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- PDB-8fn1: CryoEM structure of Go-coupled NTSR1 -

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Basic information

Entry
Database: PDB / ID: 8fn1
TitleCryoEM structure of Go-coupled NTSR1
Components
  • (Guanine nucleotide-binding protein ...) x 3
  • Neurotensin receptor type 1
  • Neurotensin/neuromedin N
  • scFv16
KeywordsMEMBRANE PROTEIN / GPCR / neurotensin receptor / allosterism / SBI-553
Function / homology
Function and homology information


response to antipsychotic drug / Peptide ligand-binding receptors / neuropeptide receptor binding / G protein-coupled neurotensin receptor activity / inositol phosphate catabolic process / positive regulation of inhibitory postsynaptic potential / symmetric synapse / response to mineralocorticoid / D-aspartate import across plasma membrane / positive regulation of gamma-aminobutyric acid secretion ...response to antipsychotic drug / Peptide ligand-binding receptors / neuropeptide receptor binding / G protein-coupled neurotensin receptor activity / inositol phosphate catabolic process / positive regulation of inhibitory postsynaptic potential / symmetric synapse / response to mineralocorticoid / D-aspartate import across plasma membrane / positive regulation of gamma-aminobutyric acid secretion / L-glutamate import across plasma membrane / mu-type opioid receptor binding / positive regulation of arachidonic acid secretion / neuron spine / corticotropin-releasing hormone receptor 1 binding / regulation of respiratory gaseous exchange / neuropeptide hormone activity / digestive tract development / negative regulation of systemic arterial blood pressure / hyperosmotic response / negative regulation of release of sequestered calcium ion into cytosol / G alpha (q) signalling events / positive regulation of glutamate secretion / positive regulation of inositol phosphate biosynthetic process / temperature homeostasis / dopamine receptor signaling pathway / response to corticosterone / response to lipid / regulation of membrane depolarization / cellular response to lithium ion / detection of temperature stimulus involved in sensory perception of pain / neuropeptide signaling pathway / response to axon injury / G protein-coupled serotonin receptor binding / axon terminus / transport vesicle / response to amphetamine / blood vessel diameter maintenance / cellular response to dexamethasone stimulus / adult locomotory behavior / cellular response to nerve growth factor stimulus / positive regulation of release of sequestered calcium ion into cytosol / dendritic shaft / response to cocaine / liver development / muscle contraction / learning / G-protein beta/gamma-subunit complex binding / Olfactory Signaling Pathway / Activation of the phototransduction cascade / visual learning / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Thromboxane signalling through TP receptor / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / G-protein activation / G protein-coupled acetylcholine receptor signaling pathway / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / Prostacyclin signalling through prostacyclin receptor / Glucagon signaling in metabolic regulation / G beta:gamma signalling through CDC42 / terminal bouton / cytoplasmic side of plasma membrane / ADP signalling through P2Y purinoceptor 12 / G beta:gamma signalling through BTK / Sensory perception of sweet, bitter, and umami (glutamate) taste / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / photoreceptor disc membrane / Adrenaline,noradrenaline inhibits insulin secretion / Glucagon-type ligand receptors / Vasopressin regulates renal water homeostasis via Aquaporins / G alpha (z) signalling events / cellular response to catecholamine stimulus / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / ADORA2B mediated anti-inflammatory cytokines production / adenylate cyclase-activating dopamine receptor signaling pathway / ADP signalling through P2Y purinoceptor 1 / G beta:gamma signalling through PI3Kgamma / cellular response to prostaglandin E stimulus / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / sensory perception of taste / GPER1 signaling / G-protein beta-subunit binding / heterotrimeric G-protein complex / Inactivation, recovery and regulation of the phototransduction cascade / extracellular vesicle / G alpha (12/13) signalling events / signaling receptor complex adaptor activity / Thrombin signalling through proteinase activated receptors (PARs) / response to estradiol / retina development in camera-type eye / GTPase binding / Ca2+ pathway / phospholipase C-activating G protein-coupled receptor signaling pathway / G alpha (i) signalling events / fibroblast proliferation / perikaryon / G alpha (s) signalling events / G alpha (q) signalling events
Similarity search - Function
Neurotensin/neuromedin N / Neurotensin/neuromedin N precursor / Neurotensin receptor / Neurotensin type 1 receptor / G-protein alpha subunit, group I / 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 ...Neurotensin/neuromedin N / Neurotensin/neuromedin N precursor / Neurotensin receptor / Neurotensin type 1 receptor / G-protein alpha subunit, group I / 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 subunit / G-protein gamma subunit domain profile. / GGL domain / G-protein gamma-like domain superfamily / 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 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) / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Guanine nucleotide-binding protein G(o) subunit alpha / Neurotensin/neuromedin N / Neurotensin receptor type 1 / Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
Spodoptera frugiperda (fall armyworm)
Homo sapiens (human)
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.88 Å
AuthorsKrumm, B.E. / DiBerto, J.F. / Olsen, R.H.J. / Kang, H. / Slocum, S.T. / Zhang, S. / Strachan, R.T. / Fay, J.F. / Roth, B.L.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)0000-0002-0561-6520 United States
CitationJournal: Biochemistry / Year: 2023
Title: Neurotensin Receptor Allosterism Revealed in Complex with a Biased Allosteric Modulator.
Authors: Brian E Krumm / Jeffrey F DiBerto / Reid H J Olsen / Hye Jin Kang / Samuel T Slocum / Shicheng Zhang / Ryan T Strachan / Xi-Ping Huang / Lauren M Slosky / Anthony B Pinkerton / Lawrence S ...Authors: Brian E Krumm / Jeffrey F DiBerto / Reid H J Olsen / Hye Jin Kang / Samuel T Slocum / Shicheng Zhang / Ryan T Strachan / Xi-Ping Huang / Lauren M Slosky / Anthony B Pinkerton / Lawrence S Barak / Marc G Caron / Terry Kenakin / Jonathan F Fay / Bryan L Roth /
Abstract: The NTSR1 neurotensin receptor (NTSR1) is a G protein-coupled receptor (GPCR) found in the brain and peripheral tissues with neurotensin (NTS) being its endogenous peptide ligand. In the brain, NTS ...The NTSR1 neurotensin receptor (NTSR1) is a G protein-coupled receptor (GPCR) found in the brain and peripheral tissues with neurotensin (NTS) being its endogenous peptide ligand. In the brain, NTS modulates dopamine neuronal activity, induces opioid-independent analgesia, and regulates food intake. Recent studies indicate that biasing NTSR1 toward β-arrestin signaling can attenuate the actions of psychostimulants and other drugs of abuse. Here, we provide the cryoEM structures of NTSR1 ternary complexes with heterotrimeric Gq and GoA with and without the brain-penetrant small-molecule SBI-553. In functional studies, we discovered that SBI-553 displays complex allosteric actions exemplified by negative allosteric modulation for G proteins that are Gα subunit selective and positive allosteric modulation and agonism for β-arrestin translocation at NTSR1. Detailed structural analysis of the allosteric binding site illuminated the structural determinants for biased allosteric modulation of SBI-553 on NTSR1.
History
DepositionDec 26, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 29, 2023Provider: repository / Type: Initial release
Revision 1.1Apr 19, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
F: Neurotensin/neuromedin N
A: Neurotensin receptor type 1
B: Guanine nucleotide-binding protein G(o) subunit alpha
C: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
D: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2
E: scFv16


