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- PDB-9xhi: Structure of the CCL21-CCR7-Gi-scFv16 complex -

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Entry
Database: PDB / ID: 9xhi
TitleStructure of the CCL21-CCR7-Gi-scFv16 complex
Components
  • (Guanine nucleotide-binding protein ...) x 2
  • Antibody fragment scFv16
  • C-C chemokine receptor type 7,Non structural polyprotein
  • C-C motif chemokine 21
KeywordsMEMBRANE PROTEIN / chemokine / chemokine receptor / GPCR
Function / homology
Function and homology information


mesangial cell-matrix adhesion / dendritic cell dendrite assembly / negative regulation of dendritic cell dendrite assembly / positive regulation of hypersensitivity / chemokine (C-C motif) ligand 19 binding / chemokine (C-C motif) ligand 21 binding / C-C motif chemokine 19 receptor activity / C-C motif chemokine 21 receptor activity / : / regulation of dendritic cell dendrite assembly ...mesangial cell-matrix adhesion / dendritic cell dendrite assembly / negative regulation of dendritic cell dendrite assembly / positive regulation of hypersensitivity / chemokine (C-C motif) ligand 19 binding / chemokine (C-C motif) ligand 21 binding / C-C motif chemokine 19 receptor activity / C-C motif chemokine 21 receptor activity / : / regulation of dendritic cell dendrite assembly / myeloid dendritic cell chemotaxis / CCL19-activated CCR7 signaling pathway / CCL21-activated CCR7 signaling pathway / CCR7 chemokine receptor binding / positive regulation of immunological synapse formation / positive regulation of T cell costimulation / positive regulation of humoral immune response / chemokine (C-C motif) ligand 21 signaling pathway / lymphocyte migration into lymph node / mature conventional dendritic cell differentiation / positive regulation of dendritic cell antigen processing and presentation / Adenylate cyclase inhibitory pathway / positive regulation of myeloid dendritic cell chemotaxis / negative regulation of leukocyte tethering or rolling / establishment of T cell polarity / positive regulation of dendritic cell chemotaxis / regulation of interleukin-1 beta production / chemokine receptor binding / positive regulation of phospholipase C/protein kinase C signal transduction / immunological synapse formation / positive regulation of T cell chemotaxis / CCR chemokine receptor binding / regulation of type II interferon production / positive regulation of glycoprotein biosynthetic process / positive regulation of pseudopodium assembly / negative regulation of interleukin-12 production / negative thymic T cell selection / negative regulation of dendritic cell apoptotic process / positive regulation of chemotaxis / ruffle organization / cellular response to chemokine / C-C chemokine receptor activity / eosinophil chemotaxis / positive regulation of T cell receptor signaling pathway / positive regulation of cell adhesion mediated by integrin / regulation of Cdc42 protein signal transduction / positive regulation of neutrophil chemotaxis / chemokine activity / positive regulation of filopodium assembly / Chemokine receptors bind chemokines / positive regulation of cell motility / ADP signalling through P2Y purinoceptor 12 / Adrenaline,noradrenaline inhibits insulin secretion / dendritic cell chemotaxis / Extra-nuclear estrogen signaling / response to nitric oxide / positive regulation of cell-matrix adhesion / G alpha (i) signalling events / cellular response to cytokine stimulus / positive regulation of actin filament polymerization / homeostasis of number of cells / positive regulation of T cell migration / positive regulation of Rac protein signal transduction / T cell costimulation / release of sequestered calcium ion into cytosol / positive regulation of interleukin-12 production / cell maturation / positive regulation of protein localization to cell cortex / positive regulation of cell adhesion / G protein-coupled serotonin receptor binding / cellular response to forskolin / regulation of mitotic spindle organization / chemokine-mediated signaling pathway / cell chemotaxis / calcium-mediated signaling / positive regulation of receptor-mediated endocytosis / positive regulation of JNK cascade / G protein-coupled receptor binding / G protein-coupled receptor activity / G-protein beta/gamma-subunit complex binding / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / Olfactory Signaling Pathway / Activation of the phototransduction cascade / G protein-coupled acetylcholine receptor signaling pathway / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Thromboxane signalling through TP receptor / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / G-protein activation / Glucagon signaling in metabolic regulation / Prostacyclin signalling through prostacyclin receptor / G beta:gamma signalling through CDC42 / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / G beta:gamma signalling through BTK / photoreceptor disc membrane / ADP signalling through P2Y purinoceptor 12 / Sensory perception of sweet, bitter, and umami (glutamate) taste / Glucagon-type ligand receptors / GDP binding
