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- PDB-9dnm: Structure of rat beta-arrestin 1 bound to allosteric inhibitor -

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Basic information

Entry
Database: PDB / ID: 9dnm
TitleStructure of rat beta-arrestin 1 bound to allosteric inhibitor
Components
  • Beta-arrestin-1,Soluble cytochrome b562
  • anti-BRIL Fab Heavy chain
  • anti-BRIL Fab Light chain
KeywordsSIGNALING PROTEIN/Immune System / GPCR signaling / arrestin / allostery / SIGNALING PROTEIN / SIGNALING PROTEIN-Immune System complex
Function / homology
Function and homology information


V2 vasopressin receptor binding / regulation of inositol trisphosphate biosynthetic process / alpha-1A adrenergic receptor binding / follicle-stimulating hormone receptor binding / TGFBR3 regulates TGF-beta signaling / sensory perception of touch / G alpha (s) signalling events / alpha-1B adrenergic receptor binding / follicle-stimulating hormone signaling pathway / protein phosphorylated amino acid binding ...V2 vasopressin receptor binding / regulation of inositol trisphosphate biosynthetic process / alpha-1A adrenergic receptor binding / follicle-stimulating hormone receptor binding / TGFBR3 regulates TGF-beta signaling / sensory perception of touch / G alpha (s) signalling events / alpha-1B adrenergic receptor binding / follicle-stimulating hormone signaling pathway / protein phosphorylated amino acid binding / angiotensin receptor binding / Lysosome Vesicle Biogenesis / Golgi Associated Vesicle Biogenesis / AP-2 adaptor complex binding / Ub-specific processing proteases / MAP2K and MAPK activation / positive regulation of smooth muscle cell apoptotic process / Cargo recognition for clathrin-mediated endocytosis / clathrin adaptor activity / negative regulation of interleukin-8 production / Clathrin-mediated endocytosis / regulation of G protein-coupled receptor signaling pathway / G protein-coupled receptor internalization / arrestin family protein binding / Thrombin signalling through proteinase activated receptors (PARs) / mitogen-activated protein kinase kinase binding / response to morphine / clathrin binding / positive regulation of Rho protein signal transduction / stress fiber assembly / pseudopodium / negative regulation of interleukin-6 production / cysteine-type endopeptidase inhibitor activity involved in apoptotic process / positive regulation of insulin secretion involved in cellular response to glucose stimulus / positive regulation of receptor internalization / negative regulation of Notch signaling pathway / phototransduction / clathrin-coated pit / insulin-like growth factor receptor binding / negative regulation of protein ubiquitination / GTPase activator activity / visual perception / positive regulation of protein ubiquitination / nuclear estrogen receptor binding / phosphoprotein binding / G protein-coupled receptor binding / electron transport chain / negative regulation of ERK1 and ERK2 cascade / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / endocytosis / protein transport / ubiquitin-dependent protein catabolic process / cytoplasmic vesicle / regulation of apoptotic process / basolateral plasma membrane / proteasome-mediated ubiquitin-dependent protein catabolic process / negative regulation of neuron apoptotic process / postsynaptic membrane / transmembrane transporter binding / dendritic spine / transcription coactivator activity / positive regulation of MAPK cascade / positive regulation of ERK1 and ERK2 cascade / electron transfer activity / periplasmic space / endosome / postsynaptic density / positive regulation of protein phosphorylation / protein ubiquitination / iron ion binding / response to xenobiotic stimulus / G protein-coupled receptor signaling pathway / signaling receptor binding / ubiquitin protein ligase binding / positive regulation of cell population proliferation / heme binding / regulation of DNA-templated transcription / regulation of transcription by RNA polymerase II / chromatin / negative regulation of apoptotic process / glutamatergic synapse / enzyme binding / positive regulation of transcription by RNA polymerase II / nucleus / plasma membrane / cytosol / cytoplasm
Similarity search - Function
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 ...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 / Immunoglobulin E-set
Similarity search - Domain/homology
Chem-ODN / Soluble cytochrome b562 / Beta-arrestin-1
Similarity search - Component
Biological speciesHomo sapiens (human)
Rattus norvegicus (Norway rat)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.47 Å
AuthorsPakharukova, N. / Kahsai, A.W. / Masoudi, A. / Lefkowitz, R.J.
Funding support United States, France, European Union, 6items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)HL016037 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)T32HL007101 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)HL16037-45S1 United States
Human Frontier Science Program (HFSP)LT000174/2018 France
European Molecular Biology Organization (EMBO)ALTF 1071-2017European Union
CitationJournal: bioRxiv / Year: 2024
Title: Small Molecule Modulators of β-arrestins.
Authors: Alem W Kahsai / Natalia Pakharukova / Henry Y Kwon / Kunal S Shah / Jason G Liang-Lin / Caroline T Del Real / Paul J Shim / Mason A Lee / Van A Ngo / Bowie N Shreiber / Samuel Liu / Allison ...Authors: Alem W Kahsai / Natalia Pakharukova / Henry Y Kwon / Kunal S Shah / Jason G Liang-Lin / Caroline T Del Real / Paul J Shim / Mason A Lee / Van A Ngo / Bowie N Shreiber / Samuel Liu / Allison M Schwalb / Emmanuel F Espinoza / Brittany N Thomas / Cal A Kunzle / Jeffrey S Smith / Jialu Wang / Jihee Kim / Xingdong Zhang / Howard A Rockman / Alex R B Thomsen / Lindsay A M Rein / Lei Shi / Seungkirl Ahn / Ali Masoudi / Robert J Lefkowitz
Abstract: β-arrestins (βarrs) are key regulators of G protein-coupled receptors (GPCRs), essential for modulating signaling pathways and physiological processes. While current pharmacological strategies ...β-arrestins (βarrs) are key regulators of G protein-coupled receptors (GPCRs), essential for modulating signaling pathways and physiological processes. While current pharmacological strategies target GPCR orthosteric and allosteric sites, as well as G protein transducers, comparable tools for studying βarrs are lacking. Here, we present the discovery and characterization of novel small-molecule allosteric inhibitors of βarrs through comprehensive biophysical, biochemical, pharmacological, and structural analyses. These inhibitors disrupt βarr interactions with agonist-activated GPCRs, impairing receptor internalization, desensitization, and βarr-mediated physiological functions. A cryo-EM structure of βarr1 in complex with the allosteric inhibitor Cmpd-5, complemented by molecular dynamics simulations and mutagenesis studies, reveals that Cmpd-5 binds within a cryptic cleft formed by the middle, C-, and lariat loops-a critical site for βarr activation and recruitment to GPCRs. Thus, Cmpd-5 acts as a molecular lock, hindering βarr1 activation via an allosteric mechanism. These findings introduce novel strategies and tools for probing βarr functions.
HIGHLIGHTS: Small molecule strategies for modulating βarr functions in both GPCR-dependent and independent contexts.Modulators disrupt βarr interaction with GPCRs, impairing their critical ...HIGHLIGHTS: Small molecule strategies for modulating βarr functions in both GPCR-dependent and independent contexts.Modulators disrupt βarr interaction with GPCRs, impairing their critical functions.Cryo-EM structures reveal the allosteric inhibitor Cmpd-5 binding to a cryptic pocket between the N and C domains in the central crest of βarr1, inhibiting its activation.Structural analyses, including cryo-EM, MD simulations, and mutagenesis, reveal a unique βarr1 conformation induced by Cmpd-5, shedding light on its mechanism of allosteric inhibition.
History
DepositionSep 17, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 22, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
H: anti-BRIL Fab Heavy chain
L: anti-BRIL Fab Light chain
A: Beta-arrestin-1,Soluble cytochrome b562
hetero molecules


