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- PDB-9d3e: Cryo-EM structure of CCR6 bound by SQA1 and OXM2 -

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Basic information

Entry
Database: PDB / ID: 9d3e
TitleCryo-EM structure of CCR6 bound by SQA1 and OXM2
Components
  • (anti-BRIL Fab ...) x 3
  • Human CCR6
KeywordsMEMBRANE PROTEIN / GPCR / Antagonist / CCR6 / BRIL
Function / homology: / CHOLESTEROL / Chem-EBX
Function and homology information
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.02 Å
AuthorsWasilko, D.J. / Wu, H.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nat Commun / Year: 2024
Title: Structural basis for CCR6 modulation by allosteric antagonists.
Authors: David Jonathan Wasilko / Brian S Gerstenberger / Kathleen A Farley / Wei Li / Jennifer Alley / Mark E Schnute / Ray J Unwalla / Jorge Victorino / Kimberly K Crouse / Ru Ding / Parag V ...Authors: David Jonathan Wasilko / Brian S Gerstenberger / Kathleen A Farley / Wei Li / Jennifer Alley / Mark E Schnute / Ray J Unwalla / Jorge Victorino / Kimberly K Crouse / Ru Ding / Parag V Sahasrabudhe / Fabien Vincent / Richard K Frisbie / Alpay Dermenci / Andrew Flick / Chulho Choi / Gary Chinigo / James J Mousseau / John I Trujillo / Philippe Nuhant / Prolay Mondal / Vincent Lombardo / Daniel Lamb / Barbara J Hogan / Gurdeep Singh Minhas / Elena Segala / Christine Oswald / Ian W Windsor / Seungil Han / Mathieu Rappas / Robert M Cooke / Matthew F Calabrese / Gabriel Berstein / Atli Thorarensen / Huixian Wu /
Abstract: The CC chemokine receptor 6 (CCR6) is a potential target for chronic inflammatory diseases. Previously, we reported an active CCR6 structure in complex with its cognate chemokine CCL20, revealing the ...The CC chemokine receptor 6 (CCR6) is a potential target for chronic inflammatory diseases. Previously, we reported an active CCR6 structure in complex with its cognate chemokine CCL20, revealing the molecular basis of CCR6 activation. Here, we present two inactive CCR6 structures in ternary complexes with different allosteric antagonists, CCR6/SQA1/OXM1 and CCR6/SQA1/OXM2. The oxomorpholine analogues, OXM1 and OXM2 are highly selective CCR6 antagonists which bind to an extracellular pocket and disrupt the receptor activation network. An energetically favoured U-shaped conformation in solution that resembles the bound form is observed for the active analogues. SQA1 is a squaramide derivative with close-in analogues reported as antagonists of chemokine receptors including CCR6. SQA1 binds to an intracellular pocket which overlaps with the G protein site, stabilizing a closed pocket that is a hallmark of inactive GPCRs. Minimal communication between the two allosteric pockets is observed, in contrast to the prevalent allosteric cooperativity model of GPCRs. This work highlights the versatility of GPCR antagonism by small molecules, complementing previous knowledge of CCR6 activation, and sheds light on drug discovery targeting CCR6.
History
DepositionAug 9, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 11, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Human CCR6
H: anti-BRIL Fab Heavy chain
K: anti-BRIL Fab Nanobody
L: anti-BRIL Fab Light chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)125,7757
Polymers124,5544
Non-polymers1,2223
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, SEC, SDS-PAGE
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 1 types, 1 molecules A

#1: Protein Human CCR6


Mass: 54623.020 Da / Num. of mol.: 1 / Mutation: L47A, L86A, V96A, S140A, R159A, F236A, G295A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CCR6 / Production host: Spodoptera frugiperda (fall armyworm)

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Antibody , 3 types, 3 molecules HKL

#2: Antibody anti-BRIL Fab Heavy chain


Mass: 28832.098 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)
#3: Antibody anti-BRIL Fab Nanobody


Mass: 15755.214 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#4: Antibody anti-BRIL Fab Light chain


Mass: 25343.348 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)

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Non-polymers , 3 types, 3 molecules

#5: Chemical ChemComp-CLR / CHOLESTEROL


Mass: 386.654 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C27H46O
#6: Chemical ChemComp-EBX / 4-[[3,4-bis(oxidanylidene)-2-[[(1~{R})-1-(4-propan-2-ylfuran-2-yl)propyl]amino]cyclobuten-1-yl]amino]-~{N},~{N}-dimethyl-3-oxidanyl-pyridine-2-carboxamide


Mass: 426.466 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C22H26N4O5 / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-A1A1W / N-{[(2R)-4-(bicyclo[1.1.1]pentan-1-yl)-5-oxomorpholin-2-yl]methyl}-1-[4-(trifluoromethyl)phenyl]cyclopropane-1-carboxamide


Mass: 408.414 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C21H23F3N2O3 / Feature type: SUBJECT OF INVESTIGATION

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Details

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

ComponentName: CCR6-BRIL/Fab/Nb in complex with SQA1 and OXM2 / Type: COMPLEX
Details: CCR6-BRIL/Fab/Nb complex co-purified with SQA1 and OXM2.
Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightValue: 120 kDa/nm / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
Details: 50 mM HEPES pH 7.5, 150 mM NaCl, 0.003% LMNG, 0.0003% CHS, 50 uM OXM2 and 50 uM of SQA1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: CCR6-BRIL/Fab/Nb complex co-purified with SQA1 and OXM2.
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
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 magnification: 130000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 400 nm / C2 aperture diameter: 100 µm
Image recordingElectron dose: 45 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1cryoSPARC3.3.1particle selection
2EPU2.12.1.2782RELimage acquisition
4cryoSPARC3.3.1CTF correction
9cryoSPARC3.3.1initial Euler assignment
10cryoSPARC3.3.1final Euler assignment
11cryoSPARC3.3.1classification
12cryoSPARC3.3.13D reconstruction
13PHENIX1.20_4459:model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 10786639
3D reconstructionResolution: 3.02 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 368745 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0047338
ELECTRON MICROSCOPYf_angle_d0.8379981
ELECTRON MICROSCOPYf_dihedral_angle_d5.0751048
ELECTRON MICROSCOPYf_chiral_restr0.0491131
ELECTRON MICROSCOPYf_plane_restr0.0071277

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