Theoretical massNumber of molelcules
Total (without water)148,0606
Polymers148,0606
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Guanine nucleotide-binding protein ... , 3 types, 3 molecules BCD

#3: Protein Guanine nucleotide-binding protein G(o) subunit alpha


Mass: 25451.166 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Spodoptera frugiperda (fall armyworm), (gene. exp.) Homo sapiens (human)
Gene: GNAO1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P09471
#4: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Transducin beta chain 1


Mass: 39418.086 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNB1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P62873
#5: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / G gamma-I


Mass: 7861.143 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GNG2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P59768

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Protein/peptide / Protein / Antibody , 3 types, 3 molecules FAE

#1: Protein/peptide Neurotensin/neuromedin N


Mass: 819.007 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Rattus norvegicus (Norway rat) / References: UniProt: P20068
#2: Protein Neurotensin receptor type 1 / NT-R-1 / NTR1 / High-affinity levocabastine-insensitive neurotensin receptor / NTRH


Mass: 45842.098 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Ntsr1, Ntsr / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P20789
#6: Antibody scFv16


Mass: 28668.922 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Spodoptera frugiperda (fall armyworm)

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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: Ternary complex of NTSR1 MiniGo heterotrimer with scFv16
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.147 MDa / Experimental value: NO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5 / Details: 20mM Hepes, 0.1M NaCl, 0.00075% LMNG, 0.00075% GDN
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3200 nm / Nominal defocus min: 100 nm
Image recordingElectron dose: 44 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.88 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 560249 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0038905
ELECTRON MICROSCOPYf_angle_d0.42912071
ELECTRON MICROSCOPYf_dihedral_angle_d9.593173
ELECTRON MICROSCOPYf_chiral_restr0.0391377
ELECTRON MICROSCOPYf_plane_restr0.0031524

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