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- EMDB-60578: Cryo-EM structure of neurotensin receptor 1 in complex with beta-... -

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Entry
Database: EMDB / ID: EMD-60578
TitleCryo-EM structure of neurotensin receptor 1 in complex with beta-arrestin1 and SBI-553 (complex 3)
Map data
Sample
  • Complex: NTSR1-beta-arrestin1 complex
    • Protein or peptide: Beta-arrestin-1
    • Protein or peptide: Fab30 heavy chain
    • Protein or peptide: Fab30 light chain
    • Protein or peptide: Soluble cytochrome b562,Neurotensin receptor type 1
    • Protein or peptide: neurotensin peptide 8-13
  • Ligand: [(2R)-2-octanoyloxy-3-[oxidanyl-[(1R,2R,3S,4R,5R,6S)-2,3,6-tris(oxidanyl)-4,5-diphosphonooxy-cyclohexyl]oxy-phosphoryl]oxy-propyl] octanoate
  • Ligand: 2-[{2-(1-fluorocyclopropyl)-4-[4-(2-methoxyphenyl)piperidin-1-yl]quinazolin-6-yl}(methyl)amino]ethan-1-ol
Keywordsneurotensin receptor / beta-arrestin1 / phosphorylation / intracellular agonist / SBI-553 / MEMBRANE PROTEIN
Function / homology
Function and homology information


