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- EMDB-46533: Cryo-EM structure of CCR6 bound by SQA1 and OXM2 -

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Entry
Database: EMDB / ID: EMD-46533
TitleCryo-EM structure of CCR6 bound by SQA1 and OXM2
Map dataCryo-EM structure of SQA1 and OXM2 bound to CCR6
Sample
  • Complex: CCR6-BRIL/Fab/Nb in complex with SQA1 and OXM2
    • Protein or peptide: Human CCR6
    • Protein or peptide: anti-BRIL Fab Heavy chain
    • Protein or peptide: anti-BRIL Fab Nanobody
    • Protein or peptide: anti-BRIL Fab Light chain
  • Ligand: CHOLESTEROL
  • Ligand: 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
  • Ligand: N-{[(2R)-4-(bicyclo[1.1.1]pentan-1-yl)-5-oxomorpholin-2-yl]methyl}-1-[4-(trifluoromethyl)phenyl]cyclopropane-1-carboxamide
KeywordsGPCR / Antagonist / CCR6 / BRIL / MEMBRANE PROTEIN
Biological speciesHomo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.02 Å
AuthorsWasilko DJ / Wu H
Funding support1 items
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, 2024-
Header (metadata) releaseSep 11, 2024-
Map releaseSep 11, 2024-
UpdateSep 11, 2024-
Current statusSep 11, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_46533.png

Downloads & links

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Map

FileDownload / File: emd_46533.map.gz / Format: CCP4 / Size: 536.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM structure of SQA1 and OXM2 bound to CCR6
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.65 Å/pix.
x 520 pix.
= 340.08 Å		0.65 Å/pix.
x 520 pix.
= 340.08 Å		0.65 Å/pix.
x 520 pix.
= 340.08 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.654 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.097
Minimum - Maximum-0.9828191 - 1.2833651
Average (Standard dev.)-0.0002960359 (±0.014006503)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions520520520
Spacing520520520
CellA=B=C: 340.08 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: EM half map 1

Fileemd_46533_half_map_1.map
AnnotationEM half map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: EM half map 2

Fileemd_46533_half_map_2.map
AnnotationEM half map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : CCR6-BRIL/Fab/Nb in complex with SQA1 and OXM2

EntireName: CCR6-BRIL/Fab/Nb in complex with SQA1 and OXM2
Components
  • Complex: CCR6-BRIL/Fab/Nb in complex with SQA1 and OXM2
    • Protein or peptide: Human CCR6
    • Protein or peptide: anti-BRIL Fab Heavy chain
    • Protein or peptide: anti-BRIL Fab Nanobody
    • Protein or peptide: anti-BRIL Fab Light chain
  • Ligand: CHOLESTEROL
  • Ligand: 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
  • Ligand: N-{[(2R)-4-(bicyclo[1.1.1]pentan-1-yl)-5-oxomorpholin-2-yl]methyl}-1-[4-(trifluoromethyl)phenyl]cyclopropane-1-carboxamide

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Supramolecule #1: CCR6-BRIL/Fab/Nb in complex with SQA1 and OXM2

SupramoleculeName: CCR6-BRIL/Fab/Nb in complex with SQA1 and OXM2 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Details: CCR6-BRIL/Fab/Nb complex co-purified with SQA1 and OXM2.
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 120 kDa/nm

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Macromolecule #1: Human CCR6

MacromoleculeName: Human CCR6 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 54.62302 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MKTIIALSYI FCLVFADYKD DDDAKLQTMH HHHHHHHHHE NLYFQGGTSV DSEMLLCSLQ EVRQFSRAFV PIAYSLICVF GLLGNILVV ITFAFYKKAR SMTDVYLANM AIADILFALT LPFWAVSHAT GAWVFSNATC KLLKGIYAIN FNCGMWLLTC I AMDRYIAI ...String:
MKTIIALSYI FCLVFADYKD DDDAKLQTMH HHHHHHHHHE NLYFQGGTSV DSEMLLCSLQ EVRQFSRAFV PIAYSLICVF GLLGNILVV ITFAFYKKAR SMTDVYLANM AIADILFALT LPFWAVSHAT GAWVFSNATC KLLKGIYAIN FNCGMWLLTC I AMDRYIAI VQATKSFRLR SATLPRSKII CLVVWGLSVI ISSSTFVFNQ KYNTQGSDVC EPKYQTVSEP IRWKLLMLGL EL LFGFFIP LMFMIFCYTA IVKTLRRQLA DLEDNWETLN DNLKVIEKAD NAAQVKDALT KMRAAALDAQ KATPPKLEDK SPD SPEMKD FRHGFDILVG QIDDALKLAN EGKVKEAQAA AEQLKTTRNA YIQKYLERAR STLQKEVKAI RVIIAVVLVF LACQ IPHNM VLLVTAANLG KMNRSCQSEK LIAYTKTVTE VLAFLHCCLN PVLYAFIGQK FRNYFLKILK DLWCVRRKYK SSGFS