Similarity search - Function
CC chemokine receptor 7 / Chemokine CC, DCCL motif-cointaining domain / Chemokine receptor family / Chemokine beta/gamma/delta / Intercrine alpha family (small cytokine C-X-C) (chemokine CXC). / Chemokine interleukin-8-like domain / Chemokine interleukin-8-like superfamily / Small cytokines (intecrine/chemokine), interleukin-8 like / : / Calycin ...CC chemokine receptor 7 / Chemokine CC, DCCL motif-cointaining domain / Chemokine receptor family / Chemokine beta/gamma/delta / Intercrine alpha family (small cytokine C-X-C) (chemokine CXC). / Chemokine interleukin-8-like domain / Chemokine interleukin-8-like superfamily / Small cytokines (intecrine/chemokine), interleukin-8 like / : / Calycin / G-protein alpha subunit, group I / G protein alpha subunit, helical insertion / G protein alpha subunit / Guanine nucleotide binding protein (G-protein), alpha subunit / G-protein alpha subunit / G-alpha domain profile. / G-protein, gamma subunit / G-protein gamma subunit domain profile. / G-protein gamma-like domain / G-protein gamma-like domain superfamily / GGL domain / G protein gamma subunit-like motifs / GGL domain / G protein beta WD-40 repeat protein / Guanine nucleotide-binding protein, beta subunit / G-protein, beta subunit / G-protein coupled receptors family 1 signature. / 7 transmembrane receptor (rhodopsin family) / G protein-coupled receptor, rhodopsin-like / GPCR, rhodopsin-like, 7TM / G-protein coupled receptors family 1 profile. / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
CHOLESTEROL / Non structural polyprotein / C-C motif chemokine 21 / C-C chemokine receptor type 7 / Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 / Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Guanine nucleotide-binding protein G(i) subunit alpha-1
Similarity search - Component
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsTsutsumi, N. / Nishikawa, K. / Fujiyoshi, Y.
Funding support Japan, 3items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)24K01965 Japan
Japan Society for the Promotion of Science (JSPS)24K21935 Japan
Japan Society for the Promotion of Science (JSPS)22K20632 Japan
CitationJournal: Proc Natl Acad Sci U S A / Year: 2026
Title: Structural insights into biased signaling at chemokine receptor CCR7.
Authors: Kotaro Tanaka / Kouki Nishikawa / Yuki Shiimura / Yoshinori Fujiyoshi / Naotaka Tsutsumi /
Abstract: CC chemokine receptor 7 (CCR7), which orchestrates adaptive immunity, exhibits a phenomenon known as biased agonism. CCL19 induces robust G-protein signaling and β-arrestin recruitment, leading to ...CC chemokine receptor 7 (CCR7), which orchestrates adaptive immunity, exhibits a phenomenon known as biased agonism. CCL19 induces robust G-protein signaling and β-arrestin recruitment, leading to transient signaling. In contrast, CCL21 preferentially activates G-protein pathways with minimal arrestin engagement, resulting in sustained signaling and differential functional outcomes. Here, we present the cryo-EM structures of the human CCR7-G complex with either CCL19 or CCL21. The structures reveal that while both engage a conserved orthosteric pocket, they adopt markedly distinct binding poses. Notably, the compact 30s loop of CCL21 inserts deeply into the receptor's extracellular vestibule, whereas the corresponding loop of CCL19 rests atop extracellular loop 2. Molecular dynamics simulations further reveal that these distinct binding modes induce differential intracellular dynamics, linked to the rotameric state of Y83 at the intracellular end of transmembrane helix 1. We demonstrate that CCL19 stabilizes a flexible conformational ensemble with a highly dynamic helix 8, creating a lateral opening favorable for GPCR kinase engagement. Conversely, CCL21 restricts this flexibility, locking the receptor in a state that precludes kinase interaction while maintaining G-protein coupling. Corroborated by functional data, these findings provide key insights into the structural basis of biased agonism at CCR7 and establish a foundation for rational design of pathway-selective immunomodulators.
History
DepositionNov 1, 2025Deposition site: PDBJ / Processing site: PDBJ
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
L: C-C motif chemokine 21
R: C-C chemokine receptor type 7,Non structural polyprotein
A: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2,Guanine nucleotide-binding protein G(i) subunit alpha-1
B: Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
C: Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2,Guanine nucleotide-binding protein G(i) subunit alpha-1
D: Antibody fragment scFv16
hetero molecules


