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- PDB-6u1n: GPCR-Beta arrestin structure in lipid bilayer -

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Basic information

Entry
Database: PDB / ID: 6u1n
TitleGPCR-Beta arrestin structure in lipid bilayer
Components
  • Beta-arrestin-1
  • Fab30 heavy chain
  • Fab30 light chain
  • Muscarinic acetylcholine receptor M2, Vasopressin V2 receptor chimera
KeywordsSIGNALING PROTEIN/IMMUNE SYSTEM / Arrestin / GPCR / complex / signaling / SIGNALING PROTEIN-IMMUNE SYSTEM complex
Function / homology
Function and homology information


V2 vasopressin receptor binding / alpha-1A adrenergic receptor binding / follicle-stimulating hormone receptor binding / TGFBR3 regulates TGF-beta signaling / renal water retention / Defective AVP does not bind AVPR2 and causes neurohypophyseal diabetes insipidus (NDI) / sensory perception of touch / G alpha (s) signalling events / Vasopressin-like receptors / regulation of systemic arterial blood pressure by vasopressin ...V2 vasopressin receptor binding / alpha-1A adrenergic receptor binding / follicle-stimulating hormone receptor binding / TGFBR3 regulates TGF-beta signaling / renal water retention / Defective AVP does not bind AVPR2 and causes neurohypophyseal diabetes insipidus (NDI) / sensory perception of touch / G alpha (s) signalling events / Vasopressin-like receptors / regulation of systemic arterial blood pressure by vasopressin / vasopressin receptor activity / regulation of inositol trisphosphate biosynthetic process / follicle-stimulating hormone signaling pathway / protein phosphorylated amino acid binding / alpha-1B adrenergic receptor binding / Lysosome Vesicle Biogenesis / Muscarinic acetylcholine receptors / G protein-coupled acetylcholine receptor activity / angiotensin receptor binding / AP-2 adaptor complex binding / phospholipase C-activating G protein-coupled acetylcholine receptor signaling pathway / Ub-specific processing proteases / cholinergic synapse / MAP2K and MAPK activation / Golgi Associated Vesicle Biogenesis / hemostasis / telencephalon development / Cargo recognition for clathrin-mediated endocytosis / clathrin adaptor activity / Clathrin-mediated endocytosis / negative regulation of interleukin-8 production / regulation of smooth muscle contraction / adenylate cyclase-inhibiting G protein-coupled acetylcholine receptor signaling pathway / regulation of G protein-coupled receptor signaling pathway / cysteine-type endopeptidase inhibitor activity involved in apoptotic process / G protein-coupled receptor internalization / arrestin family protein binding / mitogen-activated protein kinase kinase binding / Thrombin signalling through proteinase activated receptors (PARs) / response to morphine / clathrin binding / stress fiber assembly / positive regulation of Rho protein signal transduction / regulation of heart contraction / positive regulation of vasoconstriction / pseudopodium / positive regulation of systemic arterial blood pressure / negative regulation of interleukin-6 production / immunoglobulin complex / positive regulation of intracellular signal transduction / positive regulation of receptor internalization / endocytic vesicle / negative regulation of Notch signaling pathway / phototransduction / G protein-coupled receptor signaling pathway, coupled to cyclic nucleotide second messenger / positive regulation of insulin secretion involved in cellular response to glucose stimulus / activation of adenylate cyclase activity / cellular response to hormone stimulus / insulin-like growth factor receptor binding / clathrin-coated pit / response to cytokine / negative regulation of protein ubiquitination / presynaptic modulation of chemical synaptic transmission / GTPase activator activity / positive regulation of protein ubiquitination / nuclear estrogen receptor binding / phosphoprotein binding / clathrin-coated endocytic vesicle membrane / G protein-coupled receptor binding / negative regulation of ERK1 and ERK2 cascade / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / response to virus / positive regulation of protein phosphorylation / endocytosis / G protein-coupled acetylcholine receptor signaling pathway / Vasopressin regulates renal water homeostasis via Aquaporins / Cargo recognition for clathrin-mediated endocytosis / nervous system development / protein transport / presynapse / Clathrin-mediated endocytosis / cytoplasmic vesicle / ubiquitin-dependent protein catabolic process / regulation of apoptotic process / G alpha (i) signalling events / G alpha (s) signalling events / basolateral plasma membrane / chemical synaptic transmission / molecular adaptor activity / dendritic spine / adaptive immune response / proteasome-mediated ubiquitin-dependent protein catabolic process / negative regulation of neuron apoptotic process / transmembrane transporter binding / postsynaptic membrane / transcription coactivator activity / positive regulation of ERK1 and ERK2 cascade / endosome / positive regulation of MAPK cascade / postsynaptic density
