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- PDB-6tko: Phosphorylated turkey beta1 adrenoceptor with bound agonist formo... -

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Basic information

Entry
Database: PDB / ID: 6tko
TitlePhosphorylated turkey beta1 adrenoceptor with bound agonist formoterol coupled to arrestin-2 in lipid nanodisc.
Components
  • Beta-1 adrenergic receptor
  • Beta-arrestin-1Arrestin
  • Fab30 heavy chain
  • Fab30 light chain
KeywordsSIGNALING PROTEIN / GPCR / Arrestin / Complex / Nanodisc
Function / homology
Function and homology information


beta1-adrenergic receptor activity / angiotensin receptor binding / positive regulation of heart contraction / regulation of circadian sleep/wake cycle, sleep / Activation of SMO / negative regulation of interleukin-8 production / arrestin family protein binding / G protein-coupled receptor internalization / enzyme inhibitor activity / Lysosome Vesicle Biogenesis ...beta1-adrenergic receptor activity / angiotensin receptor binding / positive regulation of heart contraction / regulation of circadian sleep/wake cycle, sleep / Activation of SMO / negative regulation of interleukin-8 production / arrestin family protein binding / G protein-coupled receptor internalization / enzyme inhibitor activity / Lysosome Vesicle Biogenesis / positive regulation of Rho protein signal transduction / Golgi Associated Vesicle Biogenesis / negative regulation of NF-kappaB transcription factor activity / stress fiber assembly / negative regulation of Notch signaling pathway / pseudopodium / negative regulation of interleukin-6 production / positive regulation of receptor internalization / adenylate cyclase-activating adrenergic receptor signaling pathway / clathrin-coated pit / negative regulation of protein ubiquitination / insulin-like growth factor receptor binding / visual perception / Activated NOTCH1 Transmits Signal to the Nucleus / GTPase activator activity / G protein-coupled receptor binding / Signaling by high-kinase activity BRAF mutants / MAP2K and MAPK activation / cytoplasmic vesicle membrane / positive regulation of GTPase activity / 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 / Signaling by BRAF and RAF1 fusions / Thrombin signalling through proteinase activated receptors (PARs) / protein transport / Cargo recognition for clathrin-mediated endocytosis / Clathrin-mediated endocytosis / ubiquitin-dependent protein catabolic process / G alpha (s) signalling events / cytoplasmic vesicle / proteasome-mediated ubiquitin-dependent protein catabolic process / transcription coactivator activity / positive regulation of ERK1 and ERK2 cascade / protein ubiquitination / nuclear body / early endosome / Ub-specific processing proteases / positive regulation of protein phosphorylation / lysosomal membrane / Golgi membrane / ubiquitin protein ligase binding / chromatin / regulation of transcription by RNA polymerase II / signal transduction / positive regulation of transcription by RNA polymerase II / nucleoplasm / membrane / identical protein binding / nucleus / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Beta 1 adrenoceptor / 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 ...Beta 1 adrenoceptor / 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 / Adrenoceptor family / Serpentine type 7TM GPCR chemoreceptor Srsx / 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-H98 / Beta-1 adrenergic receptor / Beta-arrestin-1
Similarity search - Component
Biological speciesMeleagris gallopavo (turkey)
Homo sapiens (human)
Phage display vector pTDisp (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsLee, Y. / Tate, C.G.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
European Research Council (ERC)EMPSI 339995 United Kingdom
Medical Research Council (MRC, United Kingdom)MRC U105197215 United Kingdom
Citation
Journal: Nature / Year: 2020
Title: Molecular basis of β-arrestin coupling to formoterol-bound β-adrenoceptor.
Authors: Yang Lee / Tony Warne / Rony Nehmé / Shubhi Pandey / Hemlata Dwivedi-Agnihotri / Madhu Chaturvedi / Patricia C Edwards / Javier García-Nafría / Andrew G W Leslie / Arun K Shukla / Christopher G Tate /
Abstract: The β-adrenoceptor (βAR) is a G-protein-coupled receptor (GPCR) that couples to the heterotrimeric G protein G. G-protein-mediated signalling is terminated by phosphorylation of the C terminus of ...The β-adrenoceptor (βAR) is a G-protein-coupled receptor (GPCR) that couples to the heterotrimeric G protein G. G-protein-mediated signalling is terminated by phosphorylation of the C terminus of the receptor by GPCR kinases (GRKs) and by coupling of β-arrestin 1 (βarr1, also known as arrestin 2), which displaces G and induces signalling through the MAP kinase pathway. The ability of synthetic agonists to induce signalling preferentially through either G proteins or arrestins-known as biased agonism-is important in drug development, because the therapeutic effect may arise from only one signalling cascade, whereas the other pathway may mediate undesirable side effects. To understand the molecular basis for arrestin coupling, here we determined the cryo-electron microscopy structure of the βAR-βarr1 complex in lipid nanodiscs bound to the biased agonist formoterol, and the crystal structure of formoterol-bound βAR coupled to the G-protein-mimetic nanobody Nb80. βarr1 couples to βAR in a manner distinct to that of G coupling to βAR-the finger loop of βarr1 occupies a narrower cleft on the intracellular surface, and is closer to transmembrane helix H7 of the receptor when compared with the C-terminal α5 helix of G. The conformation of the finger loop in βarr1 is different from that adopted by the finger loop of visual arrestin when it couples to rhodopsin. βAR coupled to βarr1 shows considerable differences in structure compared with βAR coupled to Nb80, including an inward movement of extracellular loop 3 and the cytoplasmic ends of H5 and H6. We observe weakened interactions between formoterol and two serine residues in H5 at the orthosteric binding site of βAR, and find that formoterol has a lower affinity for the βAR-βarr1 complex than for the βAR-G complex. The structural differences between these complexes of βAR provide a foundation for the design of small molecules that could bias signalling in the β-adrenoceptors.
#1: Journal: Biorxiv / Year: 2020
Title: Molecular determinants of beta-arrestin coupling to formoterol-bound beta1-adrenoceptor.
Authors: Lee, Y. / Warne, T. / Nehme, R. / Pandey, S. / Dwivedi-Agnihotri, H. / Edwards, P.C. / Garcia-Nafria, J. / Leslie, A.G.W. / Shukla, A.K. / Tate, C.G.
History
DepositionNov 28, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 17, 2020Provider: repository / Type: Initial release
Revision 1.1Jul 8, 2020Group: Database references / Category: citation / citation_author
Revision 1.2Aug 12, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: Beta-1 adrenergic receptor
B: Beta-arrestin-1
H: Fab30 heavy chain
L: Fab30 light chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)133,5895
Polymers133,2444
Non-polymers3441
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: immunoprecipitation, IMAC pulldown of the complex using the His6-tag on Fab30., gel filtration, Retention volume on Superdex 200 Increase column consistent with expected Stokes diameter of 10-12 nm.
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area9400 Å2
ΔGint-68 kcal/mol
Surface area46370 Å2
MethodPISA

