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- PDB-9hlw: Prostate Specific Membrane Antigen (PSMA) in complex with nanobody7 -

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Basic information

Entry
Database: PDB / ID: 9hlw
TitleProstate Specific Membrane Antigen (PSMA) in complex with nanobody7
Components
  • Glutamate carboxypeptidase 2
  • Nano body 7
KeywordsMEMBRANE PROTEIN / Nanobodies their Prostate Specific Membrane Antigen PSMA prostate cancer
Function / homology
Function and homology information


Ac-Asp-Glu binding / tetrahydrofolyl-poly(glutamate) polymer binding / glutamate carboxypeptidase II / folic acid-containing compound metabolic process / C-terminal protein deglutamylation / Aspartate and asparagine metabolism / dipeptidase activity / carboxypeptidase activity / metallocarboxypeptidase activity / peptidase activity ...Ac-Asp-Glu binding / tetrahydrofolyl-poly(glutamate) polymer binding / glutamate carboxypeptidase II / folic acid-containing compound metabolic process / C-terminal protein deglutamylation / Aspartate and asparagine metabolism / dipeptidase activity / carboxypeptidase activity / metallocarboxypeptidase activity / peptidase activity / cell surface / proteolysis / extracellular exosome / metal ion binding / membrane / plasma membrane / cytoplasm
Similarity search - Function
Transferrin receptor-like, dimerisation domain / Transferrin receptor-like, dimerisation domain superfamily / Glutamate carboxypeptidase 2-like / Transferrin receptor-like dimerisation domain / PA domain superfamily / PA domain / PA domain / Peptidase M28 / Peptidase family M28
Similarity search - Domain/homology
Glutamate carboxypeptidase 2
Similarity search - Component
Biological speciesHomo sapiens (human)
Camelus dromedarius (Arabian camel)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.66 Å
AuthorsAlon, G. / Papo, N. / Zarivach, R. / Zalk, R.
Funding support United States, United Kingdom, Israel, 6items
OrganizationGrant numberCountry
Other private846497
United States - Israel Binational Science Foundation (BSF)2019303 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM144393 United States
Rosetrees Trust(OoR2022/100004 United Kingdom
Israel Science Foundation1615/19 Israel
Worldwide Cancer Research20-0238 United Kingdom
CitationJournal: Int J Biol Macromol / Year: 2025
Title: Structural analysis of nanobody interactions with their prostate-specific membrane antigen binding epitopes.
Authors: Gal Alon-Zchut / Ran Zalk / Truc T Huynh / Michael R Zalutsky / Yossi Weizmann / Raz Zarivach / Niv Papo /
Abstract: Prostate-specific membrane antigen (PSMA), overexpressed in prostate cancer, is a promising target for diagnostics and therapy. However, the monoclonal antibodies in current use for PSMA targeting ...Prostate-specific membrane antigen (PSMA), overexpressed in prostate cancer, is a promising target for diagnostics and therapy. However, the monoclonal antibodies in current use for PSMA targeting and inhibition have suboptimal activities due to their poor tissue and cell penetration and slow normal tissue clearance. Potentially superior alternatives are nanobodies (NBs), the single-chain variable domains of heavy-chain antibodies derived from camelids. The advantages of NBs include small size (~15 kDa), ability to bind hidden epitopes, and rapid clearance. In contrast to most known PSMA inhibitors, which bind to the same catalytic site in PMSA, NBs can bind to different PSMA epitopes, facilitating heterovalent binding strategies that could enhance their therapeutic and diagnostic potential. The objective of this study was to map these binding epitopes and hence to acquire an atomic-resolution understanding of NB-PMSA binding by investigating the structural interactions between PSMA and three NBs (NB7, NB8, and NB37). Using cryo-electron microscopy to generate high-resolution structures of NB-PSMA complexes, we found that NB7 had the highest affinity for PSMA due to a larger interface and to stabilizing interactions, including salt bridges and π-π stacking. Notably, we also found that NB7 and NB8 can bind simultaneously to different PSMA epitopes without interfering with the function of PSMA (which is still not completely known), opening the way for the development of theranostic applications for prostate cancer treatment and imaging. Importantly, NB7 binds specifically to human PSMA but not to murine PSMA, due to key amino acid differences responsible for its species specificity.
History
DepositionDec 5, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 16, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Glutamate carboxypeptidase 2
E: Glutamate carboxypeptidase 2
H: Nano body 7
Q: Nano body 7
hetero molecules


Theoretical massNumber of molelcules
Total (without water)206,97725
Polymers200,9864
Non-polymers5,99021
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 / Antibody , 2 types, 4 molecules AEHQ

#1: Protein Glutamate carboxypeptidase 2 / Cell growth-inhibiting gene 27 protein / Folate hydrolase 1 / Folylpoly-gamma-glutamate ...Cell growth-inhibiting gene 27 protein / Folate hydrolase 1 / Folylpoly-gamma-glutamate carboxypeptidase / FGCP / Glutamate carboxypeptidase II / GCPII / Membrane glutamate carboxypeptidase / mGCP / N-acetylated-alpha-linked acidic dipeptidase I / NAALADase I / Prostate-specific membrane antigen / PSM / PSMA / Pteroylpoly-gamma-glutamate carboxypeptidase


Mass: 84426.445 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: FOLH1, FOLH, NAALAD1, PSM, PSMA, GIG27 / Production host: Escherichia coli (E. coli) / References: UniProt: Q04609, glutamate carboxypeptidase II
#2: Antibody Nano body 7


Mass: 16066.593 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Camelus dromedarius (Arabian camel) / Production host: Escherichia coli (E. coli)

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Sugars , 4 types, 14 molecules

#3: 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.: 2
Source method: isolated from a genetically manipulated source
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
#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.: 2
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 alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2- ...alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 910.823 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-3[DManpa1-6]DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/3,5,4/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3-3/a4-b1_b4-c1_c3-d1_c6-e1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{}[(6+1)][a-D-Manp]{}}}}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.: 8 / Source method: obtained synthetically / Formula: C8H15NO6 / Feature type: SUBJECT OF INVESTIGATION
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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Non-polymers , 3 types, 7 molecules

#7: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn
#8: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca
#9: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl

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Details

Has ligand of interestY
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: PSMA in complex with nanobody 7 / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 230 kDa/nm / Experimental value: NO
Source (natural)Organism: Camelus (mammal)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE / Details: Homemade plunger, manually blotted for 3 seconds.

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 500 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 30 e/Å2 / Film or detector model: TFS FALCON 4i (4k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.20.1_4487 / Category: model refinement
CTF correctionType: NONE
3D reconstructionResolution: 2.66 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 478371 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 1Z8L

1z8l


Accession code: 1Z8L / Source name: PDB / Type: experimental model
RefinementHighest resolution: 2.66 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00313726
ELECTRON MICROSCOPYf_angle_d0.63218619
ELECTRON MICROSCOPYf_dihedral_angle_d9.2722006
ELECTRON MICROSCOPYf_chiral_restr0.0422019
ELECTRON MICROSCOPYf_plane_restr0.0042394

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