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

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Basic information

Entry
Database: PDB / ID: 7ufg
TitleCryo-EM structure of PAPP-A in complex with IGFBP5
Components
  • Insulin-like growth factor-binding protein 5
  • Pappalysin-1
KeywordsHYDROLASE/Hormone / Metalloprotease / 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 / insulin-like growth factor binding protein complex / insulin-like growth factor ternary complex / negative regulation of insulin-like growth factor receptor signaling pathway / mammary gland involution / negative regulation of growth / negative regulation of smooth muscle cell migration ...negative regulation of muscle tissue development / negative regulation of skeletal muscle hypertrophy / pappalysin-1 / regulation of insulin-like growth factor receptor signaling pathway / insulin-like growth factor binding protein complex / insulin-like growth factor ternary complex / negative regulation of insulin-like growth factor receptor signaling pathway / mammary gland involution / negative regulation of growth / negative regulation of smooth muscle cell migration / insulin-like growth factor II binding / insulin-like growth factor I binding / type B pancreatic cell proliferation / 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 / fibronectin binding / negative regulation of osteoblast differentiation / 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 / cellular response to cAMP / Post-translational protein phosphorylation / regulation of cell growth / negative regulation of smooth muscle cell proliferation / female pregnancy / metalloendopeptidase activity / Regulation of Insulin-like Growth Factor (IGF) transport and uptake by Insulin-like Growth Factor Binding Proteins (IGFBPs) / metallopeptidase activity / osteoblast differentiation / glucose homeostasis / cell surface receptor signaling pathway / cell population proliferation / positive regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / negative regulation of translation / intracellular signal transduction / receptor ligand activity / endoplasmic reticulum lumen / signal transduction / proteolysis / extracellular space / extracellular region / zinc ion binding / nucleus
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 / Peptidase M43, pregnancy-associated plasma-A / Pregnancy-associated plasma protein-A / LamG-like jellyroll fold / 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 / Peptidase M43, pregnancy-associated plasma-A / Pregnancy-associated plasma protein-A / LamG-like jellyroll fold / 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 / Jelly Rolls - #200 / Growth factor receptor cysteine-rich domain superfamily / Neutral zinc metallopeptidases, zinc-binding region signature. / Concanavalin A-like lectin/glucanase domain superfamily / Jelly Rolls / Sandwich / Mainly Beta
Similarity search - Domain/homology
Insulin-like growth factor-binding protein 5 / Pappalysin-1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.28 Å
AuthorsJudge, R.A. / Jain, R. / Hao, Q. / Ouch, C. / Sridar, J. / Smith, C.L. / Wang, J.C.K. / Eaton, D.
Funding support1items
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, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 28, 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
A: Pappalysin-1
B: Pappalysin-1
D: Insulin-like growth factor-binding protein 5
C: Insulin-like growth factor-binding protein 5
hetero molecules


Theoretical massNumber of molelcules
Total (without water)414,5676
Polymers414,4364
Non-polymers1312
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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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: 176341.703 Da / Num. of mol.: 2 / Mutation: E483A, S1144Y
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PAPPA / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: Q13219, pappalysin-1
#2: Protein Insulin-like growth factor-binding protein 5 / IBP-5 / IGF-binding protein 5 / IGFBP-5


Mass: 30876.279 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: IGFBP5, IBP5 / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: P24593
#3: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
Has ligand of interestN
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

ComponentName: Complex of PAPP-A with IGFBP5 / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK 293
Buffer solutionpH: 9.2 / Details: BTP (Bis-Tris-Propane) pH 9.2
SpecimenConc.: 0.25 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283 K

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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 magnification: 105000 X / Nominal defocus max: 3500 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 47.6 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 9080999
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.19.2_4158+SVNrefinement
PHENIX1.19.2_4158+SVNrefinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
4cryoSPARC3CTF correction
7PHENIXmodel fitting
12cryoSPARC33D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 9080999
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.28 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 245018 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 85.93 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002518730
ELECTRON MICROSCOPYf_angle_d0.905225559
ELECTRON MICROSCOPYf_chiral_restr0.17392815
ELECTRON MICROSCOPYf_plane_restr0.00253346
ELECTRON MICROSCOPYf_dihedral_angle_d3.44672548

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