G protein-coupled neurotensin receptor activity / inositol phosphate catabolic process / angiotensin receptor binding / symmetric synapse / D-aspartate import across plasma membrane / TGFBR3 regulates TGF-beta signaling / positive regulation of gamma-aminobutyric acid secretion / Activation of SMO / regulation of membrane depolarization / negative regulation of interleukin-8 production ...G protein-coupled neurotensin receptor activity / inositol phosphate catabolic process / angiotensin receptor binding / symmetric synapse / D-aspartate import across plasma membrane / TGFBR3 regulates TGF-beta signaling / positive regulation of gamma-aminobutyric acid secretion / Activation of SMO / regulation of membrane depolarization / negative regulation of interleukin-8 production / positive regulation of arachidonate secretion / L-glutamate import across plasma membrane / regulation of respiratory gaseous exchange / positive regulation of inhibitory postsynaptic potential / G protein-coupled receptor internalization / arrestin family protein binding / negative regulation of systemic arterial blood pressure / positive regulation of glutamate secretion / negative regulation of release of sequestered calcium ion into cytosol / Lysosome Vesicle Biogenesis / positive regulation of inositol phosphate biosynthetic process / response to lipid / temperature homeostasis / Golgi Associated Vesicle Biogenesis / positive regulation of Rho protein signal transduction / stress fiber assembly / positive regulation of cardiac muscle hypertrophy / negative regulation of NF-kappaB transcription factor activity / pseudopodium / negative regulation of interleukin-6 production / detection of temperature stimulus involved in sensory perception of pain / enzyme inhibitor activity / positive regulation of receptor internalization / negative regulation of Notch signaling pathway / neuropeptide signaling pathway / clathrin-coated pit / insulin-like growth factor receptor binding / negative regulation of protein ubiquitination / cytoplasmic vesicle membrane / GTPase activator activity / Activated NOTCH1 Transmits Signal to the Nucleus / positive regulation of release of sequestered calcium ion into cytosol / dendritic shaft / Peptide ligand-binding receptors / adult locomotory behavior / learning / G protein-coupled receptor binding / electron transport chain / Signaling by high-kinase activity BRAF mutants / MAP2K and MAPK activation / terminal bouton / G protein-coupled receptor activity / cytoplasmic side of plasma membrane / Signaling by RAF1 mutants / Signaling by moderate kinase activity BRAF mutants / Paradoxical activation of RAF signaling by kinase inactive BRAF / Signaling downstream of RAS mutants / Signaling by BRAF and RAF1 fusions / endocytic vesicle membrane / protein transport / Cargo recognition for clathrin-mediated endocytosis / Thrombin signalling through proteinase activated receptors (PARs) / Clathrin-mediated endocytosis / positive regulation of protein phosphorylation / ubiquitin-dependent protein catabolic process / cytoplasmic vesicle / G alpha (s) signalling events / chemical synaptic transmission / molecular adaptor activity / G alpha (q) signalling events / dendritic spine / perikaryon / proteasome-mediated ubiquitin-dependent protein catabolic process / transcription coactivator activity / periplasmic space / electron transfer activity / positive regulation of ERK1 and ERK2 cascade / Ub-specific processing proteases / protein ubiquitination / nuclear body / G protein-coupled receptor signaling pathway / positive regulation of apoptotic process / iron ion binding / membrane raft / Golgi membrane / lysosomal membrane / heme binding / ubiquitin protein ligase binding / positive regulation of gene expression / regulation of transcription by RNA polymerase II / protein-containing complex binding / negative regulation of apoptotic process / chromatin / cell surface / endoplasmic reticulum / Golgi apparatus / signal transduction / positive regulation of transcription by RNA polymerase II / nucleoplasm / identical protein binding
Similarity search - Function
Neurotensin receptor / Neurotensin type 1 receptor / Arrestin, conserved site / Arrestins signature. / Arrestin / Arrestin, N-terminal / Arrestin-like, N-terminal / Arrestin C-terminal-like domain / Arrestin (or S-antigen), N-terminal domain / Arrestin (or S-antigen), C-terminal domain ...Neurotensin receptor / Neurotensin type 1 receptor / Arrestin, conserved site / Arrestins signature. / Arrestin / Arrestin, N-terminal / Arrestin-like, N-terminal / Arrestin C-terminal-like domain / Arrestin (or S-antigen), N-terminal domain / Arrestin (or S-antigen), C-terminal domain / Arrestin (or S-antigen), C-terminal domain / Arrestin-like, C-terminal / 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) / Immunoglobulin E-set
Similarity search - Domain/homology
Soluble cytochrome b562 / Neurotensin receptor type 1 / Beta-arrestin-1
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.65 Å
AuthorsSun D / Li X / Yuan Q / Yin W / Xu HE / Tian C
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)T2221005 China
CitationJournal: Cell Res / Year: 2025
Title: Molecular mechanism of the arrestin-biased agonism of neurotensin receptor 1 by an intracellular allosteric modulator.
Authors: Demeng Sun / Xiang Li / Qingning Yuan / Yuanxia Wang / Pan Shi / Huanhuan Zhang / Tao Wang / Wenjing Sun / Shenglong Ling / Yuanchun Liu / Jinglin Lai / Wenqin Xie / Wanchao Yin / Lei Liu / ...Authors: Demeng Sun / Xiang Li / Qingning Yuan / Yuanxia Wang / Pan Shi / Huanhuan Zhang / Tao Wang / Wenjing Sun / Shenglong Ling / Yuanchun Liu / Jinglin Lai / Wenqin Xie / Wanchao Yin / Lei Liu / H Eric Xu / Changlin Tian /
Abstract: Biased allosteric modulators (BAMs) of G protein-coupled receptors (GPCRs) have been at the forefront of drug discovery owing to their potential to selectively stimulate therapeutically relevant ...Biased allosteric modulators (BAMs) of G protein-coupled receptors (GPCRs) have been at the forefront of drug discovery owing to their potential to selectively stimulate therapeutically relevant signaling and avoid on-target side effects. Although structures of GPCRs in complex with G protein or GRK in a BAM-bound state have recently been resolved, revealing that BAM can induce biased signaling by directly modulating interactions between GPCRs and these two transducers, no BAM-bound GPCR-arrestin complex structure has yet been determined, limiting our understanding of the full pharmacological profile of BAMs. Herein, we developed a chemical protein synthesis strategy to generate neurotensin receptor 1 (NTSR1) with defined hexa-phosphorylation at its C-terminus and resolved high-resolution cryo-EM structures (2.65-2.88 Å) of NTSR1 in complex with both β-arrestin1 and the BAM SBI-553. These structures revealed a unique "loop engagement" configuration of β-arrestin1 coupling to NTSR1 in the presence of SBI-553, markedly different from the typical "core engagement" configuration observed in the absence of BAMs. This configuration is characterized by the engagement of the intracellular loop 3 of NTSR1 with a cavity in the central crest of β-arrestin1, representing a previously unobserved, arrestin-selective conformation of GPCR. Our findings fill the critical knowledge gap regarding the regulation of GPCR-arrestin interactions and biased signaling by BAMs, which would advance the development of safer and more efficacious GPCR-targeted therapeutics.
History
DepositionJun 18, 2024-
Header (metadata) releaseApr 9, 2025-
Map releaseApr 9, 2025-
UpdateMay 28, 2025-
Current statusMay 28, 2025Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_60578.png