Theoretical massNumber of molelcules
Total (without water)103,2084
Polymers102,8423
Non-polymers3661
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Antibody anti-BRIL Fab Heavy chain


Mass: 23904.656 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
#2: Antibody anti-BRIL Fab Light chain


Mass: 23209.820 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
#3: Protein Beta-arrestin-1,Soluble cytochrome b562 / Arrestin beta-1 / Cytochrome b-562


Mass: 55727.160 Da / Num. of mol.: 1 / Mutation: M29W, H124I, R128L
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Arrb1, cybC / Production host: Escherichia coli (E. coli) / References: UniProt: P29066, UniProt: P0ABE7
#4: Chemical ChemComp-ODN / (1beta,6beta,7beta,8alpha,9beta,10alpha,13alpha,14R,16beta)-1,6,7,14-tetrahydroxy-7,20-epoxykauran-15-one


Mass: 366.449 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C20H30O6 / Feature type: SUBJECT OF INVESTIGATION
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: Beta-arrestin 1 with insertion of soluble cytochrome b562 bound to anti-BRIL Fab
Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Source (natural)Organism: Rattus norvegicus (Norway rat)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenConc.: 7.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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
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: 2400 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 57 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
4cryoSPARC4.4.1CTF correction
7Coot0.9.8.3model fitting
8ISOLDEmodel fitting
10PHENIX1.20.1-4487model refinement
14cryoSPARC4.4.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.47 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 177430 / Symmetry type: POINT
Atomic model building

3D fitting-ID: 1 / Source name: PDB / Type: experimental model

IDPDB-IDPdb chain-IDAccession codeChain-IDInitial refinement model-IDDetails
11G4M

1g4m

A1G4MA1
26WW2

6ww2

L6WW2L2
36WW2

6ww2

H6WW2H2
46WW2

6ww2

A6WW2A2soluble cytochrome b562 insert

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