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- PDB-8zyy: Cryo-EM structure of neurotensin receptor 1 in complex with beta-... -

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Basic information

Entry
Database: PDB / ID: 8zyy
TitleCryo-EM structure of neurotensin receptor 1 in complex with beta-arrestin1 and SBI-553 (complex 2)
Components
  • Beta-arrestin-1
  • Fab30 heavy chain
  • Fab30 light chain
  • Soluble cytochrome b562,Neurotensin receptor type 1
  • neurotensin peptide 8-13
KeywordsMEMBRANE PROTEIN / neurotensin receptor / beta-arrestin1 / phosphorylation / intracellular agonist / SBI-553
Function / homology
Function and homology information


G protein-coupled neurotensin receptor activity / inositol phosphate catabolic process / symmetric synapse / angiotensin receptor binding / D-aspartate import across plasma membrane / positive regulation of gamma-aminobutyric acid secretion / TGFBR3 regulates TGF-beta signaling / Activation of SMO / regulation of membrane depolarization / positive regulation of arachidonate secretion ...G protein-coupled neurotensin receptor activity / inositol phosphate catabolic process / symmetric synapse / angiotensin receptor binding / D-aspartate import across plasma membrane / positive regulation of gamma-aminobutyric acid secretion / TGFBR3 regulates TGF-beta signaling / Activation of SMO / regulation of membrane depolarization / positive regulation of arachidonate secretion / negative regulation of interleukin-8 production / 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 / negative regulation of release of sequestered calcium ion into cytosol / positive regulation of glutamate secretion / Lysosome Vesicle Biogenesis / negative regulation of NF-kappaB transcription factor activity / temperature homeostasis / positive regulation of cardiac muscle hypertrophy / stress fiber assembly / Golgi Associated Vesicle Biogenesis / response to lipid / positive regulation of inositol phosphate biosynthetic process / positive regulation of Rho protein signal transduction / pseudopodium / detection of temperature stimulus involved in sensory perception of pain / negative regulation of interleukin-6 production / positive regulation of receptor internalization / negative regulation of Notch signaling pathway / enzyme inhibitor activity / neuropeptide signaling pathway / insulin-like growth factor receptor binding / clathrin-coated pit / negative regulation of protein ubiquitination / GTPase activator activity / cytoplasmic vesicle membrane / Activated NOTCH1 Transmits Signal to the Nucleus / Peptide ligand-binding receptors / positive regulation of release of sequestered calcium ion into cytosol / dendritic shaft / adult locomotory behavior / learning / electron transport chain / G protein-coupled receptor binding / Signaling by high-kinase activity BRAF mutants / G protein-coupled receptor activity / MAP2K and MAPK activation / cytoplasmic side of plasma membrane / positive regulation of protein phosphorylation / terminal bouton / 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 / endocytic vesicle membrane / Signaling by BRAF and RAF1 fusions / Cargo recognition for clathrin-mediated endocytosis / protein transport / Clathrin-mediated endocytosis / Thrombin signalling through proteinase activated receptors (PARs) / cytoplasmic vesicle / ubiquitin-dependent protein catabolic process / perikaryon / G alpha (s) signalling events / G alpha (q) signalling events / chemical synaptic transmission / molecular adaptor activity / dendritic spine / 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 / positive regulation of apoptotic process / G protein-coupled receptor signaling pathway / membrane raft / iron ion binding / Golgi membrane / lysosomal membrane / heme binding / ubiquitin protein ligase binding / positive regulation of gene expression / regulation of transcription by RNA polymerase II / negative regulation of apoptotic process / chromatin / protein-containing complex binding / 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
Chem-PIO / Chem-SRW / Soluble cytochrome b562 / Neurotensin receptor type 1 / Beta-arrestin-1
Similarity search - Component
Biological speciesHomo sapiens (human)
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.83 Å
AuthorsSun, D. / Li, X. / Yuan, Q. / Yin, W. / Xu, H.E. / Tian, C.
Funding support China, 1items
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, 2024Deposition site: PDBJ / Processing site: PDBC
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: Beta-arrestin-1
D: Fab30 heavy chain
L: neurotensin peptide 8-13
Q: Fab30 light chain
R: Soluble cytochrome b562,Neurotensin receptor type 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)162,5027
Polymers161,3055
Non-polymers1,1972
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 2 molecules BR

#1: Protein Beta-arrestin-1 / Arrestin beta-1 / Non-visual arrestin-2


Mass: 45224.598 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ARRB1, ARR1 / Production host: Escherichia coli (E. coli) / References: UniProt: P49407
#5: Protein Soluble cytochrome b562,Neurotensin receptor type 1 / Cytochrome b-562 / NT-R-1 / NTR1 / High-affinity levocabastine-insensitive neurotensin receptor / NTRH


Mass: 62097.848 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli), (gene. exp.) Homo sapiens (human)
Gene: cybC, NTSR1, NTRR
Production host: Mammalian expression vector Flag-MCS-pcDNA3.1 (others)
References: UniProt: P0ABE7, UniProt: P30989

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Protein/peptide , 1 types, 1 molecules L

#3: Protein/peptide neurotensin peptide 8-13


Mass: 819.007 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)

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Antibody , 2 types, 2 molecules DQ

#2: Antibody Fab30 heavy chain


Mass: 27727.010 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Spodoptera frugiperda (fall armyworm)
#4: Antibody Fab30 light chain


Mass: 25436.492 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Spodoptera frugiperda (fall armyworm)

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Non-polymers , 2 types, 2 molecules

#6: Chemical 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 / dioctanoyl l-alpha-phosphatidyl-d-myo-inositol 4,5-diphosphate


Mass: 746.566 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C25H49O19P3
#7: Chemical ChemComp-SRW / 2-[{2-(1-fluorocyclopropyl)-4-[4-(2-methoxyphenyl)piperidin-1-yl]quinazolin-6-yl}(methyl)amino]ethan-1-ol


Mass: 450.548 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C26H31FN4O2 / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY
Has protein modificationY

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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: NTSR1-beta-arrestin1 complex / Type: COMPLEX
Details: NTSR1 in complex with beat-arrestin1 and the arrestin-biased intracellular agonist SBI-553
Entity ID: #1-#5 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Mammalian expression vector Flag-MCS-pcDNA3.1 (others)
Cell: HEK293
Buffer solutionpH: 7.4
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.83 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 148610 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0048405
ELECTRON MICROSCOPYf_angle_d0.63111499
ELECTRON MICROSCOPYf_dihedral_angle_d7.7841224
ELECTRON MICROSCOPYf_chiral_restr0.0421359
ELECTRON MICROSCOPYf_plane_restr0.0061448

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