Downloads & links

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Map

FileDownload / File: emd_60578.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

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AxesZ (Sec.)Y (Row.)X (Col.)
0.82 Å/pix.
x 360 pix.
= 296.64 Å		0.82 Å/pix.
x 360 pix.
= 296.64 Å		0.82 Å/pix.
x 360 pix.
= 296.64 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.824 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.143
Minimum - Maximum-2.2480078 - 3.560984
Average (Standard dev.)-0.00036568058 (±0.053710017)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 296.64 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_60578_half_map_1.map
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Half map: #2

Fileemd_60578_half_map_2.map
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Sample components

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Entire : NTSR1-beta-arrestin1 complex

EntireName: NTSR1-beta-arrestin1 complex
Components
  • Complex: NTSR1-beta-arrestin1 complex
    • Protein or peptide: Beta-arrestin-1
    • Protein or peptide: Fab30 heavy chain
    • Protein or peptide: Fab30 light chain
    • Protein or peptide: Soluble cytochrome b562,Neurotensin receptor type 1
    • Protein or peptide: neurotensin peptide 8-13
  • Ligand: [(2R)-2-octanoyloxy-3-[oxidanyl-[(1R,2R,3S,4R,5R,6S)-2,3,6-tris(oxidanyl)-4,5-diphosphonooxy-cyclohexyl]oxy-phosphoryl]oxy-propyl] octanoate
  • 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-beta-arrestin1 complex

SupramoleculeName: NTSR1-beta-arrestin1 complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Details: NTSR1 in complex with beat-arrestin1 and the arrestin-biased intracellular agonist SBI-553
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Beta-arrestin-1

MacromoleculeName: Beta-arrestin-1 / type: protein_or_peptide / ID: 1
Details: Human beta-arrestin1 was truncated at residues 382 to remove autoinhibition and added His tag at its N-terminus.
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 45.224598 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MGSHHHHHHH HGSLEVLFQG PGDKGTRVFK KASPNGKLTV YLGKRDFVDH IDLVDPVDGV VLVDPEYLKE RRVYVTLTCA FRYGREDLD VLGLTFRKDL FVANVQSFPP APEDKKPLTR LQERLIKKLG EHAYPFTFEI PPNLPCSVTL QPGPEDTGKA C GVDYEVKA ...String:
MGSHHHHHHH HGSLEVLFQG PGDKGTRVFK KASPNGKLTV YLGKRDFVDH IDLVDPVDGV VLVDPEYLKE RRVYVTLTCA FRYGREDLD VLGLTFRKDL FVANVQSFPP APEDKKPLTR LQERLIKKLG EHAYPFTFEI PPNLPCSVTL QPGPEDTGKA C GVDYEVKA FCAENLEEKI HKRNSVRLVI RKVQYAPERP GPQPTAETTR QFLMSDKPLH LEASLDKEIY YHGEPISVNV HV TNNTNKT VKKIKISVRQ YADICLFNTA QYKCPVAMEE ADDTVAPSST FCKVYTLTPF LANNREKRGL ALDGKLKHED TNL ASSTLL REGANREILG IIVSYKVKVK LVVSRGGLLG DLASSDVAVE LPFTLMHPKP KEEPPHREVP ENETPVDTNL IELD TN

UniProtKB: Beta-arrestin-1

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Macromolecule #2: Fab30 heavy chain

MacromoleculeName: Fab30 heavy chain / type: protein_or_peptide / ID: 2
Details: The heavy chain of antibody fragment Fab30 wasmodified with a GP64 secretion signal peptide at the N-terminus, and His tag at the C-terminus.
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 27.72701 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MVSAIVLYVL LAAAAHSAFA EISEVQLVES GGGLVQPGGS LRLSCAASGF NVYSSSIHWV RQAPGKGLEW VASISSYYGY TYYADSVKG RFTISADTSK NTAYLQMNSL RAEDTAVYYC ARSRQFWYSG LDYWGQGTLV TVSSASTKGP SVFPLAPSSK S TSGGTAAL ...String:
MVSAIVLYVL LAAAAHSAFA EISEVQLVES GGGLVQPGGS LRLSCAASGF NVYSSSIHWV RQAPGKGLEW VASISSYYGY TYYADSVKG RFTISADTSK NTAYLQMNSL RAEDTAVYYC ARSRQFWYSG LDYWGQGTLV TVSSASTKGP SVFPLAPSSK S TSGGTAAL GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA VLQSSGLYSL SSVVTVPSSS LGTQTYICNV NHKPSNTKVD KK VEPKSCD KTAAAHHHHH HHH

