoxyphenyl)piperidin-1-yl]...

MacromoleculeName: 2-[{2-(1-fluorocyclopropyl)-4-[4-(2-methoxyphenyl)piperidin-1-yl]quinazolin-6-yl}(methyl)amino]ethan-1-ol
type: ligand / ID: 9 / Number of copies: 1 / Formula: SRW
Molecular weightTheoretical: 450.548 Da
Chemical component information

ChemComp-SRW:
2-[{2-(1-fluorocyclopropyl)-4-[4-(2-methoxyphenyl)piperidin-1-yl]quinazolin-6-yl}(methyl)amino]ethan-1-ol

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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

BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: DARK FIELD / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.2 µm
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.07 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 474232

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