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- PDB-8a7e: PAPP-A dimer in complex with its inhibitor STC2 -

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Basic information

Entry
Database: PDB / ID: 8a7e
TitlePAPP-A dimer in complex with 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 / regulation of store-operated calcium entry / response to vitamin D / negative regulation of multicellular organism growth / decidualization / endoplasmic reticulum unfolded protein response / embryo implantation / Post-translational protein phosphorylation / female pregnancy ...regulation of hormone biosynthetic process / pappalysin-1 / regulation of store-operated calcium entry / response to vitamin D / negative regulation of multicellular organism growth / decidualization / endoplasmic reticulum unfolded protein response / embryo implantation / Post-translational protein phosphorylation / female pregnancy / response to peptide hormone / hormone activity / metalloendopeptidase activity / Regulation of Insulin-like Growth Factor (IGF) transport and uptake by Insulin-like Growth Factor Binding Proteins (IGFBPs) / intracellular calcium ion homeostasis / metallopeptidase activity / response to oxidative stress / cellular response to hypoxia / cell surface receptor signaling pathway / endoplasmic reticulum lumen / negative regulation of gene expression / heme binding / perinuclear region of cytoplasm / enzyme binding / endoplasmic reticulum / Golgi apparatus / protein homodimerization activity / proteolysis / extracellular space / extracellular region / zinc ion binding
Similarity search - Function
Stanniocalcin / Stanniocalcin family / Pappalysin-1/2 / Myxococcus cysteine-rich repeat / Peptidase M43, pregnancy-associated plasma-A / Pregnancy-associated plasma protein-A / LamG-like jellyroll fold / LamG-like jellyroll fold domain / Concanavalin A-like lectin/glucanases superfamily / Notch domain ...Stanniocalcin / Stanniocalcin family / Pappalysin-1/2 / Myxococcus cysteine-rich repeat / Peptidase M43, pregnancy-associated plasma-A / Pregnancy-associated plasma protein-A / LamG-like jellyroll fold / LamG-like jellyroll fold domain / 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/CCP/SCR domain profile. / Sushi/SCR/CCP superfamily / 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: 5.02 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
Revision 1.1Oct 23, 2024Group: Data collection / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)380,90422
Polymers380,1324
Non-polymers77218
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, gel filtration, immunoprecipitation, mass spectrometry
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Stanniocalcin-2 / STC-2 / Stanniocalcin-related protein / STC-related protein / STCRP


Mass: 18922.953 Da / Num. of mol.: 2
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
#2: 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 / Organ: Ubiquitous / 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
#3: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 16 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
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

Component
IDNameTypeDetailsEntity IDParent-IDSource
1PAPP-A dimer in complex with 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
2Pregnancy-associated plasma protein ACOMPLEXHomodimer#21RECOMBINANT
3Stanniocalcin-2COMPLEXHomodimer#11RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.5 MDaYES
210.4 MDaYES
310.1 MDaYES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDCellular locationTissue
21Homo sapiens (human)9606extracellularubiquitous
32Homo sapiens (human)9606extracellularubiquitous
43Homo sapiens (human)9606extracellularubiquitous
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDCellPlasmid
21Homo sapiens (human)9606embryonic kidney cellspcDNA
32Homo sapiens (human)9606embryonic kidney cellspcDNA
43Homo sapiens (human)9606embryonic kidney cellspcDNA
Buffer solutionpH: 7.4
Details: HEPES buffer 20 mM Hepes pH 7.5 100 mM NaCl, 1mM CaCl
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHepesHepes1
2100 mMNaClNaCl1
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: Blot time 4.5 sec

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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 FIELD / 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
110.858GATAN K3 BIOQUANTUM (6k x 4k)110060
210.9159GATAN K3 BIOQUANTUM (6k x 4k)132144

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Processing

EM software
IDNameVersionCategory
1cryoSPARC3particle selection
2EPUimage acquisition
4cryoSPARC3.3CTF correction
7Cootmodel fitting
11cryoSPARC3classification
12PHENIX1.23D reconstruction
13PHENIXmodel refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 601179 / Details: Topaz train and extraction
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 5.02 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 3 / Details: Composite map / Num. of class averages: 3 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Details: Initial model source: alphafold2 models PAPP-A AF-Q13219-F1 STC2 AF-O76061-F1

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