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- PDB-6y9b: Cryo-EM structure of trimeric human STEAP1 bound to three Fab120.... -

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Basic information

Entry
Database: PDB / ID: 6y9b
TitleCryo-EM structure of trimeric human STEAP1 bound to three Fab120.545 fragments
Components
  • Fab120.545 heavy chain
  • Fab120.545 light chain
  • Metalloreductase STEAP1
KeywordsMEMBRANE PROTEIN / Integral membrane protein Heme-binding protein Oxidoreductase Antibody-antigen complex
Function / homology
Function and homology information


cell-cell junction / endosome membrane / endosome / heme binding / membrane / metal ion binding / plasma membrane
Similarity search - Function
Ferric reductase transmembrane component-like domain / Ferric reductase like transmembrane component
Similarity search - Domain/homology
PROTOPORPHYRIN IX CONTAINING FE / 1,2-DIMYRISTOYL-SN-GLYCERO-3-PHOSPHATE / STEAP1 protein
Similarity search - Component
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.97 Å
AuthorsOosterheert, W. / Gros, P.
Funding support Netherlands, 1items
OrganizationGrant numberCountry
Netherlands Organisation for Scientific Research (NWO)731.015.201 Netherlands
CitationJournal: J Biol Chem / Year: 2020
Title: Cryo-electron microscopy structure and potential enzymatic function of human six-transmembrane epithelial antigen of the prostate 1 (STEAP1).
Authors: Wout Oosterheert / Piet Gros /
Abstract: Six-transmembrane epithelial antigen of the prostate 1 (STEAP1) is an integral membrane protein that is highly up-regulated on the cell surface of several human cancers, making it a promising ...Six-transmembrane epithelial antigen of the prostate 1 (STEAP1) is an integral membrane protein that is highly up-regulated on the cell surface of several human cancers, making it a promising therapeutic target to manage these diseases. It shares sequence homology with three enzymes (STEAP2-STEAP4) that catalyze the NADPH-dependent reduction of iron(III). However, STEAP1 lacks an intracellular NADPH-binding domain and does not exhibit cellular ferric reductase activity. Thus, both the molecular function of STEAP1 and its role in cancer progression remain elusive. Here, we present a ∼3.0-Å cryo-EM structure of trimeric human STEAP1 bound to three antigen-binding fragments (Fabs) of the clinically used antibody mAb120.545. The structure revealed that STEAP1 adopts a reductase-like conformation and interacts with the Fabs through its extracellular helices. Enzymatic assays in human cells revealed that STEAP1 promotes iron(III) reduction when fused to the intracellular NADPH-binding domain of its family member STEAP4, suggesting that STEAP1 functions as a ferric reductase in STEAP heterotrimers. Our work provides a foundation for deciphering the molecular mechanisms of STEAP1 and may be useful in the design of new therapeutic strategies to target STEAP1 in cancer.
History
DepositionMar 6, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 20, 2020Provider: repository / Type: Initial release
Revision 1.1May 27, 2020Group: Database references / Category: citation
Item: _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Jul 22, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.title / _citation_author.identifier_ORCID

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Structure visualization

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Assembly

Deposited unit
C: Metalloreductase STEAP1
L: Fab120.545 light chain
H: Fab120.545 heavy chain
I: Fab120.545 heavy chain
J: Fab120.545 heavy chain
M: Fab120.545 light chain
N: Fab120.545 light chain
A: Metalloreductase STEAP1
B: Metalloreductase STEAP1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)287,65815
Polymers284,0339
Non-polymers3,6256
Water0
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area32390 Å2
ΔGint-228 kcal/mol
Surface area57530 Å2
MethodPISA

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Components

#1: Protein Metalloreductase STEAP1 / Six-transmembrane epithelial antigen of prostate 1


Mass: 44183.652 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: STEAP1, PRSS24, STEAP / Plasmid: pUPE 2959
Details (production host): Plasmid contains C-terminal Strep3-tag
Cell line (production host): HEK293 GNT1- / Organ (production host): kidney / Production host: Homo sapiens (human) / Tissue (production host): embryonic kidney
References: UniProt: Q9UHE8, Oxidoreductases; Oxidizing metal ions; With NAD+ or NADP+ as acceptor
#2: Antibody Fab120.545 light chain


