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- EMDB-15219: PAPP-A dimer in complex with endogenous STC2 inhibitor. -

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Basic information

Entry
Database: EMDB / ID: EMD-15219
TitlePAPP-A dimer in complex with endogenous STC2 inhibitor.
Map dataPrimary, sharpened map of the PAPP-A dimer in complex with STC2 dimer. Refinement focused on the dimerization region
Sample
  • Complex: PAPP-A dimer in complex with its endogenous inhibitor STC2 dimer
    • Complex: Pregnancy-associated plasma protein A
      • Protein or peptide: PAPP-A
    • Complex: Stanniocalcin-2
      • Protein or peptide: Stanniocalcin 2
KeywordsMetzincin metalloprotease Inhibitor complex / HYDROLASE
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.02 Å
AuthorsKobbero SD / Oxvig C / Gajhede M / Boesen T
Funding support Denmark, 1 items
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, 2022-
Header (metadata) releaseNov 2, 2022-
Map releaseNov 2, 2022-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15219.png

Downloads & links

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Map

FileDownload / File: emd_15219.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPrimary, sharpened map of the PAPP-A dimer in complex with STC2 dimer. Refinement focused on the dimerization region
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.19 Å/pix.
x 256 pix.
= 303.6 Å		1.19 Å/pix.
x 256 pix.
= 303.6 Å		1.19 Å/pix.
x 256 pix.
= 303.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.18594 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.07
Minimum - Maximum-0.11998253 - 0.22352219
Average (Standard dev.)0.0012110415 (±0.01281748)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 303.59998 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Raw, unsharpened map of the PAPP-A dimer in...

Fileemd_15219_additional_1.map
AnnotationRaw, unsharpened map of the PAPP-A dimer in complex with STC2 dimer. Refinement focused on the dimerization region
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A of the PAPP-A dimer in...

Fileemd_15219_half_map_1.map
AnnotationHalf map A of the PAPP-A dimer in complex with STC2 dimer. Refinement focused on the dimerization region
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B of the PAPP-A dimer in...

Fileemd_15219_half_map_2.map
AnnotationHalf map B of the PAPP-A dimer in complex with STC2 dimer. Refinement focused on the dimerization region
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : PAPP-A dimer in complex with its endogenous inhibitor STC2 dimer

EntireName: PAPP-A dimer in complex with its endogenous inhibitor STC2 dimer
Components
  • Complex: PAPP-A dimer in complex with its endogenous inhibitor STC2 dimer
    • Complex: Pregnancy-associated plasma protein A
      • Protein or peptide: PAPP-A
    • Complex: Stanniocalcin-2
      • Protein or peptide: Stanniocalcin 2

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Supramolecule #1: PAPP-A dimer in complex with its endogenous inhibitor STC2 dimer

SupramoleculeName: PAPP-A dimer in complex with its endogenous inhibitor STC2 dimer
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Inhibited proteolytic complex generated by harvest of serum media, purifying on a nickel column followed by negative affinity purification and size-exclusion chromatography.
Molecular weightTheoretical: 100 KDa

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Supramolecule #2: Pregnancy-associated plasma protein A

SupramoleculeName: Pregnancy-associated plasma protein A / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1 / Details: Homodimer
Source (natural)Organism: Homo sapiens (human) / Tissue: ubiquitous / Location in cell: extracellular

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Supramolecule #3: Stanniocalcin-2

SupramoleculeName: Stanniocalcin-2 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2 / Details: Homodimer
Source (natural)Organism: Homo sapiens (human) / Tissue: ubiquitous / Location in cell: extracellular