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Macromolecule #3: Fab30 light chain

MacromoleculeName: Fab30 light chain / type: protein_or_peptide / ID: 3
Details: The heavy chain of antibody fragment Fab30 wasmodified with a GP64 secretion signal peptide at the N-terminus
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 25.436492 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MVSAIVLYVL LAAAAHSAFA SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSG TDFTLTISSL QPEDFATYYC QQYKYVPVTF GQGTKVEIKR TVAAPSVFIF PPSDSQLKSG TASVVCLLNN F YPREAKVQ ...String:
MVSAIVLYVL LAAAAHSAFA SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSG TDFTLTISSL QPEDFATYYC QQYKYVPVTF GQGTKVEIKR TVAAPSVFIF PPSDSQLKSG TASVVCLLNN F YPREAKVQ WKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC

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Macromolecule #4: Soluble cytochrome b562,Neurotensin receptor type 1

MacromoleculeName: Soluble cytochrome b562,Neurotensin receptor type 1 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 62.097848 KDa
Recombinant expressionOrganism: Mammalian expression vector Flag-MCS-pcDNA3.1 (others)
SequenceString: MKTIIALSYI FCLVFAGSAD LEDNWETLND NLKVIEKADN AAQVKDALTK MRAAALDAQK ATPPKLEDKS PDSPEMKDFR HGFDILVGQ IDDALKLANE GKVKEAQAAA EQLKTTRNAY IQKYLASGSL EVLFQGPMRL NSSAPGTPGT PAADPFQRAQ A GLEEALLA ...String:
MKTIIALSYI FCLVFAGSAD LEDNWETLND NLKVIEKADN AAQVKDALTK MRAAALDAQK ATPPKLEDKS PDSPEMKDFR HGFDILVGQ IDDALKLANE GKVKEAQAAA EQLKTTRNAY IQKYLASGSL EVLFQGPMRL NSSAPGTPGT PAADPFQRAQ A GLEEALLA PGFGNASGNA SERVLAAPSS ELDVNTDIYS KVLVTAVYLA LFVVGTVGNT VTAFTLARKK SLQSLQSTVH YH LGSLALS DLLTLLLAMP VELYNFIWVH HPWAFGDAGC RGYYFLRDAC TYATALNVAS LSVERYLALC HPFKAKTLMS RSR TKKFIS AIWLASALLA VPMLFTMGEQ NRSADGQHAG GLVCTPTIHT ATVKVVIQVN TFMSFIFPMV VISVLNTIIA NKLT VMVRQ AAEQGQVCTV GGEHSTFSMA IEPGRVQALR HGVRVLRAVV IAFVVCWLPY HVRRLMFCYI SDEQWTPFLY DFYHY FYMV TNALFYVSST INPILYNLVS ANFRHIFIAT LACLCPVWRR RRKRPCFNAF SRKCD(SEP)V(SEP)(SEP)N H (TPO)L(SEP)(SEP)NATR ETLY

UniProtKB: Soluble cytochrome b562, Neurotensin receptor type 1

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Macromolecule #5: neurotensin peptide 8-13

MacromoleculeName: neurotensin peptide 8-13 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 819.007 Da
SequenceString:
RRPYIL

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Macromolecule #6: [(2R)-2-octanoyloxy-3-[oxidanyl-[(1R,2R,3S,4R,5R,6S)-2,3,6-tris(o...

MacromoleculeName: [(2R)-2-octanoyloxy-3-[oxidanyl-[(1R,2R,3S,4R,5R,6S)-2,3,6-tris(oxidanyl)-4,5-diphosphonooxy-cyclohexyl]oxy-phosphoryl]oxy-propyl] octanoate
type: ligand / ID: 6 / Number of copies: 1 / Formula: PIO
Molecular weightTheoretical: 746.566 Da
Chemical component information

ChemComp-PIO:
[(2R)-2-octanoyloxy-3-[oxidanyl-[(1R,2R,3S,4R,5R,6S)-2,3,6-tris(oxidanyl)-4,5-diphosphonooxy-cyclohexyl]oxy-phosphoryl]oxy-propyl] octanoate

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Macromolecule #7: 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: 7 / 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

Concentration1.0 mg/mL
BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.65 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 5006335
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION

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