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- PDB-9yc6: Mutant human uPAR bound to the Fab fragment of the targeted cance... -

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Basic information

Entry
Database: PDB / ID: 9yc6
TitleMutant human uPAR bound to the Fab fragment of the targeted cancer therapeutic antibody FL1
Components
  • (Anti-uPAR antibody FL1 Fab ...) x 2
  • Urokinase plasminogen activator surface receptor
KeywordsIMMUNE SYSTEM / Complex / Targeted cancer therapy / Monoclonal anti-uPAR antibody FL1 / Antibody-drug conjugate
Function / homology
Function and homology information


urokinase plasminogen activator receptor activity / Attachment of GPI anchor to uPAR / positive regulation of homotypic cell-cell adhesion / urokinase plasminogen activator signaling pathway / regulation of plasminogen activation / regulation of fibrinolysis / protein complex involved in cell-matrix adhesion / serine-type endopeptidase complex / Dissolution of Fibrin Clot / positive regulation of epidermal growth factor receptor signaling pathway ...urokinase plasminogen activator receptor activity / Attachment of GPI anchor to uPAR / positive regulation of homotypic cell-cell adhesion / urokinase plasminogen activator signaling pathway / regulation of plasminogen activation / regulation of fibrinolysis / protein complex involved in cell-matrix adhesion / serine-type endopeptidase complex / Dissolution of Fibrin Clot / positive regulation of epidermal growth factor receptor signaling pathway / extrinsic component of membrane / positive regulation of DNA binding / negative regulation of intrinsic apoptotic signaling pathway / positive regulation of release of cytochrome c from mitochondria / regulation of proteolysis / regulation of cell adhesion / specific granule membrane / cell projection / chemotaxis / positive regulation of protein phosphorylation / blood coagulation / signaling receptor activity / endoplasmic reticulum lumen / protein domain specific binding / signaling receptor binding / external side of plasma membrane / focal adhesion / Neutrophil degranulation / endoplasmic reticulum membrane / negative regulation of apoptotic process / enzyme binding / cell surface / signal transduction / extracellular region / membrane / plasma membrane
Similarity search - Function
CD59 antigen, conserved site / Ly-6 / u-PAR domain signature. / u-PAR/Ly-6 domain / Ly-6 antigen / uPA receptor -like domain / Ly-6 antigen/uPA receptor-like / Snake toxin-like superfamily
Similarity search - Domain/homology
Urokinase plasminogen activator surface receptor
Similarity search - Component
Biological speciesMus musculus (house mouse)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.8 Å
AuthorsAnane, R.F. / Whisstock, J.C. / Engelholm, L.H. / Law, R.H.P. / Ploug, M.
Funding support Australia, 4items
OrganizationGrant numberCountry
Other private
Other governmentR231-A13832 R352-A20542
Australian Research Council (ARC)FL180100019 Australia
National Health and Medical Research Council (NHMRC, Australia)APP2000728 Australia
CitationJournal: Protein Sci / Year: 2026
Title: Structural basis for uPAR binding to an antibody developed for targeted cancer therapy. Mechanistic insights into flexibility, ligand recognition, and molecular imaging.
Authors: Rex F Anane / Anni Kumari / Hari Venugopal / Sylvain Trépout / James C Whisstock / Lars H Engelholm / Ruby H P Law / Michael Ploug /
Abstract: The urokinase-type plasminogen activator receptor (uPAR) is currently gaining momentum as a promising molecular target for treatment of various solid cancers. For patient stratification, we developed ...The urokinase-type plasminogen activator receptor (uPAR) is currently gaining momentum as a promising molecular target for treatment of various solid cancers. For patient stratification, we developed a high-affinity uPAR-targeting peptide (AE105) detecting primary cancer lesions as well as occult metastasis by positron emission tomography (PET) imaging. uPAR-targeting by AE105 is also used for optical imaging in fluorescence-guided surgery of, for example, head-and-neck cancers. Recently, we showed that a monoclonal anti-uPAR antibody (FL1), in the form of an antibody-drug conjugate (FL1-ADC), efficiently eradicate pancreatic ductal carcinomas in surrogate mouse models leading to long-term remissions. In the current study, we solved high-resolution cryo-EM structures of FL1 in complex with two different conformational states of uPAR. Combined with comprehensive kinetic data from surface plasmon resonance studies, our cryo-EM structures provide essential insights into how FL1 binding impacts the interdomain flexibility of uPAR by restricting the movement of its N-terminal LU domain. This constraint from the bound FL1 drives uPAR into its open conformation, which leads to a pronounced reduction in the binding affinity for both its natural protease ligand (300-fold) and the PET imaging probe AE105 (25-fold). Collectively, these consequences of FL1-binding on uPAR conformation are considered beneficial for both targeted cancer treatment with FL1-ADCs and for the accompanying evaluation of treatment efficacy by longitudinal AE105-based PET imaging.
History
DepositionSep 18, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 4, 2026Provider: repository / Type: Initial release
Revision 1.0Feb 4, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Feb 4, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Feb 4, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Feb 4, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Feb 4, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Feb 4, 2026Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Feb 4, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
L: Anti-uPAR antibody FL1 Fab light chain
U: Urokinase plasminogen activator surface receptor
H: Anti-uPAR antibody FL1 Fab heavy chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)80,6597
Polymers79,2053
Non-polymers1,4534
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 , 1 types, 1 molecules U

