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- EMDB-47042: Structure of rat beta-arrestin 1 bound to allosteric inhibitor -

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Basic information

Entry
Database: EMDB / ID: EMD-47042
TitleStructure of rat beta-arrestin 1 bound to allosteric inhibitor
Map dataMap sharpened with DeepEMhancer
Sample
  • Complex: Beta-arrestin 1 with insertion of soluble cytochrome b562 bound to anti-BRIL Fab
    • Protein or peptide: anti-BRIL Fab Heavy chain
    • Protein or peptide: anti-BRIL Fab Light chain
    • Protein or peptide: Beta-arrestin-1,Soluble cytochrome b562
  • Ligand: (1beta,6beta,7beta,8alpha,9beta,10alpha,13alpha,14R,16beta)-1,6,7,14-tetrahydroxy-7,20-epoxykauran-15-one
KeywordsGPCR 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 / Lysosome Vesicle Biogenesis / angiotensin receptor binding / Golgi Associated Vesicle Biogenesis / AP-2 adaptor complex binding / Ub-specific processing proteases / MAP2K and MAPK activation / 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 / cysteine-type endopeptidase inhibitor activity involved in apoptotic process / Thrombin signalling through proteinase activated receptors (PARs) / response to morphine / mitogen-activated protein kinase kinase binding / clathrin binding / positive regulation of Rho protein signal transduction / stress fiber assembly / pseudopodium / negative regulation of interleukin-6 production / 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 / 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 / positive regulation of protein phosphorylation / ubiquitin-dependent protein catabolic process / cytoplasmic vesicle / basolateral plasma membrane / regulation of apoptotic process / dendritic spine / negative regulation of neuron apoptotic process / proteasome-mediated ubiquitin-dependent protein catabolic process / transmembrane transporter binding / postsynaptic membrane / transcription coactivator activity / periplasmic space / electron transfer activity / positive regulation of ERK1 and ERK2 cascade / positive regulation of MAPK cascade / postsynaptic density / endosome / protein ubiquitination / G protein-coupled receptor signaling pathway / iron ion binding / response to xenobiotic stimulus / signaling receptor binding / positive regulation of cell population proliferation / heme binding / ubiquitin protein ligase binding / regulation of DNA-templated transcription / regulation of transcription by RNA polymerase II / negative regulation of apoptotic process / chromatin / 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
Soluble cytochrome b562 / Beta-arrestin-1
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.47 Å
AuthorsPakharukova N / Kahsai AW / Masoudi A / Lefkowitz RJ
Funding support United States, France, European Union, 6 items
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, 2024-
Header (metadata) releaseJan 22, 2025-
Map releaseJan 22, 2025-
UpdateJan 22, 2025-
Current statusJan 22, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_47042.png

Downloads & links

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Map

FileDownload / File: emd_47042.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap sharpened with DeepEMhancer
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.38 Å/pix.
x 300 pix.
= 414.72 Å		1.38 Å/pix.
x 300 pix.
= 414.72 Å		1.38 Å/pix.
x 300 pix.
= 414.72 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.3824 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.04
Minimum - Maximum-0.014654089 - 1.9611895
Average (Standard dev.)0.00028479786 (±0.009683817)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 414.72 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half Map B

Fileemd_47042_half_map_1.map
AnnotationHalf Map B
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: Half Map A

Fileemd_47042_half_map_2.map
AnnotationHalf Map A
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Beta-arrestin 1 with insertion of soluble cytochrome b562 bound t...

EntireName: Beta-arrestin 1 with insertion of soluble cytochrome b562 bound to anti-BRIL Fab
Components
  • Complex: Beta-arrestin 1 with insertion of soluble cytochrome b562 bound to anti-BRIL Fab
    • Protein or peptide: anti-BRIL Fab Heavy chain
    • Protein or peptide: anti-BRIL Fab Light chain
    • Protein or peptide: Beta-arrestin-1,Soluble cytochrome b562
  • Ligand: (1beta,6beta,7beta,8alpha,9beta,10alpha,13alpha,14R,16beta)-1,6,7,14-tetrahydroxy-7,20-epoxykauran-15-one

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Supramolecule #1: Beta-arrestin 1 with insertion of soluble cytochrome b562 bound t...

