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- PDB-6gwp: Crystal Structure of Stabilized Active Plasminogen Activator Inhi... -

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Basic information

Entry
Database: PDB / ID: 6gwp
TitleCrystal Structure of Stabilized Active Plasminogen Activator Inhibitor-1 (PAI-1-stab) in Complex with Two Inhibitory Nanobodies (VHH-2g-42, VHH-2w-64)
Components
  • Plasminogen Activator Inhibitor-1
  • VHH-2g-42
  • VHH-2w-64
KeywordsHYDROLASE / plasminogen activator inhibitor-1 / PAI-1 / PAI-1-stab / serpin / protease inhibitor / serine protease inhibitor / nanobody / antibody fragment / protein complex
Function / homology
Function and homology information


positive regulation of leukotriene production involved in inflammatory response / dentinogenesis / negative regulation of smooth muscle cell-matrix adhesion / peptidase inhibitor complex / positive regulation of coagulation / Regulation of MITF-M-dependent genes involved in extracellular matrix, focal adhesion and epithelial-to-mesenchymal transition / negative regulation of vascular wound healing / negative regulation of smooth muscle cell migration / negative regulation of endopeptidase activity / negative regulation of wound healing ...positive regulation of leukotriene production involved in inflammatory response / dentinogenesis / negative regulation of smooth muscle cell-matrix adhesion / peptidase inhibitor complex / positive regulation of coagulation / Regulation of MITF-M-dependent genes involved in extracellular matrix, focal adhesion and epithelial-to-mesenchymal transition / negative regulation of vascular wound healing / negative regulation of smooth muscle cell migration / negative regulation of endopeptidase activity / negative regulation of wound healing / positive regulation of odontoblast differentiation / negative regulation of cell adhesion mediated by integrin / negative regulation of plasminogen activation / regulation of signaling receptor activity / positive regulation of monocyte chemotaxis / Dissolution of Fibrin Clot / replicative senescence / negative regulation of blood coagulation / positive regulation of blood coagulation / negative regulation of fibrinolysis / ECM proteoglycans / negative regulation of endothelial cell apoptotic process / negative regulation of extrinsic apoptotic signaling pathway via death domain receptors / serine protease inhibitor complex / fibrinolysis / BMAL1:CLOCK,NPAS2 activates circadian expression / negative regulation of cell migration / platelet alpha granule lumen / positive regulation of interleukin-8 production / serine-type endopeptidase inhibitor activity / SMAD2/SMAD3:SMAD4 heterotrimer regulates transcription / positive regulation of receptor-mediated endocytosis / positive regulation of inflammatory response / positive regulation of angiogenesis / Platelet degranulation / cellular response to lipopolysaccharide / protease binding / : / defense response to Gram-negative bacterium / angiogenesis / signaling receptor binding / extracellular space / extracellular exosome / extracellular region / plasma membrane
Similarity search - Function
Antithrombin; Chain I, domain 2 / Antithrombin, subunit I, domain 2 / Alpha-1-antitrypsin; domain 1 / Alpha-1-antitrypsin, domain 1 / Serpin, conserved site / Serpins signature. / Serpin superfamily, domain 2 / Serpin family / Serpin domain / Serpin superfamily ...Antithrombin; Chain I, domain 2 / Antithrombin, subunit I, domain 2 / Alpha-1-antitrypsin; domain 1 / Alpha-1-antitrypsin, domain 1 / Serpin, conserved site / Serpins signature. / Serpin superfamily, domain 2 / Serpin family / Serpin domain / Serpin superfamily / Serpin superfamily, domain 1 / Serpin (serine protease inhibitor) / SERine Proteinase INhibitors / Roll / 2-Layer Sandwich / Mainly Beta / Alpha Beta
Similarity search - Domain/homology
Plasminogen activator inhibitor 1
Similarity search - Component
Biological speciesHomo sapiens (human)
Vicugna pacos (alpaca)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.28 Å
AuthorsSillen, M. / Weeks, S.D. / Strelkov, S.V. / Declerck, P.J.
Funding support Belgium, 2items
OrganizationGrant numberCountry
Research Foundation - FlandersG072915N Belgium
Research Foundation - Flanders11ZQ916N Belgium
CitationJournal: J.Thromb.Haemost. / Year: 2020
Title: Molecular mechanism of two nanobodies that inhibit PAI-1 activity reveals a modulation at distinct stages of the PAI-1/plasminogen activator interaction.
Authors: Sillen, M. / Weeks, S.D. / Zhou, X. / Komissarov, A.A. / Florova, G. / Idell, S. / Strelkov, S.V. / Declerck, P.J.
History
DepositionJun 25, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 1, 2020Provider: repository / Type: Initial release
Revision 1.1Mar 11, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year / _citation_author.identifier_ORCID
Revision 1.2Jan 17, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.3Nov 13, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Plasminogen Activator Inhibitor-1
B: VHH-2g-42
C: VHH-2w-64