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Macromolecule #1: PAPP-A

MacromoleculeName: PAPP-A / type: protein_or_peptide / ID: 1 / Details: Pre-pro-peptide / Enantiomer: LEVO / EC number: pappalysin-1
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MRLWSWVLHL GLLSAALGCG LAERPRRARR DPRAGRPPRP AAGPATCATR AARGRRASPP PPPPPGGAW EAVRVPRRRQ QREARGATEE PSPPSRALYF SGRGEQLRLR ADLELPRDAF T LQVWLRAE GGQRSPAVIT GLYDKCSYIS RDRGWVVGIH TISDQDNKDP ...String:
MRLWSWVLHL GLLSAALGCG LAERPRRARR DPRAGRPPRP AAGPATCATR AARGRRASPP PPPPPGGAW EAVRVPRRRQ QREARGATEE PSPPSRALYF SGRGEQLRLR ADLELPRDAF T LQVWLRAE GGQRSPAVIT GLYDKCSYIS RDRGWVVGIH TISDQDNKDP RYFFSLKTDR AR QVTTINA HRSYLPGQWV YLAATYDGQF MKLYVNGAQV ATSGEQVGGI FSPLTQKCKV LML GGSALN HNYRGYIEHF SLWKVARTQR EILSDMETHG AHTALPQLLL QENWDNVKHA WSPM KDGSS PKVEFSNAHG FLLDTSLEPP LCGQTLCDNT EVIASYNQLS SFRQPKVVRY RVVNL YEDD HKNPTVTREQ VDFQHHQLAE AFKQYNISWE LDVLEVSNSS LRRRLILANC DISKIG DEN CDPECNHTLT GHDGGDCRHL RHPAFVKKQH NGVCDMDCNY ERFNFDGGEC CDPEITN VT QTCFDPDSPH RAYLDVNELK NILKLDGSTH LNIFFAKSSE EELAGVATWP WDKEALMH L GGIVLNPSFY GMPGHTHTMI HEIGHSLGLY HVFRGISEIQ SCSDPCMETE PSFETGDLC NDTNPAPKHK SCGDPGPGND TCGFHSFFNT PYNNFMSYAD DDCTDSFTPN QVARMHCYLD LVYQGWQPS RKPAPVALAP QVLGHTTDSV TLEWFPPIDG HFFERELGSA CHLCLEGRIL V QYASNASS PMPCSPSGHW SPREAEGHPD VEQPCKSSVR TWSPNSAVNP HTVPPACPEP QG CYLELEF LYPLVPESLT IWVTFVSTDW DSSGAVNDIK LLAVSGKNIS LGPQNVFCDV PLT IRLWDV GEEVYGIQIY TLDEHLEIDA AMLTSTADTP LCLQCKPLKY KVVRDPPLQM DVAS ILHLN RKFVDMDLNL GSVYQYWVIT ISGTEESEPS PAVTYIHGSG YCGDGIIQKD QGEQC DDMN KINGDGCSLF CRQEVSFNCI DEPSRCYFHD GDGVCEEFEQ KTSIKDCGVY TPQGFL DQW ASNASVSHQD QQCPGWVIIG QPAASQVCRT KVIDLSEGIS QHAWYPCTIS YPYSQLA QT TFWLRAYFSQ PMVAAAVIVH LVTDGTYYGD QKQETISVQL LDTKDQSHDL GLHVLSCR N NPLIIPVVHD LSQPFYHSQA VRVSFSSPLV AISGVALRSF DNFDPVTLSS CQRGETYSP AEQSCVHFAC EKTDCPELAV ENASLNCSSS DRYHGAQCTV SCRTGYVLQI RRDDELIKSQ TGPSVTVTC TEGKWNKQVA CEPVDCSIPD HHQVYAASFS CPEGTTFGSQ CSFQCRHPAQ L KGNNSLLT CMEDGLWSFP EALCELMCLA PPPVPNADLQ TARCRENKHK VGSFCKYKCK PG YHVPGSS RKSKKRAFKT QCTQDGSWQE GACVPVTCDP PPPKFHGLYQ CTNGFQFNSE CRI KCEDSD ASQGLGSNVI HCRKDGTWNG SFHVCQEMQG QCSVPNELNS NLKLQCPDGY AIGS ECATS CLDHNSESII LPMNVTVRDI PHWLNPTRVE RVVCTAGLKW YPHPALIHCV KGCEP FMGD NYCDAINNRA FCNYDGGDCC TSTVKTKKVT PFPMSCDLQG DCACRDPQAQ EHSRKD LRG YSHG

GENBANK: GENBANK: AL691426

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Macromolecule #2: Stanniocalcin 2

MacromoleculeName: Stanniocalcin 2 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: TDATNPPEGP QDRSSQQKGR LSLQNTAEIQ HCLVNAGDVG CGVFECFENN SCEIRGLHGI CMTFLHNAG KFDAQGKSFI KDALKCKAHA LRHRFGCISR KCPAIREMVS QLQRECYLKH D LCAAAQEN TRVIVEMIHF KDLLLHEPYV DLVNLLLTCG EEVKEAITHS ...String:
TDATNPPEGP QDRSSQQKGR LSLQNTAEIQ HCLVNAGDVG CGVFECFENN SCEIRGLHGI CMTFLHNAG KFDAQGKSFI KDALKCKAHA LRHRFGCISR KCPAIREMVS QLQRECYLKH D LCAAAQEN TRVIVEMIHF KDLLLHEPYV DLVNLLLTCG EEVKEAITHS VQVQCEQNWG SL CSILSFC TSAIQKPPTA PPERQPQVDR TKLSRAHHGE AGHHLPEPSS RETGRGAKGE RGS KSHPNA HARGRVGGLG AQGPSGSSEW EDEQSEYSDI RR

GENBANK: GENBANK: AF031036

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.6 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
20.0 mMHepesHepes
100.0 mMNaClSodium chlorideNaClSodium chloride
1.0 mMCaClCaCl

Details: HEPES buffer 20 mM Hepes pH 7.5 100 mM NaCl, 1mM CaCl
GridModel: C-flat-2/2 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 277.15 K / Instrument: LEICA EM GP / Details: Blot time 4.5 sec.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.6 µm
Image recording#0 - Image recording ID: 1 / #0 - Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / #0 - Number grids imaged: 1 / #0 - Number real images: 32144 / #0 - Average exposure time: 0.91 sec. / #0 - Average electron dose: 59.0 e/Å2 / #1 - Image recording ID: 2 / #1 - Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / #1 - Number grids imaged: 1 / #1 - Number real images: 10060 / #1 - Average exposure time: 0.8 sec. / #1 - Average electron dose: 58.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: OTHER
Final 3D classificationNumber classes: 4 / Software - Name: cryoSPARC (ver. 3)
Final angle assignmentType: OTHER
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 5.02 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 31119
Image recording ID1
FSC plot (resolution estimation)

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