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- EMDB-72760: Human uPAR bound to the Fab fragment of targeted cancer therapeut... -

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Basic information

Entry
Database: EMDB / ID: EMD-72760
TitleHuman uPAR bound to the Fab fragment of targeted cancer therapeutic antibody FL1
Map data
Sample
  • Complex: Binary complex of human uPAR and Fab fragment of anti-uPAR antibody FL1
    • Protein or peptide: Urokinase plasminogen activator surface receptor
    • Protein or peptide: Anti-uPAR antibody FL1 Fab heavy chain
    • Protein or peptide: Anti-uPAR antibody FL1 Fab light chain
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
KeywordsComplex / Targeted cancer therapy / Monoclonal anti-uPAR antibody FL1 / Antibody-drug conjugate / IMMUNE SYSTEM
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 speciesHomo sapiens (human) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.94 Å
AuthorsAnane RF / Whisstock JC / Engelholm LH / Law RHP / Ploug M
Funding support Denmark, Australia, 4 items
OrganizationGrant numberCountry
Other private Denmark
Other governmentR231-A13832 and R352-A20542 Denmark
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, 2025-
Header (metadata) releaseFeb 4, 2026-
Map releaseFeb 4, 2026-
UpdateFeb 4, 2026-
Current statusFeb 4, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_72760.png

Downloads & links

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Map

FileDownload / File: emd_72760.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.82 Å/pix.
x 320 pix.
= 262.4 Å		0.82 Å/pix.
x 320 pix.
= 262.4 Å		0.82 Å/pix.
x 320 pix.
= 262.4 Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.82 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-1.6133835 - 2.8104153
Average (Standard dev.)0.0027221134 (±0.038525105)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 262.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_72760_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_72760_half_map_2.map
Projections & Slices
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Histogram

Histogram (log scale)

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Sample components

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Entire : Binary complex of human uPAR and Fab fragment of anti-uPAR antibo...

EntireName: Binary complex of human uPAR and Fab fragment of anti-uPAR antibody FL1
Components
  • Complex: Binary complex of human uPAR and Fab fragment of anti-uPAR antibody FL1
    • Protein or peptide: Urokinase plasminogen activator surface receptor
    • Protein or peptide: Anti-uPAR antibody FL1 Fab heavy chain
    • Protein or peptide: Anti-uPAR antibody FL1 Fab light chain
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: Binary complex of human uPAR and Fab fragment of anti-uPAR antibo...

SupramoleculeName: Binary complex of human uPAR and Fab fragment of anti-uPAR antibody FL1
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Details: Full-length human uPAR, and full-length antibody FL1 (IgG) were mixed to form the protein complex which was used for EM sample.
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Urokinase plasminogen activator surface receptor

MacromoleculeName: Urokinase plasminogen activator surface receptor / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 31.501342 KDa
Recombinant expressionOrganism: Drosophila melanogaster (fruit fly)
SequenceString: LRCMQCKTNG DCRVEECALG QDLCRTTIVR LWEEGEELEL VEKSCTHSEK TNRTLSYRTG LKITSLTEVV CGLDLCNQGN SGRAVTYSR SRYLECISCG SSDMSCERGR HQSLQCRSPE EQCLDVVTHW IQEGEEGRPK DDRHLRGCGY LPGCPGSNGF H NNDTFHFL ...String:
LRCMQCKTNG DCRVEECALG QDLCRTTIVR LWEEGEELEL VEKSCTHSEK TNRTLSYRTG LKITSLTEVV CGLDLCNQGN SGRAVTYSR SRYLECISCG SSDMSCERGR HQSLQCRSPE EQCLDVVTHW IQEGEEGRPK DDRHLRGCGY LPGCPGSNGF H NNDTFHFL KCCNTTKCNE GPILELENLP QNGRQCYSCK GNSTHGCSSE ETFLIDCRGP MNQCLVATGT HEPKNQSYMV RG CATASMC QHAHLGDAFS MNHIDVSCCT KSGCNHPDLD VQYRSG

UniProtKB: Urokinase plasminogen activator surface receptor

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Macromolecule #2: Anti-uPAR antibody FL1 Fab heavy chain

