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- PDB-3hkj: Crystal structure of human thrombin mutant W215A/E217A in complex... -

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Basic information

Entry
Database: PDB / ID: 3hkj
TitleCrystal structure of human thrombin mutant W215A/E217A in complex with the extracellular fragment of human PAR1
Components
  • Proteinase-activated receptor 1
  • Thrombin heavy chain
  • Thrombin light chain
KeywordsHYDROLASE / Serine protease / Acute phase / Blood coagulation / Cleavage on pair of basic residues / Disease mutation / Disulfide bond / Gamma-carboxyglutamic acid / Glycoprotein / Kringle / Protease / Secreted / Zymogen / Cell membrane / G-protein coupled receptor / Membrane / Phosphoprotein / Receptor / Transducer / Transmembrane
Function / homology
Function and homology information


negative regulation of renin secretion into blood stream / negative regulation of glomerular filtration / dendritic cell homeostasis / establishment of synaptic specificity at neuromuscular junction / thrombin-activated receptor signaling pathway / thrombin-activated receptor activity / platelet dense tubular network / cell-cell junction maintenance / regulation of interleukin-1 beta production / platelet dense granule organization ...negative regulation of renin secretion into blood stream / negative regulation of glomerular filtration / dendritic cell homeostasis / establishment of synaptic specificity at neuromuscular junction / thrombin-activated receptor signaling pathway / thrombin-activated receptor activity / platelet dense tubular network / cell-cell junction maintenance / regulation of interleukin-1 beta production / platelet dense granule organization / connective tissue replacement involved in inflammatory response wound healing / trans-synaptic signaling by endocannabinoid, modulating synaptic transmission / regulation of sensory perception of pain / positive regulation of smooth muscle contraction / positive regulation of calcium ion transport / : / positive regulation of Rho protein signal transduction / positive regulation of lipid kinase activity / cytolysis by host of symbiont cells / positive regulation of phospholipase C-activating G protein-coupled receptor signaling pathway / thrombospondin receptor activity / Defective factor XII causes hereditary angioedema / thrombin / : / regulation of blood coagulation / neutrophil-mediated killing of gram-negative bacterium / ligand-gated ion channel signaling pathway / Defective F8 cleavage by thrombin / Platelet Aggregation (Plug Formation) / negative regulation of astrocyte differentiation / negative regulation of platelet activation / positive regulation of collagen biosynthetic process / G-protein alpha-subunit binding / negative regulation of cytokine production involved in inflammatory response / anatomical structure morphogenesis / positive regulation of blood coagulation / negative regulation of fibrinolysis / Gamma-carboxylation of protein precursors / Transport of gamma-carboxylated protein precursors from the endoplasmic reticulum to the Golgi apparatus / Common Pathway of Fibrin Clot Formation / Removal of aminoterminal propeptides from gamma-carboxylated proteins / positive regulation of vasoconstriction / homeostasis of number of cells within a tissue / release of sequestered calcium ion into cytosol / fibrinolysis / regulation of cytosolic calcium ion concentration / Intrinsic Pathway of Fibrin Clot Formation / Peptide ligand-binding receptors / positive regulation of GTPase activity / positive regulation of release of sequestered calcium ion into cytosol / Regulation of Complement cascade / acute-phase response / positive regulation of interleukin-8 production / negative regulation of proteolysis / Cell surface interactions at the vascular wall / G protein-coupled receptor activity / lipopolysaccharide binding / positive regulation of receptor signaling pathway via JAK-STAT / growth factor activity / neuromuscular junction / positive regulation of insulin secretion / regulation of synaptic plasticity / caveola / platelet activation / : / response to wounding / positive regulation of protein localization to nucleus / Golgi lumen / positive regulation of interleukin-6 production / Regulation of Insulin-like Growth Factor (IGF) transport and uptake by Insulin-like Growth Factor Binding Proteins (IGFBPs) / positive regulation of reactive oxygen species metabolic process / blood coagulation / antimicrobial humoral immune response mediated by antimicrobial peptide / G-protein beta-subunit binding / late endosome / Thrombin signalling through proteinase activated receptors (PARs) / heparin binding / regulation of cell shape / phospholipase C-activating G protein-coupled receptor signaling pathway / positive regulation of cytosolic calcium ion concentration / positive regulation of cell growth / G alpha (q) signalling events / collagen-containing extracellular matrix / postsynaptic membrane / positive regulation of canonical NF-kappaB signal transduction / blood microparticle / negative regulation of neuron apoptotic process / response to lipopolysaccharide / positive regulation of MAPK cascade / positive regulation of ERK1 and ERK2 cascade / positive regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / early endosome / cell surface receptor signaling pathway / positive regulation of cell migration / inflammatory response / positive regulation of protein phosphorylation / G protein-coupled receptor signaling pathway / negative regulation of cell population proliferation / endoplasmic reticulum lumen / serine-type endopeptidase activity