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Components

#1: Protein Beta-1 adrenergic receptor / / Beta-1 adrenoreceptor / Beta-T


Mass: 37275.395 Da / Num. of mol.: 1
Mutation: S32G M44C M90V V103C C116L E130W D322K F327A F338M C358A
Source method: isolated from a genetically manipulated source
Details: Relative to wild-type sequence: construct is truncated at the N-terminus; and in ICL3; its C-terminus is truncated at C358A and fused with a linker to a series of phosphorylated residues ...Details: Relative to wild-type sequence: construct is truncated at the N-terminus; and in ICL3; its C-terminus is truncated at C358A and fused with a linker to a series of phosphorylated residues (ARGRPLPETGGGDE[pS]A[pT][pT]A[pS][pS][pS]LAKDTSS).
Source: (gene. exp.) Meleagris gallopavo (turkey) / Gene: ADRB1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P07700
#2: Protein Beta-arrestin-1 / Arrestin / Arrestin beta-1 / Non-visual arrestin-2


Mass: 47021.332 Da / Num. of mol.: 1 / Mutation: M1G L68C R169E
Source method: isolated from a genetically manipulated source
Details: Additional N-terminal Gly residue, in place of starting Met, remaining from proteolytic cleavage.
Source: (gene. exp.) Homo sapiens (human) / Gene: ARRB1, ARR1 / Production host: Escherichia coli (E. coli) / References: UniProt: P49407
#3: Antibody Fab30 heavy chain


Mass: 25512.354 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Phage display vector pTDisp (others) / Production host: Escherichia coli (E. coli)
#4: Antibody Fab30 light chain


Mass: 23435.064 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Phage display vector pTDisp (others) / Production host: Escherichia coli (E. coli)
#5: Chemical ChemComp-H98 / ~{N}-[5-[(1~{R})-2-[[(2~{R})-1-(4-methoxyphenyl)propan-2-yl]amino]-1-oxidanyl-ethyl]-2-oxidanyl-phenyl]methanamide / Formoterol


