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- PDB-8a7d: Partial dimer complex of PAPP-A and its inhibitor STC2 -

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Basic information

Entry
Database: PDB / ID: 8a7d
TitlePartial dimer complex of PAPP-A and its inhibitor STC2
Components
  • Pappalysin-1
  • Stanniocalcin-2
KeywordsHYDROLASE / Metzincin metalloprotease Inhibitor complex
Function / homology
Function and homology information


regulation of hormone biosynthetic process / pappalysin-1 / response to follicle-stimulating hormone / regulation of store-operated calcium entry / response to vitamin D / protein metabolic process / negative regulation of multicellular organism growth / response to dexamethasone / decidualization / endoplasmic reticulum unfolded protein response ...regulation of hormone biosynthetic process / pappalysin-1 / response to follicle-stimulating hormone / regulation of store-operated calcium entry / response to vitamin D / protein metabolic process / negative regulation of multicellular organism growth / response to dexamethasone / decidualization / endoplasmic reticulum unfolded protein response / embryo implantation / female pregnancy / Post-translational protein phosphorylation / protein catabolic process / intracellular calcium ion homeostasis / hormone activity / metalloendopeptidase activity / response to peptide hormone / Regulation of Insulin-like Growth Factor (IGF) transport and uptake by Insulin-like Growth Factor Binding Proteins (IGFBPs) / metallopeptidase activity / cellular response to hypoxia / response to oxidative stress / cell surface receptor signaling pathway / endoplasmic reticulum lumen / negative regulation of gene expression / heme binding / perinuclear region of cytoplasm / Golgi apparatus / enzyme binding / endoplasmic reticulum / protein homodimerization activity / proteolysis / extracellular space / zinc ion binding / extracellular region
Similarity search - Function
Stanniocalcin / Stanniocalcin family / Pappalysin-1/2 / Myxococcus cysteine-rich repeat / LamG-like jellyroll fold / LamG-like jellyroll fold domain / Peptidase M43, pregnancy-associated plasma-A / Pregnancy-associated plasma protein-A / Concanavalin A-like lectin/glucanases superfamily / Notch domain ...Stanniocalcin / Stanniocalcin family / Pappalysin-1/2 / Myxococcus cysteine-rich repeat / LamG-like jellyroll fold / LamG-like jellyroll fold domain / Peptidase M43, pregnancy-associated plasma-A / Pregnancy-associated plasma protein-A / Concanavalin A-like lectin/glucanases superfamily / Notch domain / Domain found in Notch and Lin-12 / Sushi repeat (SCR repeat) / Domain abundant in complement control proteins; SUSHI repeat; short complement-like repeat (SCR) / Sushi/SCR/CCP domain / Sushi/SCR/CCP superfamily / Sushi/CCP/SCR domain profile. / Metallopeptidase, catalytic domain superfamily / Neutral zinc metallopeptidases, zinc-binding region signature. / Concanavalin A-like lectin/glucanase domain superfamily
Similarity search - Domain/homology
Stanniocalcin-2 / Pappalysin-1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.06 Å
AuthorsKobbero, S.D. / Gajhede, M. / Mirza, O.A. / Boesen, T. / Oxvig, C.
Funding support Denmark, 1items
OrganizationGrant numberCountry
Danish Council for Independent Research Denmark
CitationJournal: Nat Commun / Year: 2022
Title: Structure of the proteolytic enzyme PAPP-A with the endogenous inhibitor stanniocalcin-2 reveals its inhibitory mechanism.
Authors: Sara Dam Kobberø / Michael Gajhede / Osman Asghar Mirza / Søren Kløverpris / Troels Rønn Kjær / Jakob Hauge Mikkelsen / Thomas Boesen / Claus Oxvig /
Abstract: The metzincin metalloproteinase PAPP-A plays a key role in the regulation of insulin-like growth factor (IGF) signaling by specific cleavage of inhibitory IGF binding proteins (IGFBPs). Using single- ...The metzincin metalloproteinase PAPP-A plays a key role in the regulation of insulin-like growth factor (IGF) signaling by specific cleavage of inhibitory IGF binding proteins (IGFBPs). Using single-particle cryo-electron microscopy (cryo-EM), we here report the structure of PAPP-A in complex with its endogenous inhibitor, stanniocalcin-2 (STC2), neither of which have been reported before. The highest resolution (3.1 Å) was obtained for the STC2 subunit and the N-terminal approximately 1000 residues of the PAPP-A subunit. The 500 kDa 2:2 PAPP-A·STC2 complex is a flexible multidomain ensemble with numerous interdomain contacts. In particular, a specific disulfide bond between the subunits of STC2 and PAPP-A prevents dissociation, and interactions between STC2 and a module located in the very C-terminal end of the PAPP-A subunit prevent binding of its main substrate, IGFBP-4. While devoid of activity towards IGFBP-4, the active site cleft of the catalytic domain is accessible in the inhibited PAPP-A·STC2 complex, as shown by its ability to hydrolyze a synthetic peptide derived from IGFBP-4. Relevant to multiple human pathologies, this unusual mechanism of proteolytic inhibition may support the development of specific pharmaceutical agents, by which IGF signaling can be indirectly modulated.
History
DepositionJun 20, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 2, 2022Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
C: Pappalysin-1
P: Stanniocalcin-2
Q: Pappalysin-1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)363,26220
Polymers361,1063
Non-polymers2,15617
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area5720 Å2
ΔGint-74 kcal/mol
Surface area64030 Å2
MethodPISA

