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- EMDB-26475: Structure of a metalloprotease in complex with its substrate. -

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Basic information

Entry
Database: EMDB / ID: EMD-26475
TitleStructure of a metalloprotease in complex with its substrate.
Map dataFull map
Sample
  • Complex: Complex of PAPP-A with IGFBP5
    • Protein or peptide: Pappalysin-1
    • Protein or peptide: Insulin-like growth factor-binding protein 5
  • Ligand: ZINC ION
KeywordsMetalloprotease / Metal-binding / HYDROLASE / HYDROLASE-Hormone complex
Function / homology
Function and homology information


negative regulation of muscle tissue development / negative regulation of skeletal muscle hypertrophy / pappalysin-1 / regulation of insulin-like growth factor receptor signaling pathway / response to follicle-stimulating hormone / insulin-like growth factor binding protein complex / insulin-like growth factor ternary complex / negative regulation of insulin-like growth factor receptor signaling pathway / negative regulation of smooth muscle cell migration / mammary gland involution ...negative regulation of muscle tissue development / negative regulation of skeletal muscle hypertrophy / pappalysin-1 / regulation of insulin-like growth factor receptor signaling pathway / response to follicle-stimulating hormone / insulin-like growth factor binding protein complex / insulin-like growth factor ternary complex / negative regulation of insulin-like growth factor receptor signaling pathway / negative regulation of smooth muscle cell migration / mammary gland involution / negative regulation of growth / insulin-like growth factor II binding / type B pancreatic cell proliferation / insulin-like growth factor I binding / positive regulation of vascular associated smooth muscle cell migration / response to growth hormone / positive regulation of insulin-like growth factor receptor signaling pathway / hair follicle morphogenesis / lung alveolus development / response to dexamethasone / fibronectin binding / cellular response to organic cyclic compound / negative regulation of osteoblast differentiation / cellular response to cAMP / positive regulation of vascular associated smooth muscle cell proliferation / striated muscle cell differentiation / insulin-like growth factor receptor signaling pathway / negative regulation of cell migration / female pregnancy / regulation of cell growth / Post-translational protein phosphorylation / negative regulation of smooth muscle cell proliferation / protein catabolic process / metalloendopeptidase activity / osteoblast differentiation / Regulation of Insulin-like Growth Factor (IGF) transport and uptake by Insulin-like Growth Factor Binding Proteins (IGFBPs) / metallopeptidase activity / glucose homeostasis / negative regulation of translation / positive regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / cell surface receptor signaling pathway / intracellular signal transduction / endoplasmic reticulum lumen / signal transduction / proteolysis / extracellular space / zinc ion binding / extracellular region
Similarity search - Function
Insulin-like growth factor binding protein 5 / Insulin-like growth factor-binding protein family 1-6, chordata / Insulin-like growth factor binding protein, N-terminal, Cys-rich conserved site / Insulin-like growth factor-binding protein (IGFBP) N-terminal domain signature. / Pappalysin-1/2 / Myxococcus cysteine-rich repeat / LamG-like jellyroll fold / Peptidase M43, pregnancy-associated plasma-A / Pregnancy-associated plasma protein-A / LamG-like jellyroll fold domain ...Insulin-like growth factor binding protein 5 / Insulin-like growth factor-binding protein family 1-6, chordata / Insulin-like growth factor binding protein, N-terminal, Cys-rich conserved site / Insulin-like growth factor-binding protein (IGFBP) N-terminal domain signature. / Pappalysin-1/2 / Myxococcus cysteine-rich repeat / LamG-like jellyroll fold / Peptidase M43, pregnancy-associated plasma-A / Pregnancy-associated plasma protein-A / LamG-like jellyroll fold domain / Insulin-like growth factor binding protein / Insulin-like growth factor-binding protein, IGFBP / Insulin-like growth factor-binding protein (IGFBP) N-terminal domain profile. / Insulin growth factor-binding protein homologues / Concanavalin A-like lectin/glucanases superfamily / Thyroglobulin type-1 repeat signature. / Thyroglobulin type-1 / Thyroglobulin type-1 superfamily / Thyroglobulin type-1 repeat / Thyroglobulin type-1 domain profile. / Thyroglobulin type I repeats. / 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 / Growth factor receptor cysteine-rich domain superfamily / Neutral zinc metallopeptidases, zinc-binding region signature. / Concanavalin A-like lectin/glucanase domain superfamily
Similarity search - Domain/homology
Insulin-like growth factor-binding protein 5 / Pappalysin-1
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.28 Å
AuthorsJudge RA / Jain R / Hao Q / Ouch C / Sridar J / Smith CL / Wang JCK / Eaton D
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nat Commun / Year: 2022
Title: Structure of the PAPP-A complex reveals mechanism of substrate recognition.
Authors: Russell A Judge / Janani Sridar / Kathryn Tunyasuvunakool / Rinku Jain / John C K Wang / Christna Ouch / Jun Xu / Amirhossein Mafi / Aaron H Nile / Clint Remarcik / Corey L Smith / Crystal ...Authors: Russell A Judge / Janani Sridar / Kathryn Tunyasuvunakool / Rinku Jain / John C K Wang / Christna Ouch / Jun Xu / Amirhossein Mafi / Aaron H Nile / Clint Remarcik / Corey L Smith / Crystal Ghosh / Chen Xu / Vincent Stoll / John Jumper / Amoolya H Singh / Dan Eaton / Qi Hao /
Abstract: Insulin-like growth factor (IGF) signaling is highly conserved and tightly regulated by proteases including Pregnancy-Associated Plasma Protein A (PAPP-A). PAPP-A and its paralog PAPP-A2 are ...Insulin-like growth factor (IGF) signaling is highly conserved and tightly regulated by proteases including Pregnancy-Associated Plasma Protein A (PAPP-A). PAPP-A and its paralog PAPP-A2 are metalloproteases that mediate IGF bioavailability through cleavage of IGF binding proteins (IGFBPs). Here, we present single-particle cryo-EM structures of the catalytically inactive mutant PAPP-A (E483A) in complex with a peptide from its substrate IGFBP5 (PAPP-A) and also in its substrate-free form, by leveraging the power of AlphaFold to generate a high quality predicted model as a starting template. We show that PAPP-A is a flexible trans-dimer that binds IGFBP5 via a 25-amino acid anchor peptide which extends into the metalloprotease active site. This unique IGFBP5 anchor peptide that mediates the specific PAPP-A-IGFBP5 interaction is not found in other PAPP-A substrates. Additionally, we illustrate the critical role of the PAPP-A central domain as it mediates both IGFBP5 recognition and trans-dimerization. We further demonstrate that PAPP-A trans-dimer formation and distal inter-domain interactions are both required for efficient proteolysis of IGFBP4, but dispensable for IGFBP5 cleavage. Together the structural and biochemical studies reveal the mechanism of PAPP-A substrate binding and selectivity.
History
DepositionMar 22, 2022-
Header (metadata) releaseSep 28, 2022-
Map releaseSep 28, 2022-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26475.png