Theoretical massNumber of molelcules
Total (without water)68,5913
Polymers68,5913
Non-polymers00
Water1,15364
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: SAXS
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2820 Å2
ΔGint-13 kcal/mol
Surface area26090 Å2
MethodPISA
Unit cell
Length a, b, c (Å)43.681, 70.797, 98.513
Angle α, β, γ (deg.)90.000, 97.480, 90.000
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein Plasminogen Activator Inhibitor-1


Mass: 42751.008 Da / Num. of mol.: 1 / Mutation: N150H-K154T-Q301P-Q319L-M354I
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Plasmid: pETHSUK2 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Rosetta2 pLysS / References: UniProt: P05121
#2: Antibody VHH-2g-42


Mass: 12752.254 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vicugna pacos (alpaca) / Plasmid: pETHSUK2 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Rosetta2 pLysS
#3: Antibody VHH-2w-64


Mass: 13087.387 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vicugna pacos (alpaca) / Plasmid: pETHSUK2 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Rosetta2 pLysS
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 64 / Source method: isolated from a natural source / Formula: H2O
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.2 Å3/Da / Density % sol: 44.14 % / Mosaicity: 0.17 °
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 6.5
Details: 0.1 M Bis-Tris, 17 % w/v PEG 3350, 3 % v/v methanol

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: MASSIF-3 / Wavelength: 0.9677 Å
DetectorType: DECTRIS EIGER X 4M / Detector: PIXEL / Date: Jul 21, 2017
Details: CRLs and a half-Kirkpatrick-Baez (KB) geometry as the vertical and horizontal focusing systems
RadiationMonochromator: Si (111) monochromator / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9677 Å / Relative weight: 1
ReflectionResolution: 2.18→97.674 Å / Num. obs: 30312 / % possible obs: 97.5 % / Redundancy: 3.8 % / Biso Wilson estimate: 46.45 Å2 / CC1/2: 0.993 / Rmerge(I) obs: 0.102 / Rpim(I) all: 0.062 / Rrim(I) all: 0.119 / Net I/σ(I): 9.2 / Num. measured all: 115963
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. measured allNum. unique obsCC1/2Rpim(I) allRrim(I) allNet I/σ(I) obs% possible all
2.18-2.33.91.6181688243610.7190.9911.9071.496.8
6.9-97.6743.80.05391110400.9950.030.05923.699.5

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassification
PHENIX1.10.1_2155refinement
XDSJune 1, 2017 (BUILT=20170923)data reduction
Aimless0.5.32data scaling
PHASER2.7.17phasing
PDB_EXTRACT3.24data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1DB2, 5JA8, 5JA9

1db2

5ja8

5ja9


Resolution: 2.28→97.674 Å / SU ML: 0.33 / Cross valid method: THROUGHOUT / σ(F): 0.31 / Phase error: 28.89 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.225 1362 5.13 %
Rwork0.2029 25191 -
obs0.2041 26553 97.15 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 138.83 Å2 / Biso mean: 61.2692 Å2 / Biso min: 27.51 Å2
Refinement stepCycle: final / Resolution: 2.28→97.674 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4686 0 0 64 4750
Biso mean---49.24 -
Num. residues----604
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0094791
X-RAY DIFFRACTIONf_angle_d1.2226502
X-RAY DIFFRACTIONf_chiral_restr0.076728
X-RAY DIFFRACTIONf_plane_restr0.005838
X-RAY DIFFRACTIONf_dihedral_angle_d14.4741725
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 10

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.28-2.36150.3741390.33062526266598
2.3615-2.45610.30461160.27742587270399
2.4561-2.56790.29741300.272553268399
2.5679-2.70330.33811610.2812467262897
2.7033-2.87270.33611190.26652531265098
2.8727-3.09450.25051300.25142534266497
3.0945-3.40590.24051200.23062502262296
3.4059-3.89880.24151440.20982287243189
3.8988-4.9120.17431550.14862564271999
4.912-97.76370.16441480.150226402788100
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
11.241-0.51171.29411.4155-0.76253.3182-0.1504-0.18210.1602-0.0430.10750.0716-0.4007-0.42870.03920.28430.02510.02890.3935-0.04530.3749-30.2576-8.7577-21.7112
24.39420.9162-2.44761.38990.37553.56440.0853-0.195-0.0686-0.0046-0.0698-0.1919-0.04910.34870.00070.36050.0461-0.06040.32120.05340.3546-16.0126-4.584-57.4799
31.202-0.9478-0.67353.90920.47834.61540.1303-0.2332-0.15490.03960.0939-0.20451.10770.0319-0.23730.52660.0129-0.08650.41450.04880.3849-6.2848-39.7695-10.4141
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1(chain A and resseq 6:379)A6 - 379
2X-RAY DIFFRACTION2(chain B and resseq 1:115)B1 - 115
3X-RAY DIFFRACTION3(chain C and resseq 1:121)C1 - 121

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