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- PDB-8esv: Structure of human ADAM10-Tspan15 complex bound to 11G2 vFab -

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Basic information

Entry
Database: PDB / ID: 8esv
TitleStructure of human ADAM10-Tspan15 complex bound to 11G2 vFab
Components
  • 11G2 Fab Heavy Chain
  • 11G2 Fab Light Chain
  • Disintegrin and metalloproteinase domain-containing protein 10
  • Tetraspanin-15
KeywordsMEMBRANE PROTEIN / Protease / Metalloprotease / Tetraspanin / Sheddase / Adhesion
Function / homology
Function and homology information


regulation of membrane protein ectodomain proteolysis / ADAM10 endopeptidase / constitutive protein ectodomain proteolysis / regulation of vasculature development / epidermal growth factor receptor ligand maturation / monocyte activation / metalloendopeptidase activity involved in amyloid precursor protein catabolic process / postsynapse organization / protein catabolic process at postsynapse / : ...regulation of membrane protein ectodomain proteolysis / ADAM10 endopeptidase / constitutive protein ectodomain proteolysis / regulation of vasculature development / epidermal growth factor receptor ligand maturation / monocyte activation / metalloendopeptidase activity involved in amyloid precursor protein catabolic process / postsynapse organization / protein catabolic process at postsynapse / : / Constitutive Signaling by NOTCH1 t(7;9)(NOTCH1:M1580_K2555) Translocation Mutant / regulation of Notch signaling pathway / perinuclear endoplasmic reticulum / pore complex assembly / positive regulation of T cell chemotaxis / tetraspanin-enriched microdomain / NOTCH4 Activation and Transmission of Signal to the Nucleus / metallodipeptidase activity / negative regulation of cell adhesion / adherens junction organization / regulation of postsynapse organization / regulation of neurotransmitter receptor localization to postsynaptic specialization membrane / clathrin-coated vesicle / Golgi-associated vesicle / cochlea development / Signaling by EGFR / negative regulation of Notch signaling pathway / pore complex / amyloid precursor protein catabolic process / tertiary granule membrane / protein maturation / membrane protein ectodomain proteolysis / Collagen degradation / EPH-ephrin mediated repulsion of cells / extracellular matrix disassembly / response to tumor necrosis factor / specific granule membrane / Notch signaling pathway / Constitutive Signaling by NOTCH1 HD Domain Mutants / NOTCH2 Activation and Transmission of Signal to the Nucleus / Degradation of the extracellular matrix / Activated NOTCH1 Transmits Signal to the Nucleus / synaptic membrane / protein localization to plasma membrane / integrin-mediated signaling pathway / NOTCH3 Activation and Transmission of Signal to the Nucleus / Post-translational protein phosphorylation / adherens junction / protein processing / metalloendopeptidase activity / Constitutive Signaling by NOTCH1 PEST Domain Mutants / Constitutive Signaling by NOTCH1 HD+PEST Domain Mutants / SH3 domain binding / metallopeptidase activity / Regulation of Insulin-like Growth Factor (IGF) transport and uptake by Insulin-like Growth Factor Binding Proteins (IGFBPs) / integrin binding / cell junction / cell-cell signaling / late endosome membrane / positive regulation of cell growth / endopeptidase activity / in utero embryonic development / postsynaptic density / molecular adaptor activity / nuclear body / positive regulation of cell migration / Amyloid fiber formation / endoplasmic reticulum lumen / axon / Golgi membrane / protein phosphorylation / negative regulation of gene expression / signaling receptor binding / intracellular membrane-bounded organelle / focal adhesion / glutamatergic synapse / dendrite / Neutrophil degranulation / positive regulation of cell population proliferation / protein kinase binding / Golgi apparatus / enzyme binding / cell surface / protein homodimerization activity / extracellular exosome / membrane / metal ion binding / nucleus / plasma membrane / cytosol / cytoplasm
Similarity search - Function
: / ADAM10, cysteine-rich domain / Tetraspanin, animals / Tetraspanin, EC2 domain superfamily / Tetraspanin/Peripherin / ADAM10/ADAM17 catalytic domain / Tetraspanin family / Metallo-peptidase family M12B Reprolysin-like / Disintegrin / Disintegrin domain profile. ...: / ADAM10, cysteine-rich domain / Tetraspanin, animals / Tetraspanin, EC2 domain superfamily / Tetraspanin/Peripherin / ADAM10/ADAM17 catalytic domain / Tetraspanin family / Metallo-peptidase family M12B Reprolysin-like / Disintegrin / Disintegrin domain profile. / Homologues of snake disintegrins / Disintegrin domain / Disintegrin domain superfamily / Peptidase M12B, ADAM/reprolysin / ADAM type metalloprotease domain profile. / Metallopeptidase, catalytic domain superfamily / Neutral zinc metallopeptidases, zinc-binding region signature.
Similarity search - Domain/homology
Chem-BAT / CHOLESTEROL HEMISUCCINATE / Disintegrin and metalloproteinase domain-containing protein 10 / Tetraspanin-15
Similarity search - Component
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsLipper, C.H. / Blacklow, S.C.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)R35 CA220340 United States
Other privateGift from Edward B. Goodnow
CitationJournal: Cell / Year: 2023
Title: Structural basis for membrane-proximal proteolysis of substrates by ADAM10.
Authors: Colin H Lipper / Emily D Egan / Khal-Hentz Gabriel / Stephen C Blacklow /
Abstract: The endopeptidase ADAM10 is a critical catalyst for the regulated proteolysis of key drivers of mammalian development, physiology, and non-amyloidogenic cleavage of APP as the primary α-secretase. ...The endopeptidase ADAM10 is a critical catalyst for the regulated proteolysis of key drivers of mammalian development, physiology, and non-amyloidogenic cleavage of APP as the primary α-secretase. ADAM10 function requires the formation of a complex with a C8-tetraspanin protein, but how tetraspanin binding enables positioning of the enzyme active site for membrane-proximal cleavage remains unknown. We present here a cryo-EM structure of a vFab-ADAM10-Tspan15 complex, which shows that Tspan15 binding relieves ADAM10 autoinhibition and acts as a molecular measuring stick to position the enzyme active site about 20 Å from the plasma membrane for membrane-proximal substrate cleavage. Cell-based assays of N-cadherin shedding establish that the positioning of the active site by the interface between the ADAM10 catalytic domain and the bound tetraspanin influences selection of the preferred cleavage site. Together, these studies reveal the molecular mechanism underlying ADAM10 proteolysis at membrane-proximal sites and offer a roadmap for its modulation in disease.
History
DepositionOct 14, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 14, 2023Provider: repository / Type: Initial release
Revision 1.1Aug 9, 2023Group: Database references / Category: citation
Item: _citation.journal_abbrev / _citation.journal_id_CSD ..._citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Aug 16, 2023Group: Data collection / Category: chem_comp_atom / chem_comp_bond
Revision 1.3Aug 30, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Disintegrin and metalloproteinase domain-containing protein 10
B: Tetraspanin-15
H: 11G2 Fab Heavy Chain
L: 11G2 Fab Light Chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)146,65212
Polymers143,5394
Non-polymers3,1138
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 2 molecules AB