Similarity search - Function
Thrombin receptor / Protease-activated receptor / Prothrombin/thrombin / Thrombin light chain / Thrombin light chain domain superfamily / : / Thrombin light chain / Kringle domain / Kringle / Kringle, conserved site ...Thrombin receptor / Protease-activated receptor / Prothrombin/thrombin / Thrombin light chain / Thrombin light chain domain superfamily / : / Thrombin light chain / Kringle domain / Kringle / Kringle, conserved site / Kringle superfamily / Kringle domain signature. / Kringle domain profile. / Kringle domain / Vitamin K-dependent carboxylation/gamma-carboxyglutamic (GLA) domain / Gamma-carboxyglutamic acid-rich (GLA) domain / Gamma-carboxyglutamic acid-rich (GLA) domain superfamily / Vitamin K-dependent carboxylation domain. / Gla domain profile. / Domain containing Gla (gamma-carboxyglutamate) residues. / Kringle-like fold / G-protein coupled receptors family 1 signature. / G protein-coupled receptor, rhodopsin-like / GPCR, rhodopsin-like, 7TM / G-protein coupled receptors family 1 profile. / 7 transmembrane receptor (rhodopsin family) / Serine proteases, trypsin family, histidine active site / Serine proteases, trypsin family, serine active site / Peptidase S1A, chymotrypsin family / Serine proteases, trypsin family, histidine active site. / Serine proteases, trypsin family, serine active site. / Serine proteases, trypsin domain profile. / Trypsin-like serine protease / Serine proteases, trypsin domain / Trypsin / Trypsin-like serine proteases / Thrombin, subunit H / Peptidase S1, PA clan, chymotrypsin-like fold / Peptidase S1, PA clan / Beta Barrel / Mainly Beta
Similarity search - Domain/homology
Prothrombin / Proteinase-activated receptor 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.6 Å
AuthorsGandhi, P.S. / Page, M.J. / Chen, Z. / Bush-Pelc, L. / Di Cera, E.
Citation
Journal: J.Biol.Chem. / Year: 2009
Title: Mechanism of the Anticoagulant Activity of Thrombin Mutant W215A/E217A.
Authors: Gandhi, P.S. / Page, M.J. / Chen, Z. / Bush-Pelc, L. / Di Cera, E.
#1: Journal: J.Biol.Chem. / Year: 2004
Title: The anticoagulant thrombin mutant W215A/E217A has a collapsed primary specificity pocket.
Authors: Pineda, A.O. / Chen, Z.W. / Caccia, S. / Cantwell, A.M. / Savvides, S.N. / Waksman, G. / Mathews, F.S. / Di Cera, E.
History
DepositionMay 23, 2009Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 7, 2009Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Non-polymer description / Version format compliance
Revision 1.2Jul 29, 2020Group: Data collection / Derived calculations / Structure summary
Category: chem_comp / entity ...chem_comp / entity / pdbx_chem_comp_identifier / pdbx_entity_nonpoly / struct_conn / struct_site / struct_site_gen
Item: _chem_comp.name / _chem_comp.type ..._chem_comp.name / _chem_comp.type / _entity.pdbx_description / _pdbx_entity_nonpoly.name / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.pdbx_role
Description: Carbohydrate remediation / Provider: repository / Type: Remediation
Revision 1.3Oct 13, 2021Group: Database references / Structure summary / Category: chem_comp / database_2 / struct_ref_seq_dif
Item: _chem_comp.pdbx_synonyms / _database_2.pdbx_DOI ..._chem_comp.pdbx_synonyms / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ref_seq_dif.details
Revision 1.4Sep 6, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Thrombin light chain
B: Thrombin heavy chain
C: Proteinase-activated receptor 1
D: Thrombin light chain
E: Thrombin heavy chain
F: Proteinase-activated receptor 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)72,2988
Polymers71,8566
Non-polymers4422
Water2,774154
1
A: Thrombin light chain
B: Thrombin heavy chain
C: Proteinase-activated receptor 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)36,1494
Polymers35,9283
Non-polymers2211
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2190 Å2
ΔGint-7.5 kcal/mol
Surface area15040 Å2
MethodPISA
2
D: Thrombin light chain
E: Thrombin heavy chain
F: Proteinase-activated receptor 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)36,1494
Polymers35,9283
Non-polymers2211
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2090 Å2
ΔGint-7.8 kcal/mol
Surface area14340 Å2
MethodPISA
Unit cell
Length a, b, c (Å)53.140, 61.783, 67.834
Angle α, β, γ (deg.)98.78, 110.28, 92.95
Int Tables number1
Space group name H-MP1
DetailsTHE BIOLOGICAL ASSEMBLY CONSISTS OF A, B AND C CHAINS.