Downloads & links

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Map

FileDownload / File: emd_26475.map.gz / Format: CCP4 / Size: 325 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFull map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 440 pix.
= 365.2 Å		0.83 Å/pix.
x 440 pix.
= 365.2 Å		0.83 Å/pix.
x 440 pix.
= 365.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.2305
Minimum - Maximum-1.6446083 - 2.6908176
Average (Standard dev.)0.0009787463 (±0.038532022)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions440440440
Spacing440440440
CellA=B=C: 365.19998 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half map B

Fileemd_26475_half_map_1.map
AnnotationHalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A

Fileemd_26475_half_map_2.map
AnnotationHalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of PAPP-A with IGFBP5

EntireName: Complex of PAPP-A with IGFBP5
Components
  • Complex: Complex of PAPP-A with IGFBP5
    • Protein or peptide: Pappalysin-1
    • Protein or peptide: Insulin-like growth factor-binding protein 5
  • Ligand: ZINC ION

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Supramolecule #1: Complex of PAPP-A with IGFBP5

SupramoleculeName: Complex of PAPP-A with IGFBP5 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Pappalysin-1

MacromoleculeName: Pappalysin-1 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: pappalysin-1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 176.341703 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: REARGATEEP SPPSRALYFS GRGEQLRLRA DLELPRDAFT LQVWLRAEGG QRSPAVITGL YDKCSYISRD RGWVVGIHTI SDQDNKDPR YFFSLKTDRA RQVTTINAHR SYLPGQWVYL AATYDGQFMK LYVNGAQVAT SGEQVGGIFS PLTQKCKVLM L GGSALNHN ...String:
REARGATEEP SPPSRALYFS GRGEQLRLRA DLELPRDAFT LQVWLRAEGG QRSPAVITGL YDKCSYISRD RGWVVGIHTI SDQDNKDPR YFFSLKTDRA RQVTTINAHR SYLPGQWVYL AATYDGQFMK LYVNGAQVAT SGEQVGGIFS PLTQKCKVLM L GGSALNHN YRGYIEHFSL WKVARTQREI LSDMETHGAH TALPQLLLQE NWDNVKHAWS PMKDGSSPKV EFSNAHGFLL DT SLEPPLC GQTLCDNTEV IASYNQLSSF RQPKVVRYRV VNLYEDDHKN PTVTREQVDF QHHQLAEAFK QYNISWELDV LEV SNSSLR RRLILANCDI SKIGDENCDP ECNHTLTGHD GGDCRHLRHP AFVKKQHNGV CDMDCNYERF NFDGGECCDP EITN VTQTC FDPDSPHRAY LDVNELKNIL KLDGSTHLNI FFAKSSEEEL AGVATWPWDK EALMHLGGIV LNPSFYGMPG HTHTM IHAI GHSLGLYHVF RGISEIQSCS DPCMETEPSF ETGDLCNDTN PAPKHKSCGD PGPGNDTCGF HSFFNTPYNN FMSYAD DDC TDSFTPNQVA RMHCYLDLVY QGWQPSRKPA PVALAPQVLG HTTDSVTLEW FPPIDGHFFE RELGSACHLC LEGRILV QY ASNASSPMPC SPSGHWSPRE AEGHPDVEQP CKSSVRTWSP NSAVNPHTVP PACPEPQGCY LELEFLYPLV PESLTIWV T FVSTDWDSSG AVNDIKLLAV SGKNISLGPQ NVFCDVPLTI RLWDVGEEVY GIQIYTLDEH LEIDAAMLTS TADTPLCLQ CKPLKYKVVR DPPLQMDVAS ILHLNRKFVD MDLNLGSVYQ YWVITISGTE