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Macromolecule #2: anti-BRIL Fab Heavy chain

MacromoleculeName: anti-BRIL Fab Heavy chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 28.832098 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MGWSCIILFL VATATGVHSE ISEVQLVESG GGLVQPGGSL RLSCAASGFN VVDFSLHWVR QAPGKGLEWV AYISSSSGST SYADSVKGR FTISADTSKN TAYLQMNSLR AEDTAVYYCA RWGYWPGEPW WKAFDYWGQG TLVTVSSAST KGPSVFPLAP S SKSTSGGT ...String:
MGWSCIILFL VATATGVHSE ISEVQLVESG GGLVQPGGSL RLSCAASGFN VVDFSLHWVR QAPGKGLEWV AYISSSSGST SYADSVKGR FTISADTSKN TAYLQMNSLR AEDTAVYYCA RWGYWPGEPW WKAFDYWGQG TLVTVSSAST KGPSVFPLAP S SKSTSGGT AALGCLVKDY FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYI CNVNHKPSNT KV DKKVEPK SGGSENLYFQ GSHHHHHHHH HH

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Macromolecule #3: anti-BRIL Fab Nanobody

MacromoleculeName: anti-BRIL Fab Nanobody / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 15.755214 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MGHHHHHHHH HHENLYFQGS QVQLQESGGG LVQPGGSLRL SCAASGRTIS RYAMSWFRQA PGKEREFVAV ARRSGDGAFY ADSVQGRFT VSRDDAKNTV YLQMNSLKPE DTAVYYCAID SDTFYSGSYD YWGQGTQVTV SS

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Macromolecule #4: anti-BRIL Fab Light chain

MacromoleculeName: anti-BRIL Fab Light chain / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 25.343348 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MGWSCIILFL VATATGVHSS DIQMTQSPSS LSASVGDRVT ITCRASQSVS SAVAWYQQKP GKAPKLLIYS ASSLYSGVPS RFSGSRSGT DFTLTISSLQ PEDFATYYCQ QYLYYSLVTF GQGTKVEIKR TVAAPSVFIF PPSDSQLKSG TASVVCLLNN F YPREAKVQ ...String:
MGWSCIILFL VATATGVHSS DIQMTQSPSS LSASVGDRVT ITCRASQSVS SAVAWYQQKP GKAPKLLIYS ASSLYSGVPS RFSGSRSGT DFTLTISSLQ PEDFATYYCQ QYLYYSLVTF GQGTKVEIKR TVAAPSVFIF PPSDSQLKSG TASVVCLLNN F YPREAKVQ WKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRG

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Macromolecule #5: CHOLESTEROL

MacromoleculeName: CHOLESTEROL / type: ligand / ID: 5 / Number of copies: 1 / Formula: CLR
Molecular weightTheoretical: 386.654 Da
Chemical component information

ChemComp-CLR:
CHOLESTEROL

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Macromolecule #6: 4-[[3,4-bis(oxidanylidene)-2-[[(1~{R})-1-(4-propan-2-ylfuran-2-yl...

MacromoleculeName: 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
type: ligand / ID: 6 / Number of copies: 1 / Formula: EBX
Molecular weightTheoretical: 426.466 Da
Chemical component information

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

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Macromolecule #7: N-{[(2R)-4-(bicyclo[1.1.1]pentan-1-yl)-5-oxomorpholin-2-yl]methyl...

MacromoleculeName: N-{[(2R)-4-(bicyclo[1.1.1]pentan-1-yl)-5-oxomorpholin-2-yl]methyl}-1-[4-(trifluoromethyl)phenyl]cyclopropane-1-carboxamide
type: ligand / ID: 7 / Number of copies: 1 / Formula: A1A1W
Molecular weightTheoretical: 408.414 Da

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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

Concentration1 mg/mL
BufferpH: 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
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsCCR6-BRIL/Fab/Nb complex co-purified with SQA1 and OXM2.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 45.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.4 µm / Nominal magnification: 130000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 10786639
Startup modelType of model: INSILICO MODEL
Final reconstructionNumber classes used: 1 / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.02 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.1) / Number images used: 368745
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1)
Final 3D classificationNumber classes: 4 / Software - Name: cryoSPARC (ver. 3.3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL
Output model

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

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