#2: Protein Urokinase plasminogen activator surface receptor / U-PAR / uPAR / Monocyte activation antigen Mo3


Mass: 31455.377 Da / Num. of mol.: 1 / Mutation: H47C, N259C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PLAUR, MO3, UPAR / Cell line (production host): S2 / Production host: Drosophila melanogaster (fruit fly) / References: UniProt: Q03405

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Antibody , 2 types, 2 molecules LH

#1: Antibody Anti-uPAR antibody FL1 Fab light chain


Mass: 24168.818 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Cell (production host): hybridoma / Cell line (production host): hybridoma / Production host: Mus musculus (house mouse)
#3: Antibody Anti-uPAR antibody FL1 Fab heavy chain


Mass: 23581.258 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Cell (production host): hybridoma / Cell line (production host): hybridoma / Production host: Mus musculus (house mouse)

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Sugars , 3 types, 4 molecules

#4: Polysaccharide beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}LINUCSPDB-CARE
#5: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 1 / Source method: obtained synthetically
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE
#6: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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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

ComponentName: Binary complex of mutant human uPAR and Fab fragment of anti-uPAR antibody FL1
Type: COMPLEX
Details: Full-length mutant human uPAR, and full-length antibody FL1 (IgG) were mixed to form the protein complex which was used for EM sample.
Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Drosophila melanogaster (fruit fly)
Buffer solutionpH: 7.4
Details: 20mM Tris, 150mM NaCl, 2.5% Glycerol, 1mM EDTA, pH 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris(hydroxymethyl)aminomethaneC4H11NO31
2150 mMSodium chlorideNaCl1
32.5 %GlycerolC3H8O31
41 mMEDTAC10H16N2O81
SpecimenConc.: 3.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Full-length mutant human uPAR, and full-length antibody FL1 (IgG) were mixed in 2:1 molar ratio to form the protein complex which was used for EM sample.
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 165000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 12.06 e/Å2 / Film or detector model: TFS FALCON 4i (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 6669 / Details: Images were collected at a pixel size of 0.75
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 10 eV / Phase plate: VOLTA PHASE PLATE
Image scansWidth: 4092 / Height: 4092

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC4.5.3particle selection
2EPU3.9image acquisitionImages were automatically collected using EPU software
4cryoSPARC4.5.3CTF correction
7ISOLDE1.10.1model fitting
9cryoSPARC4.5.3initial Euler assignment
10cryoSPARC4.5.3final Euler assignment
11cryoSPARC4.5.3classification
12cryoSPARC4.5.33D reconstruction
19PHENIX1.21.2_5419:model refinement
Image processingDetails: Following motion correction, 4668 images were selected for particle picking
CTF correctionDetails: CTF corrections were performed after 3D reconstruction to improve the final resolution
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 8296036
Details: The 8296036 particles were picked by automated particle picking using blob picker. Several rounds of 2D classification were performed to discard particles of low quality, producing 86404 ...Details: The 8296036 particles were picked by automated particle picking using blob picker. Several rounds of 2D classification were performed to discard particles of low quality, producing 86404 particles of good quality which were used for 3D reconstruction.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 86404 / Algorithm: FOURIER SPACE / Num. of class averages: 14 / Symmetry type: POINT
Atomic model buildingB value: 92 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: cross-correlation coefficient
Details: An initial local fitting was performed with ISOLDE implemented in ChimeraX prior to real-space refinement in phenix.
Atomic model buildingPDB-ID: 3u74

3u74


Accession code: 3u74 / Chain residue range: 23-301 / Pdb chain residue range: 23-301 / Source name: PDB / Type: experimental model

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