Theoretical massNumber of molelcules
Total (without water)240,2857
Polymers239,8996
Non-polymers3871
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 2 types, 2 molecules LR

#1: Protein C-C motif chemokine 21 / 6Ckine / Beta-chemokine exodus-2 / Secondary lymphoid-tissue chemokine / SLC / Small-inducible cytokine A21


Mass: 13652.765 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: CCL21 with a C-terminal linker to an expression/purification tag and the 3C protease scar
Source: (gene. exp.) Homo sapiens (human) / Gene: CCL21, SCYA21, UNQ784/PRO1600 / Production host: Homo sapiens (human) / References: UniProt: O00585
#2: Protein C-C chemokine receptor type 7,Non structural polyprotein / C-C CKR-7 / CC-CKR-7 / CCR-7 / BLR2 / CDw197 / Epstein-Barr virus-induced G-protein coupled ...C-C CKR-7 / CC-CKR-7 / CCR-7 / BLR2 / CDw197 / Epstein-Barr virus-induced G-protein coupled receptor 1 / EBI1 / EBV-induced G-protein coupled receptor 1 / MIP-3 beta receptor


Mass: 61585.000 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: CCR7 with a C-terminal LgBit-His tag. CLR is a bound small molecule, not a part of the polypeptide.,CCR7 with a C-terminal LgBit-His tag. CLR is a bound small molecule, not a part of the polypeptide.
Source: (gene. exp.) Homo sapiens (human) / Gene: CCR7, CMKBR7, EBI1, EVI1 / Production host: Homo sapiens (human) / References: UniProt: P32248, UniProt: A0A482LYE4

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Guanine nucleotide-binding protein ... , 2 types, 3 molecules ACB

#3: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2,Guanine nucleotide-binding protein G(i) subunit alpha-1 / G gamma-I / Adenylate cyclase-inhibiting G alpha protein


Mass: 48640.371 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: chimeric protein with molecule 5 Gg2-Galpha(i)1 single chain fusion assigned a different chain ID for residue numbering,chimeric protein with molecule 5 Gg2-Galpha(i)1 single chain fusion ...Details: chimeric protein with molecule 5 Gg2-Galpha(i)1 single chain fusion assigned a different chain ID for residue numbering,chimeric protein with molecule 5 Gg2-Galpha(i)1 single chain fusion assigned a different chain ID for residue numbering
Source: (gene. exp.) Homo sapiens (human) / Gene: GNG2, GNAI1 / Production host: Homo sapiens (human)
References: UniProt: P59768, UniProt: P63097, Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement
#4: Protein Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 / Transducin beta chain 1


Mass: 40039.648 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Gb1 with a C-terminal HiBit tag. The 3C protease scar and a short linker at the N-terminus.
Source: (gene. exp.) Homo sapiens (human) / Gene: GNB1 / Production host: Homo sapiens (human) / References: UniProt: P62873

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Antibody / Non-polymers , 2 types, 2 molecules D

#5: Antibody Antibody fragment scFv16


Mass: 27340.482 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Trichoplusia ni (cabbage looper)
#6: Chemical ChemComp-CLR / CHOLESTEROL


Mass: 386.654 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C27H46O

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Details

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-IDSource
1CCL21-CCR7-Gi-scFv16COMPLEX#1-#50MULTIPLE SOURCES
2CCL21COMPLEX#11RECOMBINANT
3CCR7-GiCOMPLEX#2-#41RECOMBINANT
4scFv16COMPLEX#51RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.19 MDaNO
210.01 MDaNO
310.15 MDaNO
410.03 MDaNO
54
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
32Homo sapiens (human)9606
43Homo sapiens (human)9606
54Mus musculus (house mouse)10090
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
32Homo sapiens (human)9606
43Homo sapiens (human)9606
54Trichoplusia ni (cabbage looper)7111
Buffer solutionpH: 7.2
Buffer component
IDConc.NameBuffer-ID
110 mMHepes-Na1
2150 mMNaCl1
30.001 % (w/v)LMNG1
40.001 % (w/v)GDN1
50.0001 % (w/v)CHS1
SpecimenConc.: 4.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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 K

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Electron microscopy imaging

MicroscopyModel: JEOL CRYO ARM 300
Details: The microscope model is the JEOL's "JEM-Z320FHC", a custom-built model of JEOL CRYO ARM 300.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Calibrated magnification: 63291 X / Nominal defocus max: 1800 nm / Nominal defocus min: 1000 nm / Cs: 3.4 mm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 5968
EM imaging opticsEnergyfilter name: In-column Omega Filter / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.7particle selection
2SerialEMimage acquisition
4cryoSPARC4.7CTF correction
7Coot0.9.8.96 ELmodel fitting
9cryoSPARC4.7initial Euler assignment
10cryoSPARC4.7final Euler assignment
11cryoSPARC4.7classification
12cryoSPARC4.73D reconstruction
13PHENIX1.21.1_5286model refinement
CTF correctionDetails: CryoSPARC patch CTF estimation and global/local CTF refinements
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1537777 / Details: initial particles that appeared to be protein
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 195011 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingB value: 60 / Protocol: FLEXIBLE FIT / Space: REAL
Atomic model building

3D fitting-ID: 1

IDPDB-IDDetails (eV)Source nameTypeAccession codeInitial refinement model-ID
1CCL21-CCR7-GiAlphaFoldin silico model
27JHJ

7jhj

scFv16PDBexperimental model7JHJ2
RefinementHighest resolution: 3.2 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0039643
ELECTRON MICROSCOPYf_angle_d0.61213067
ELECTRON MICROSCOPYf_dihedral_angle_d5.4411318
ELECTRON MICROSCOPYf_chiral_restr0.0451504
ELECTRON MICROSCOPYf_plane_restr0.0041637

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