Similarity search - Function
Muscarinic acetylcholine receptor M2 / Vasopressin V2 receptor / Vasopressin receptor / Muscarinic acetylcholine receptor family / Arrestin, conserved site / Arrestins signature. / Arrestin / Arrestin, N-terminal / Arrestin-like, N-terminal / Arrestin C-terminal-like domain ...Muscarinic acetylcholine receptor M2 / Vasopressin V2 receptor / Vasopressin receptor / Muscarinic acetylcholine receptor family / 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 / : / : / Serpentine type 7TM GPCR chemoreceptor Srsx / Immunoglobulin V-Type / Immunoglobulin V-set domain / Immunoglobulin V-set domain / 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/major histocompatibility complex, conserved site / Immunoglobulins and major histocompatibility complex proteins signature. / Immunoglobulin subtype / Immunoglobulin / Immunoglobulin C-Type / Immunoglobulin E-set / Immunoglobulin C1-set / Immunoglobulin C1-set domain / Ig-like domain profile. / Immunoglobulin-like domain / Immunoglobulin-like domain superfamily / Immunoglobulin-like fold
Similarity search - Domain/homology
Chem-2CU / Muscarinic acetylcholine receptor M2 / Beta-arrestin-1 / Vasopressin V2 receptor / Ig-like domain-containing protein / Epididymis luminal protein 214
Similarity search - Component
Biological speciesHomo sapiens (human)
Rattus norvegicus (Norway rat)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsStaus, D.P. / Hu, H. / Robertson, M.J. / Kleinhenz, A.L.W. / Wingler, L.M. / Capel, W.D. / Latorraca, N.R. / Lefkowitz, R.J. / Skiniotis, G.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)HL16037 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)NS092695 United States
CitationJournal: Nature / Year: 2020
Title: Structure of the M2 muscarinic receptor-β-arrestin complex in a lipid nanodisc.
Authors: Dean P Staus / Hongli Hu / Michael J Robertson / Alissa L W Kleinhenz / Laura M Wingler / William D Capel / Naomi R Latorraca / Robert J Lefkowitz / Georgios Skiniotis /
Abstract: After activation by an agonist, G-protein-coupled receptors (GPCRs) recruit β-arrestin, which desensitizes heterotrimeric G-protein signalling and promotes receptor endocytosis. Additionally, β- ...After activation by an agonist, G-protein-coupled receptors (GPCRs) recruit β-arrestin, which desensitizes heterotrimeric G-protein signalling and promotes receptor endocytosis. Additionally, β-arrestin directly regulates many cell signalling pathways that can induce cellular responses distinct from that of G proteins. In contrast to G proteins, for which there are many high-resolution structures in complex with GPCRs, the molecular mechanisms underlying the interaction of β-arrestin with GPCRs are much less understood. Here we present a cryo-electron microscopy structure of β-arrestin 1 (βarr1) in complex with M2 muscarinic receptor (M2R) reconstituted in lipid nanodiscs. The M2R-βarr1 complex displays a multimodal network of flexible interactions, including binding of the N domain of βarr1 to phosphorylated receptor residues and insertion of the finger loop of βarr1 into the M2R seven-transmembrane bundle, which adopts a conformation similar to that in the M2R-heterotrimeric G protein complex. Moreover, the cryo-electron microscopy map reveals that the C-edge of βarr1 engages the lipid bilayer. Through atomistic simulations and biophysical, biochemical and cellular assays, we show that the C-edge is critical for stable complex formation, βarr1 recruitment, receptor internalization, and desensitization of G-protein activation. Taken together, these data suggest that the cooperative interactions of β-arrestin with both the receptor and the phospholipid bilayer contribute to its functional versatility.
History
DepositionAug 16, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 26, 2020Provider: repository / Type: Initial release
Revision 1.1Mar 4, 2020Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Mar 25, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Oct 16, 2024Group: Data collection / Database references / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