ESEPSPAVTY IHGSGYCGDG IIQKDQGEQC DDMNKINGD GCSLFCRQEV SFNCIDEPSR CYFHDGDGVC EEFEQKTSIK DCGVYTPQGF LDQWASNASV SHQDQQCPGW V IIGQPAAS QVCRTKVIDL SEGISQHAWY PCTISYPYSQ LAQTTFWLRA YFSQPMVAAA VIVHLVTDGT YYGDQKQETI SV QLLDTKD QSHDLGLHVL SCRNNPLIIP VVHDLSQPFY HSQAVRVSFS SPLVAISGVA LRSFDNFDPV TLSSCQRGET YSP AEQSCV HFACEKTDCP ELAVENAYLN CSSSDRYHGA QCTVSCRTGY VLQIRRDDEL IKSQTGPSVT VTCTEGKWNK QVAC EPVDC SIPDHHQVYA ASFSCPEGTT FGSQCSFQCR HPAQLKGNNS LLTCMEDGLW SFPEALCELM CLAPPPVPNA DLQTA RCRE NKHKVGSFCK YKCKPGYHVP GSSRKSKKRA FKTQCTQDGS WQEGACVPVT CDPPPPKFHG LYQCTNGFQF NSECRI KCE DSDASQGLGS NVIHCRKDGT WNGSFHVCQE MQGQCSVPNE LNSNLKLQCP DGYAIGSECA TSCLDHNSES IILPMNV TV RDIPHWLNPT RVERVVCTAG LKWYPHPALI HCVKGCEPFM GDNYCDAINN RAFCNYDGGD CCTSTVKTKK VTPFPMSC D LQGDCACRDP QAQEHSRKDL RGYSHGSGPT RTRPLEQKLI SEEDLAANDI LDYKDDDDKV

UniProtKB: Pappalysin-1

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Macromolecule #2: Insulin-like growth factor-binding protein 5

MacromoleculeName: Insulin-like growth factor-binding protein 5 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 30.876279 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: HHHHHHAAAL GSFVHCEPCD EKALSMCPPS PLGCELVKEP GCGCCMTCAL AEGQSCGVYT ERCAQGLRCL PRQDEEKPLH ALLHGRGVC LNEKSYREQV KIERDSREHE EPTTSEMAEE TYSPKIFRPK HTRISELKAE AVKKDRRKKL TQSKFVGGAE N TAHPRIIS ...String:
HHHHHHAAAL GSFVHCEPCD EKALSMCPPS PLGCELVKEP GCGCCMTCAL AEGQSCGVYT ERCAQGLRCL PRQDEEKPLH ALLHGRGVC LNEKSYREQV KIERDSREHE EPTTSEMAEE TYSPKIFRPK HTRISELKAE AVKKDRRKKL TQSKFVGGAE N TAHPRIIS APEMRQESEQ GPCRRHMEAS LQELKASPRM VPRAVYLPNC DRKGFYKRKQ CKPSRGRKRG ICWCVDKYGM KL PGMEYVD GDFQCHTFDS SNVEAAADYK DDDDK

UniProtKB: Insulin-like growth factor-binding protein 5

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Macromolecule #3: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 3 / Number of copies: 2 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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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

Concentration0.25 mg/mL
BufferpH: 9.2 / Details: BTP (Bis-Tris-Propane) pH 9.2
GridModel: C-flat-1.2/1.3 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 9080999 / Average electron dose: 47.6 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 105000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 9080999
Startup modelType of model: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.28 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3) / Number images used: 245018
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: OTHER
Output model

PDB-7ufg:
Cryo-EM structure of PAPP-A in complex with IGFBP5

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