#1: Protein Disintegrin and metalloproteinase domain-containing protein 10 / ADAM 10 / CDw156 / Kuzbanian protein homolog / Mammalian disintegrin-metalloprotease


Mass: 60477.340 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ADAM10, KUZ, MADM / Plasmid: pRK5M / Cell line (production host): Expi293F / Production host: Homo sapiens (human) / References: UniProt: O14672, ADAM10 endopeptidase
#2: Protein Tetraspanin-15 / / Tspan-15 / Tetraspan NET-7 / Transmembrane 4 superfamily member 15


Mass: 34740.469 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TSPAN15, NET7, TM4SF15, UNQ677/PRO1311 / Plasmid: pcDNA3.1/Hygro(+) / Cell line (production host): Expi293F / Production host: Homo sapiens (human) / References: UniProt: O95858

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Antibody , 2 types, 2 molecules HL

#3: Antibody 11G2 Fab Heavy Chain


Mass: 24135.105 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Plasmid: pFUSE-hIgG1-Fc2
Details (production host): Heavy and light chain in same vector with P2A sequence
Cell line (production host): Expi293F / Production host: Homo sapiens (human)
#4: Antibody 11G2 Fab Light Chain


Mass: 24185.803 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Plasmid: pFUSE-hIgG1-Fc2
Details (production host): Heavy and light chain in same vector with P2A sequence
Cell line (production host): Expi293F / Production host: Homo sapiens (human)

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Sugars , 3 types, 3 molecules

#5: Polysaccharide alpha-D-mannopyranose-(1-3)-alpha-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1- ...alpha-D-mannopyranose-(1-3)-alpha-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 748.682 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-3DManpa1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,4,3/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1a_1-5]/1-1-2-2/a4-b1_b4-c1_c3-d1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][a-D-Manp]{[(3+1)][a-D-Manp]{}}}}LINUCSPDB-CARE
#6: Polysaccharide alpha-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...alpha-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
DManpa1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1a_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][a-D-Manp]{}}}LINUCSPDB-CARE
#7: 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 / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 1 / 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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Non-polymers , 4 types, 5 molecules

#8: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca
#9: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#10: Chemical ChemComp-BAT / 4-(N-HYDROXYAMINO)-2R-ISOBUTYL-2S-(2-THIENYLTHIOMETHYL)SUCCINYL-L-PHENYLALANINE-N-METHYLAMIDE / BATIMASTAT / BB94 / Batimastat


Mass: 477.640 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C23H31N3O4S2 / Comment: inhibitor*YM
#11: Chemical ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C31H50O4

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Details

Has ligand of interestN

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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
1ADAM10-Tspan15-11G2 Fab complexCOMPLEX#1-#40RECOMBINANT
2Disintegrin and metalloproteinase domain-containing protein 10 (E.C.3.4.24.81), Tetraspanin-15COMPLEX#1-#21RECOMBINANT
311G2 Fab Heavy Chain, 11G2 Fab Light ChainCOMPLEX#3-#41RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Mus musculus (house mouse)10090
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDCell
12Homo sapiens (human)9606Expi293F
23Homo sapiens (human)9606Expi293F
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPES1
2150 mMsodium chlorideNaClSodium chloride1
30.03 %glyco-diosgenin (GDN)1
40.003 %cholesteryl hemisuccinate1
SpecimenConc.: 2.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K / Details: Blot for 7 seconds with a blot force of 15

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 105000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.3 sec. / Electron dose: 51.99 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 10037

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Processing

EM software
IDNameVersionCategory
1cryoSPARC3.3.1particle selection
2SerialEMimage acquisition
4cryoSPARC3.3.1CTF correction
7UCSF Chimeramodel fitting
9PHENIXmodel refinement
10cryoSPARC3.3.1initial Euler assignment
11cryoSPARC3.3.1final Euler assignment
12cryoSPARC3.3.1classification
13cryoSPARC3.3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 178031 / Symmetry type: POINT
Atomic model buildingSpace: REAL

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