SupramoleculeName: Beta-arrestin 1 with insertion of soluble cytochrome b562 bound to anti-BRIL Fab
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Rattus norvegicus (Norway rat)

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Macromolecule #1: anti-BRIL Fab Heavy chain

MacromoleculeName: anti-BRIL Fab Heavy chain / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.904656 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: EVQLVESGGG LVQPGGSLRL SCAASGFNVV DFSLHWVRQA PGKGLEWVAY ISSSSGSTSY ADSVKGRFTI SADTSKNTAY LQMNSLRAE DTAVYYCARW GYWPGEPWWK AFDYWGQGTL VTVSSASTKG PSVFPLAPSS KSTSGGTAAL GCLVKDYFPE P VTVSWNSG ...String:
EVQLVESGGG LVQPGGSLRL SCAASGFNVV DFSLHWVRQA PGKGLEWVAY ISSSSGSTSY ADSVKGRFTI SADTSKNTAY LQMNSLRAE DTAVYYCARW GYWPGEPWWK AFDYWGQGTL VTVSSASTKG PSVFPLAPSS KSTSGGTAAL GCLVKDYFPE P VTVSWNSG ALTSGVHTFP AVLQSSGLYS LSSVVTVPSS SLGTQTYICN VNHKPSNTKV DKKVEPK

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Macromolecule #2: anti-BRIL Fab Light chain

MacromoleculeName: anti-BRIL Fab Light chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.20982 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: DIQMTQSPSS LSASVGDRVT ITCRASQSVS SAVAWYQQKP GKAPKLLIYS ASSLYSGVPS RFSGSRSGTD FTLTISSLQP EDFATYYCQ QYLYYSLVTF GQGTKVEIKR TVAAPSVFIF PPSDSQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD ...String:
DIQMTQSPSS LSASVGDRVT ITCRASQSVS SAVAWYQQKP GKAPKLLIYS ASSLYSGVPS RFSGSRSGTD FTLTISSLQP EDFATYYCQ QYLYYSLVTF GQGTKVEIKR TVAAPSVFIF PPSDSQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNR

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Macromolecule #3: Beta-arrestin-1,Soluble cytochrome b562

MacromoleculeName: Beta-arrestin-1,Soluble cytochrome b562 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 55.72716 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: LGDKGTRVFK KASPNGKLTV YLGKRDFVDH IDLVDPVDGV VLVDPEYLKE RRVYVTLTCA FRYGREDLDV LGLTFRKDLF VANVQSFPP APEDKKPLTR LQERLIKKLG EHAYPFTFEI PPNLPCSVTL QPGPEDTGKA CGVDYEVKAF CAENLEEKIH K RNSVRLVI ...String:
LGDKGTRVFK KASPNGKLTV YLGKRDFVDH IDLVDPVDGV VLVDPEYLKE RRVYVTLTCA FRYGREDLDV LGLTFRKDLF VANVQSFPP APEDKKPLTR LQERLIKKLG EHAYPFTFEI PPNLPCSVTL QPGPEDTGKA CGVDYEVKAF CAENLEEKIH K RNSVRLVI RKVQYAPEAD LEDNWETLND NLKVIEKADN AAQVKDALTK MRAAALDAQK ATPPKLEDKS PDSPEMKDFR HG FDILVGQ IDDALKLANE GKVKEAQAAA EQLKTTRNAY IQKYLPTAET TRQFLMSDKP LHLEASLDKE IYYHGEPISV NVH VTNNTN KTVKKIKISV RQYADICLFN TAQYKCPVAM EEADDTVAPS STFCKVYTLT PFLANNREKR GLALDGKLKH EDTN LASST LLREGANREI LGIIVSYKVK VKLVVSRGDY KDDDDKSDVA VELPFTLMHP KPKEEPPHRE VPESETPVDT NLIEL DTND DDIVFEDFAR

UniProtKB: Beta-arrestin-1, Soluble cytochrome b562, Beta-arrestin-1

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Macromolecule #4: (1beta,6beta,7beta,8alpha,9beta,10alpha,13alpha,14R,16beta)-1,6,7...

MacromoleculeName: (1beta,6beta,7beta,8alpha,9beta,10alpha,13alpha,14R,16beta)-1,6,7,14-tetrahydroxy-7,20-epoxykauran-15-one
type: ligand / ID: 4 / Number of copies: 1 / Formula: ODN
Molecular weightTheoretical: 366.449 Da
Chemical component information

ChemComp-ODN:
(1beta,6beta,7beta,8alpha,9beta,10alpha,13alpha,14R,16beta)-1,6,7,14-tetrahydroxy-7,20-epoxykauran-15-one

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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

Concentration7.5 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 57.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

1g4m

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.47 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.4.1) / Number images used: 177430
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChainDetails

1g4m

chain_id: A, source_name: PDB, initial_model_type: experimental model

6ww2

chain_id: L, source_name: PDB, initial_model_type: experimental model

6ww2

chain_id: H, source_name: PDB, initial_model_type: experimental model

6ww2

chain_id: A, source_name: PDB, initial_model_type: experimental modelsoluble cytochrome b562 insert
Output model

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

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