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Components

#1: Protein/peptide Thrombin light chain / Coagulation factor II


Mass: 3647.075 Da / Num. of mol.: 2 / Fragment: Light chain: UNP residues 333-363
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: F2 / Cell (production host): BHK cells / Organ (production host): baby hamster kidney / Production host: Cricetulus griseus (Chinese hamster) / References: UniProt: P00734, thrombin
#2: Protein Thrombin heavy chain / Coagulation factor II


Mass: 29607.055 Da / Num. of mol.: 2 / Fragment: Heavy chain: UNP residues 364-622 / Mutation: W215A, E217A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: F2 / Cell (production host): BHK cells / Organ (production host): baby hamster kidney / Production host: Cricetulus griseus (Chinese hamster) / References: UniProt: P00734, thrombin
#3: Protein/peptide Proteinase-activated receptor 1 / PAR-1 / Thrombin receptor / Coagulation factor II receptor


Mass: 2673.862 Da / Num. of mol.: 2 / Fragment: Extracellular fragment: UNP residues 42-62
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: F2R, CF2R, PAR1, TR / Cell (production host): BHK cells / Organ (production host): baby hamster kidney / Production host: Cricetulus griseus (Chinese hamster) / References: UniProt: P25116
#4: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 154 / Source method: isolated from a natural source / Formula: H2O

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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: 2.85 Å3/Da / Density % sol: 56.92 %
Crystal growTemperature: 295 K / Method: vapor diffusion, hanging drop / pH: 7.5
Details: 100mM HEPES pH 7.5, 20% PEG 10000, VAPOR DIFFUSION, HANGING DROP, temperature 295K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 14-BM-C / Wavelength: 0.9 Å
DetectorType: ADSC QUANTUM 315 / Detector: CCD / Date: Oct 27, 2008
RadiationMonochromator: Bent Ge(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9 Å / Relative weight: 1
ReflectionResolution: 2.6→40 Å / Num. all: 24771 / Num. obs: 22864 / % possible obs: 93.4 % / Observed criterion σ(I): -3 / Redundancy: 3 % / Biso Wilson estimate: 31.7 Å2 / Rmerge(I) obs: 0.052 / Net I/σ(I): 18.5
Reflection shellResolution: 2.6→2.69 Å / Redundancy: 2.6 % / Rmerge(I) obs: 0.199 / Mean I/σ(I) obs: 4.2 / Num. unique all: 1956 / % possible all: 79.3

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Processing

Software
NameVersionClassification
ADSCQuantumdata collection
MOLREPphasing
CNS1.2refinement
HKL-2000data reduction
SCALEPACKdata scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 1TQ0

1tq0


Resolution: 2.6→37.17 Å / Rfactor Rfree error: 0.007 / Data cutoff high absF: 127702.59 / Data cutoff low absF: 0 / Isotropic thermal model: RESTRAINED / Cross valid method: THROUGHOUT / σ(F): 0 / σ(I): 0 / Stereochemistry target values: Engh & Huber
RfactorNum. reflection% reflectionSelection details
Rfree0.234 1079 4.9 %RANDOM
Rwork0.196 ---
obs0.196 22129 90.5 %-
Displacement parametersBiso mean: 40.8 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.38 Å0.29 Å
Luzzati d res low-5 Å
Luzzati sigma a0.38 Å0.32 Å
Refinement stepCycle: LAST / Resolution: 2.6→37.17 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4682 0 28 154 4864
Refine LS restraints
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONc_bond_d0.007
X-RAY DIFFRACTIONc_angle_deg1.5
X-RAY DIFFRACTIONc_dihedral_angle_d23.9
X-RAY DIFFRACTIONc_improper_angle_d0.83
LS refinement shellResolution: 2.6→2.76 Å / Rfactor Rfree error: 0.026 / Total num. of bins used: 6
RfactorNum. reflection% reflection
Rfree0.327 157 5.1 %
Rwork0.266 2898 -
obs-3039 75.1 %

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