Mass: 24344.135 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Details: Antibody fragment generated through mouse immunization
Source: (gene. exp.) Mus musculus (house mouse) / Cell line (production host): HEK293 / Production host: Homo sapiens (human)
#3: Antibody Fab120.545 heavy chain


Mass: 26149.922 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Details: Antibody fragment generated through mouse immunization
Source: (gene. exp.) Mus musculus (house mouse) / Cell line (production host): HEK293 / Production host: Homo sapiens (human)
#4: Chemical ChemComp-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME / Heme B


Mass: 616.487 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C34H32FeN4O4 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-XP4 / 1,2-DIMYRISTOYL-SN-GLYCERO-3-PHOSPHATE


Mass: 591.777 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C31H60O8P / Comment: DMPA, phospholipid*YM
Has ligand of interestY

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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
IDNameTypeDetailsEntity IDParent-IDSource
1Trimeric human six-transmembrane epithelial antigen of the prostate (STEAP1) bound to three Fab-fragments of antibody mAb120.545COMPLEXSTEAP1 was expressed in HEK293 GNT1- cells and purified in digitonin. Fab120.545 was a kind gift from Genmab BV and was produced upon our request.#1-#30RECOMBINANT
2Fab-fragment 1 of antibody mAb120.545COMPLEX#2-#31RECOMBINANT
3Homo-trimeric STEAP1, chain a, b and c.COMPLEX#11RECOMBINANT
4Fab Fragment 2 of antibody mAb120.545COMPLEX#2-#31RECOMBINANT
5Fab-fragment 3 of antibody mAb120.545COMPLEX#2-#31RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.284 MDaNO
210.052 MDaNO
310.134 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDCellular location
21Homo sapiens (human)9606plasma membrane
32house mouse (house mouse)10090secreted
43Homo sapiens (human)9606plasma membrane
54Mus musculus (house mouse)10090secreted
65Mus musculus (house mouse)10090secreted
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDCellPlasmid
21Homo sapiens (human)9606HEK293 GNT1-pUPE 2959
32Homo sapiens (human)9606HEK293
43Homo sapiens (human)9606HEK293 GNT1-pUPE 2959
54Homo sapiens (human)9606HEK293
65Homo sapiens (human)9606HEK293
Buffer solutionpH: 7.8
Details: Protein sample was subjected to size-exclusion chromatography in 20 mM Tris pH 7.8, 200 mM NaCl, 0.08% (w/v) digitonin. 1 mM FAD (final concentration) was added to the sample before vitrification.
Buffer component
IDConc.NameFormulaBuffer-ID
1200 mMsodium chlorideNaClSodium chloride1
220 mMTris1
30.08 % (w/v)digitonin1
41 mMFlavin-Adenine dinucleotide (FAD)1
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: STEAP1-Fab120.545 complex purified in digitonin. The sample was monodisperse.
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 293 K / Details: blot 4 seconds, blotforce 0

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Details: Data was collected on a 200 kV Talos Arctica, located at Utrecht University, the Netherlands.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 130000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Calibrated defocus min: 400 nm / Calibrated defocus max: 3500 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 6.5 sec. / Electron dose: 49.5 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5325
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 26 / Used frames/image: 1-26

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Processing

SoftwareName: PHENIX / Version: dev_3689: / Classification: refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
4Gctf1.18CTF correction
7UCSF Chimera1.12model fitting
9RELION3initial Euler assignment
10RELION3.1betafinal Euler assignment
11RELION3.1betaclassification
12RELION3.1beta3D reconstruction
13PHENIX1.17.1-3660model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 616302 / Details: Picking was performed in Relion.
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 2.97 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 169426 / Details: Final reconstruction was generated in Relion. / Symmetry type: POINT
Atomic model buildingB value: 100.9 / Protocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 6HCY

6hcy


Pdb chain-ID: A / Pdb chain residue range: 196-454
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00412246
ELECTRON MICROSCOPYf_angle_d0.52616701
ELECTRON MICROSCOPYf_dihedral_angle_d17.6844404
ELECTRON MICROSCOPYf_chiral_restr0.0421842
ELECTRON MICROSCOPYf_plane_restr0.0032004

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