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Assembly

Deposited unit
R: Muscarinic acetylcholine receptor M2, Vasopressin V2 receptor chimera
C: Beta-arrestin-1
H: Fab30 heavy chain
L: Fab30 light chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)151,0195
Polymers150,5814
Non-polymers4381
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Muscarinic acetylcholine receptor M2, Vasopressin V2 receptor chimera / V2R / AVPR V2 / Antidiuretic hormone receptor / Renal-type arginine vasopressin receptor


Mass: 56616.176 Da / Num. of mol.: 1 / Fragment: M2 UNP residues 2-466 + V2 UNP residues 343-371
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CHRM2, AVPR2, ADHR, DIR, DIR3, V2R / Production host: Homo sapiens (human) / References: UniProt: P08172, UniProt: P30518
#2: Protein Beta-arrestin-1 / Arrestin beta-1


Mass: 45017.180 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Arrb1 / Production host: Escherichia coli (E. coli) / References: UniProt: P29066
#3: Antibody Fab30 heavy chain


Mass: 25512.354 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) / References: UniProt: V9HW68
#4: Antibody Fab30 light chain


Mass: 23435.064 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) / References: UniProt: Q7Z3Y4
#5: Chemical ChemComp-2CU / 3-amino-5-chloro-N-cyclopropyl-4-methyl-6-[2-(4-methylpiperazin-1-yl)-2-oxoethoxy]thieno[2,3-b]pyridine-2-carboxamide / LY2119620 positive allosteric modulator of M2/M4 receptor


Mass: 437.944 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C19H24ClN5O3S
Has ligand of interestN
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

Component
IDNameTypeEntity IDParent-IDSourceDetails
1Phosphorylated human muscarinic acetylcholine receptor M2 in complex with rat beta-arrestin1, stabilized by an antibody fragment (Fab30).COMPLEX#1-#40MULTIPLE SOURCES
2Phosphorylated human muscarinic acetylcholine receptor M2COMPLEX#11RECOMBINANTPhosphorylated M2R was generated by ligating a synthetic phosphopeptide derived from the vasopressin-2-receptor (V2Rpp) using the enzyme sortase.
3Cysteine-free rat beta-arrestin 1 truncated at amino acid 393COMPLEX#21RECOMBINANT
4Antibody Fragment (Fab30)COMPLEX#3-#41RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Rattus norvegicus (Norway rat)10116
34Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Escherichia coli (E. coli)562
34Escherichia coli (E. coli)562
Buffer solutionpH: 7.4
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
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 magnification: 47169 X / Calibrated magnification: 47169 X / Nominal defocus max: 2200 nm / Nominal defocus min: 1200 nm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.15rc3_3435: / Classification: refinement
EM software
IDNameVersionCategory
1RELION3particle selection
2SerialEM3.6image acquisition
4Gctf1.06CTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10RELIONinitial Euler assignment
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 11700000
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 145618 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0056038
ELECTRON MICROSCOPYf_angle_d0.6338314
ELECTRON MICROSCOPYf_dihedral_angle_d4.8873523
ELECTRON MICROSCOPYf_chiral_restr0.042993
ELECTRON MICROSCOPYf_plane_restr0.0041060

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