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

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Basic information

Entry
Database: PDB / ID: 6gwn
TitleCrystal Structure of Stabilized Active Plasminogen Activator Inhibitor-1 (PAI-1-W175F) 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-W175F / 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 / negative regulation of smooth muscle cell migration / peptidase inhibitor complex / negative regulation of vascular wound healing / negative regulation of wound healing / positive regulation of odontoblast differentiation / negative regulation of cell adhesion mediated by integrin / negative regulation of endopeptidase activity ...positive regulation of leukotriene production involved in inflammatory response / dentinogenesis / negative regulation of smooth muscle cell-matrix adhesion / negative regulation of smooth muscle cell migration / peptidase inhibitor complex / negative regulation of vascular wound healing / negative regulation of wound healing / positive regulation of odontoblast differentiation / negative regulation of cell adhesion mediated by integrin / negative regulation of endopeptidase activity / regulation of signaling receptor activity / negative regulation of plasminogen activation / negative regulation of blood coagulation / positive regulation of monocyte chemotaxis / Dissolution of Fibrin Clot / replicative senescence / ECM proteoglycans / positive regulation of blood coagulation / negative regulation of fibrinolysis / 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 gene expression / platelet alpha granule lumen / negative regulation of cell migration / positive regulation of interleukin-8 production / SMAD2/SMAD3:SMAD4 heterotrimer regulates transcription / serine-type endopeptidase inhibitor activity / positive regulation of receptor-mediated endocytosis / positive regulation of inflammatory response / positive regulation of angiogenesis / Platelet degranulation / cellular response to lipopolysaccharide / protease binding / angiogenesis / collagen-containing extracellular matrix / defense response to Gram-negative bacterium / 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: 2.03 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 1.3Oct 16, 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)68,6203
Polymers68,6203
Non-polymers00
Water1,964109
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: SAXS
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2820 Å2
ΔGint-12 kcal/mol
Surface area24470 Å2
MethodPISA
Unit cell
Length a, b, c (Å)45.483, 71.424, 96.598
Angle α, β, γ (deg.)90.000, 100.530, 90.000
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein Plasminogen activator inhibitor 1 / PAI-1 / Endothelial plasminogen activator inhibitor / Serpin E1


Mass: 42780.031 Da / Num. of mol.: 1 / Mutation: W175F
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SERPINE1, PAI1, PLANH1 / 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.: 109 / 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: 2.25 Å3/Da / Density % sol: 45.29 %
Crystal growTemperature: 277.15 K / Method: vapor diffusion, hanging drop / pH: 6.5 / Details: 0.1 M Bis-Tris, 10 % w/v PEG 3350

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID30B / Wavelength: 0.976251 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Jul 20, 2017
Details: CRL combinations for vertical focussing and an eliptical mirror for horizontal focussing
RadiationMonochromator: Si (111) monochromator / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.976251 Å / Relative weight: 1
ReflectionResolution: 1.96→94.97 Å / Num. obs: 42665 / % possible obs: 98.3 % / Redundancy: 3.7 % / Biso Wilson estimate: 38.93 Å2 / CC1/2: 0.998 / Rmerge(I) obs: 0.059 / Rpim(I) all: 0.036 / Rrim(I) all: 0.069 / Net I/σ(I): 11.2
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) all% possible all
1.96-2.073.60.78361000.7570.4790.92196.5
6.21-94.973.30.04314090.9950.0270.05197.6

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassification
PHENIX1.10.1_2155refinement
XDSJun 1, 2017 BUILT=20170923data reduction
Aimless0.5.32data scaling
PHASER2.7.17phasing
PDB_EXTRACT3.24data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3Q02, 5JA8, 5JA9

3q02

5ja8

5ja9


Resolution: 2.03→94.97 Å / SU ML: 0.29 / Cross valid method: THROUGHOUT / σ(F): 0.32 / Phase error: 32.84
RfactorNum. reflection% reflection
Rfree0.2499 1956 5.06 %
Rwork0.2083 --
obs0.2103 38685 98.05 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 115.9 Å2 / Biso mean: 55.7968 Å2 / Biso min: 27.91 Å2
Refinement stepCycle: final / Resolution: 2.03→94.97 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4567 0 0 109 4676
Biso mean---51.87 -
Num. residues----595
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0074671
X-RAY DIFFRACTIONf_angle_d1.066346
X-RAY DIFFRACTIONf_chiral_restr0.075714
X-RAY DIFFRACTIONf_plane_restr0.006819
X-RAY DIFFRACTIONf_dihedral_angle_d13.3991659
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 14

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.03-2.08080.41081360.36342509264594
2.0808-2.13710.36881460.32822578272498
2.1371-2.19990.34021380.30832599273798
2.1999-2.2710.31951550.29592608276398
2.271-2.35210.32671590.28122600275998
2.3521-2.44630.35951420.27152586272898
2.4463-2.55770.36581380.26092640277898
2.5577-2.69250.32861150.2482629274498
2.6925-2.86120.33421230.23812671279499
2.8612-3.08210.2761400.23782628276899
3.0821-3.39230.26311400.22332661280199
3.3923-3.88320.23871340.19552639277398
3.8832-4.89240.17841340.15122682281699
4.8924-95.07370.18051560.15862699285598
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)
11.4521-0.96491.2531.5151-1.44212.261-0.04780.24130.18270.03270.0309-0.0341-0.17380.00380.02040.2818-0.0260.01860.43060.0790.3626-27.9566-8.4636-21.097
24.573-0.1468-1.67772.28631.11964.03740.04580.28830.1667-0.29990.0879-0.16360.0630.379-0.08160.40630.05260.00210.8080.19820.3846-13.0192-4.7951-55.5578
32.68610.0784-0.52915.1878-0.35463.7918-0.08870.3904-0.129-0.1681-0.0051-0.31070.37440.02250.07330.2684-0.00280.02410.4395-0.01320.2632-7.1446-41.0296-10.1184
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1chain 'A' and (resid 1 through 379 )A1 - 379
2X-RAY DIFFRACTION2chain 'B' and (resid 1 through 115 )B1 - 115
3X-RAY DIFFRACTION3chain 'C' and (resid 2 through 120 )C2 - 120

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