Mass: 344.405 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C19H24N2O4 / Feature type: SUBJECT OF INVESTIGATION / Comment: agonist*YM
Has ligand of interestY

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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
IDNameTypeDetailsEntity IDParent-IDSource
1Formoterol-bound beta1-adrenoceptor coupled to beta-arrestin-1 stabilised by Fab30.COMPLEXComplex formed in lipid nanodisc.#1-#40MULTIPLE SOURCES
2Beta-1 adrenergic receptorCOMPLEX#11RECOMBINANT
3Beta-arrestin-1ArrestinCOMPLEX#21RECOMBINANT
4Fab30 heavy chainCOMPLEX#31RECOMBINANT
5Fab30 light chainCOMPLEX#41RECOMBINANT
Molecular weightValue: 0.133 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Meleagris gallopavo (turkey)9103
23Homo sapiens (human)9606
34Phage display vector pTDisp (others)279974
45Phage display vector pTDisp (others)279974
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
13Escherichia coli (E. coli)562
22Trichoplusia ni (cabbage looper)7111
34Escherichia coli (E. coli)562
45Escherichia coli (E. coli)562
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMHEPESC8H18N2O4S1
220 mMsodium chlorideNaClSodium chloride1
32 uMFormoterol hemifumarateC21H26N2O21
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse.
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K
Details: Blotted for 2-3 seconds before plunging. Liquid ethane maintained at 92.15 K.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
EM imaging

Accelerating voltage: 300 kV / Alignment procedure: COMA FREE / C2 aperture diameter: 100 µm / Cryogen: NITROGEN / Electron source: FIELD EMISSION GUN / Illumination mode: FLOOD BEAM / Model: FEI TITAN KRIOS / Mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3000 nm / Nominal defocus min: 1200 nm / Temperature (max): 70 K / Temperature (min): 70 K / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Specimen-ID: 1

IDDetailsNominal magnification (X)
1Data collected with a stage-tilt of 30deg; two non-overlapping exposures per hole and multi-hole image-shift data acquisition strategies (3x3 holes per stage shift).105000
2Data collected with a stage-tilt of 30deg; two non-overlapping exposures per hole using image-shift.130000
Image recording
IDImaging-IDElectron dose (e/Å2)Detector modeFilm or detector model
1150COUNTINGGATAN K2 QUANTUM (4k x 4k)
2245COUNTINGGATAN K2 QUANTUM (4k x 4k)
EM imaging optics
Energyfilter nameIDImaging-IDEnergyfilter slit width (eV)
GIF Quantum SE1120
GIF Quantum SE2220

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Processing

EM software
IDNameVersionCategoryImaging-IDDetails
2SerialEM3.8.0 betaimage acquisition1
4Warp1.0.6CTF correctionInitial CTF determination
5RELION3.1CTF correctionSubsequent CTF refinement
8Coot0.8.9.2model fitting
10EPU2.3.0.79image acquisition2
11RELION3.0.7initial Euler assignment
12RELION3.1final Euler assignment
14RELION3.13D reconstruction
15PHENIX1.17.1-3660model refinement
16REFMAC5model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 10161197
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 403991
Details: Half maps were locally filtered between refinement iterations using SIDESPLITTER, an adaptation of the LAFTER algorithm that maintains gold-standard separation between the two half maps.
Symmetry type: POINT
Atomic model buildingB value: 80.6 / Protocol: OTHER / Space: REAL / Target criteria: Correlation coefficient
Details: Initial placement was done manually in Coot, followed by rigid-body refinement in PHENIX and jelly-body refinement in REFMAC5. Manual remodeling was performed in Coot and iterated with real ...Details: Initial placement was done manually in Coot, followed by rigid-body refinement in PHENIX and jelly-body refinement in REFMAC5. Manual remodeling was performed in Coot and iterated with real space refinement in PHENIX. Chemical restraints for formoterol were calculated in eLBOW using AM1 optimisation.
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-IDPdb chain residue range
16IBL

6ibl

A141-401
24JQI

4jqi

A16-356
34JQI

4jqi

H15-223
44JQI

4jqi

L11-190
54JQI

4jqi

V1356-368
RefinementStereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 80.62 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00158271
ELECTRON MICROSCOPYf_angle_d0.411411279
ELECTRON MICROSCOPYf_chiral_restr0.03921308
ELECTRON MICROSCOPYf_plane_restr0.00171413
ELECTRON MICROSCOPYf_dihedral_angle_d15.30972923

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