MacromoleculeName: Anti-uPAR antibody FL1 Fab heavy chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 23.581258 KDa
Recombinant expressionOrganism: Mus musculus (house mouse)
SequenceString: EVQLQQSGSV LVRPGTSVNL SCKASGYTFT SYWMSWVKQR PGQGLEWIGE IFPKSGRINS NENFRGKATL TVDTSSSTAY INLSSLTSE DSAVYYCSPI YSGDYGFADW GQGTLVTVSA AKTTPPSVYP LAPGSAAQTN SMVTLGCLVK GYFPEPVTVT W NSGSLSSG ...String:
EVQLQQSGSV LVRPGTSVNL SCKASGYTFT SYWMSWVKQR PGQGLEWIGE IFPKSGRINS NENFRGKATL TVDTSSSTAY INLSSLTSE DSAVYYCSPI YSGDYGFADW GQGTLVTVSA AKTTPPSVYP LAPGSAAQTN SMVTLGCLVK GYFPEPVTVT W NSGSLSSG VHTFPAVLQS DLYTLSSSVT VPSSTWPSET VTCNVAHPAS STKVDKKIVP RDC

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Macromolecule #3: Anti-uPAR antibody FL1 Fab light chain

MacromoleculeName: Anti-uPAR antibody FL1 Fab light chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 24.168818 KDa
Recombinant expressionOrganism: Mus musculus (house mouse)
SequenceString: DIVMTQTPLS LPVSLGDQAS ISCRSSQSVV SNNGKTYLEW YLQKPGQSPK LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDLG VYYCFQGSHV PWTFGGGTKL EVKRADAAPT VSIFPPSSEQ LTSGGASVVC FLNNFYPKDI NVKWKIDGSE R QNGVLNSW ...String:
DIVMTQTPLS LPVSLGDQAS ISCRSSQSVV SNNGKTYLEW YLQKPGQSPK LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDLG VYYCFQGSHV PWTFGGGTKL EVKRADAAPT VSIFPPSSEQ LTSGGASVVC FLNNFYPKDI NVKWKIDGSE R QNGVLNSW TDQDSKDSTY SMSSTLTLTK DEYERHNSYT CEATHKTSTS PIVKSFNRNE C

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Macromolecule #5: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 5 / Number of copies: 2 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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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

Concentration3.5 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
20.0 mMC4H11NO3Tris(hydroxymethyl)aminomethane
150.0 mMNaClSodium chloride
2.5 %C3H8O3Glycerol
1.0 mMC10H16N2O8EDTA

Details: 20mM Tris, 150mM NaCl, 2.5% Glycerol, 1mM EDTA, pH 7.4
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Pressure: 3.9 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
DetailsFull-length 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.

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsPhase plate: VOLTA PHASE PLATE / Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 10 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 7181 / Average exposure time: 4.92 sec. / Average electron dose: 10.7 e/Å2 / Details: Images were collected at a pixel size of 0.8234
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsFollowing motion correction, 4345 images were selected for particle picking
Particle selectionNumber selected: 7426690
Details: The 7426690 particles were picked by automated particle picking using blob picker. Several rounds of 2D classification were performed to discard particles of low quality, producing 184642 ...Details: The 7426690 particles were picked by automated particle picking using blob picker. Several rounds of 2D classification were performed to discard particles of low quality, producing 184642 particles of good quality which were used for 3D reconstruction.
CTF correctionSoftware - Name: cryoSPARC (ver. 4.5.3)
Details: CTF corrections were performed after 3D reconstruction to improve the final resolution
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionNumber classes used: 16 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.94 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.5.3) / Number images used: 184642
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.5.3)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.5.3)
Final 3D classificationNumber classes: 16 / Avg.num./class: 11540 / Software - Name: cryoSPARC (ver. 4.5.3)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

1-v4


Chain - Residue range: 114-299 / Chain - Source name: AlphaFold / Chain - Initial model type: in silico model
DetailsAn initial local fitting was performed with ISOLDE implemented in ChimeraX prior to real-space refinement in phenix.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 112 / Target criteria: cross-correlation coefficient
Output model

PDB-9yc5:
Human uPAR bound to the Fab fragment of targeted cancer therapeutic antibody FL1

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