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Components

#1: Protein Pappalysin-1 / / Insulin-like growth factor-dependent IGF-binding protein 4 protease / IGF-dependent IGFBP-4 ...Insulin-like growth factor-dependent IGF-binding protein 4 protease / IGF-dependent IGFBP-4 protease / IGFBP-4ase / Pregnancy-associated plasma protein A / PAPP-A


Mass: 171143.047 Da / Num. of mol.: 2 / Mutation: E563Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Cell: extracellular / Gene: PAPPA / Cell (production host): stem cell / Cell line (production host): HEK293T / Organ (production host): Kidney / Production host: Homo sapiens (human) / Tissue (production host): embryonic / References: UniProt: Q13219, pappalysin-1
#2: Protein Stanniocalcin-2 / / STC-2 / Stanniocalcin-related protein / STC-related protein / STCRP


Mass: 18819.811 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Cell: Extracellular / Gene: STC2 / Cell (production host): stem cell / Cell line (production host): HEK293T / Organ (production host): Kidney / Production host: Homo sapiens (human) / Tissue (production host): embryonic / References: UniProt: O76061
#3: 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.: 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
#4: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
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
1Partial PAPP-A dimer in complex with subunit of its endogenous inhibitor STC2 dimerCOMPLEXInhibited proteolytic complex generated by harvest of serum media, purifying on a nickel column followed by negative affinity purification and size-exclusion chromatography.#1-#20RECOMBINANT
2PAPP-ACOMPLEXParts of both PAPP-A subunits#11RECOMBINANT
3Subunit of Stanniocalcin-2COMPLEXOne subunit of the STC2 dimer#21RECOMBINANT
4N-terminal part of PAPP-ACOMPLEXThe first approximately 1000 residues of a PAPP-A subunit#12RECOMBINANT
5C-terminal part of PAPP-ACOMPLEXThe C-terminal 240 residues of one PAPP-A subunit#12RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.5 MDaYES
22NO
33NO
44NO
55NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDCellular locationTissue
11Homo sapiens (human)9606extracellularubiquitous
22Homo sapiens (human)9606extracellularubiquitous
33Homo sapiens (human)9606extracellularubiquitous
44Homo sapiens (human)9606extracellularubiquitous
55Homo sapiens (human)9606extracellularubiquitous
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDCellPlasmid
11Homo sapiens (human)9606embryonic kidney cellspcDNA
22Homo sapiens (human)9606embryonic kidney cellspcDNA
33Homo sapiens (human)9606embryonic kidney cellspcDNA
44Homo sapiens (human)9606embryonic kidney cellspcDNA
55Homo sapiens (human)9606embryonic kidney cellspcDNA
Buffer solutionpH: 7.4
Details: Hepes buffer, 20 mM Hepes pH 7.4 100 mM NaCl, 1 mM CaCl
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHepesHepes1
2100 mMNaCLSodium chlorideNaClSodium chloride1
31 mMCaClCaCl1
SpecimenConc.: 0.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-2/2
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 277.15 K / Details: 4 s

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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 defocus max: 1800 nm / Nominal defocus min: 600 nm
Image recording
IDImaging-IDAverage exposure time (sec.)Electron dose (e/Å2)Film or detector modelNum. of grids imagedNum. of real images
110.9159GATAN K3 BIOQUANTUM (6k x 4k)132144
210.858GATAN K3 BIOQUANTUM (6k x 4k)110060

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Processing

EM software
IDNameVersionCategoryFitting-ID
4cryoSPARC3.3CTF correction
7Cootmodel fitting1
12cryoSPARC3.33D reconstruction
13PHENIXmodel refinement1
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 6677370 / Details: Combined two datasets
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.06 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 278982 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model building
IDProtocolSpaceDetails
1RIGID BODY FITREALalphafold2 model PAPP-A AF-Q13219-F1 STC2 AF-O76061-F1
2

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