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- PDB-4mlf: Crystal structure for the complex of thrombin mutant D102N and hirudin -

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Basic information

Entry
Database: PDB / ID: 4mlf
TitleCrystal structure for the complex of thrombin mutant D102N and hirudin
Components
  • (Thrombin) x 2
  • Hirudin variant-1
KeywordsHYDROLASE/HYDROLASE INHIBITOR / Serine protease / ligand binding / hirudin / thrombin / conformational selection / HYDROLASE-HYDROLASE INHIBITOR complex
Function / homology
Function and homology information


negative regulation of serine-type peptidase activity / positive regulation of lipid kinase activity / positive regulation of phospholipase C-activating G protein-coupled receptor signaling pathway / cytolysis by host of symbiont cells / 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 ...negative regulation of serine-type peptidase activity / positive regulation of lipid kinase activity / positive regulation of phospholipase C-activating G protein-coupled receptor signaling pathway / cytolysis by host of symbiont cells / 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 / negative regulation of cytokine production involved in inflammatory response / 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 / fibrinolysis / regulation of cytosolic calcium ion concentration / Intrinsic Pathway of Fibrin Clot Formation / Peptide ligand-binding receptors / positive regulation of release of sequestered calcium ion into cytosol / acute-phase response / Regulation of Complement cascade / negative regulation of proteolysis / Cell surface interactions at the vascular wall / lipopolysaccharide binding / positive regulation of receptor signaling pathway via JAK-STAT / growth factor activity / serine-type endopeptidase inhibitor activity / positive regulation of insulin secretion / platelet activation / response to wounding / positive regulation of protein localization to nucleus / Golgi lumen / antimicrobial humoral immune response mediated by antimicrobial peptide / 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 / Thrombin signalling through proteinase activated receptors (PARs) / heparin binding / regulation of cell shape / positive regulation of cell growth / G alpha (q) signalling events / collagen-containing extracellular matrix / positive regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / cell surface receptor signaling pathway / blood microparticle / positive regulation of protein phosphorylation / G protein-coupled receptor signaling pathway / endoplasmic reticulum lumen / serine-type endopeptidase activity / signaling receptor binding / calcium ion binding / positive regulation of cell population proliferation / proteolysis / extracellular space / extracellular exosome / extracellular region / plasma membrane
Similarity search - Function
Thrombin Inhibitor (Hirudin); Chain I / Thrombin Inhibitor (Hirudin), subunit I / Thrombin inhibitor hirudin / Hirudin / Proteinase inhibitor I14, hirudin / Hirudin/antistatin / Prothrombin/thrombin / Thrombin light chain / Thrombin light chain domain superfamily / Thrombin light chain ...Thrombin Inhibitor (Hirudin); Chain I / Thrombin Inhibitor (Hirudin), subunit I / Thrombin inhibitor hirudin / Hirudin / Proteinase inhibitor I14, hirudin / Hirudin/antistatin / 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 / Distorted Sandwich / 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 domain profile. / Serine proteases, trypsin family, serine active site. / 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
ACETATE ION / Prothrombin / Hirudin variant-1
Similarity search - Component
Biological speciesHomo sapiens (human)
Hirudo medicinalis (medicinal leech)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / Resolution: 2.2 Å
AuthorsVogt, A.D. / Pozzi, N. / Chen, Z. / Di Cera, E.
Citation
Journal: Biophys.Chem. / Year: 2014
Title: Essential role of conformational selection in ligand binding.
Authors: Vogt, A.D. / Pozzi, N. / Chen, Z. / Di Cera, E.
#1: Journal: J.Biol.Chem. / Year: 2004
Title: Molecular dissection of Na+ binding to thrombin.
Authors: Pineda, A.O. / Carrell, C.J. / Bush, L.A. / Prasad, S. / Caccia, S. / Chen, Z.W. / Mathews, F.S. / Di Cera, E.
History
DepositionSep 6, 2013Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 25, 2013Provider: repository / Type: Initial release
Revision 1.1Oct 23, 2013Group: Database references
Revision 1.2Mar 5, 2014Group: Database references
Revision 1.3Jul 29, 2020Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Structure summary
Category: chem_comp / entity ...chem_comp / entity / pdbx_chem_comp_identifier / pdbx_entity_nonpoly / pdbx_struct_conn_angle / struct_conn / struct_ref_seq_dif / 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 / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.pdbx_role / _struct_conn.ptnr2_auth_seq_id / _struct_ref_seq_dif.details
Description: Carbohydrate remediation / Provider: repository / Type: Remediation
Revision 1.4Sep 20, 2023Group: Data collection / Database references ...Data collection / Database references / Refinement description / Structure summary
Category: chem_comp / chem_comp_atom ...chem_comp / chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _chem_comp.pdbx_synonyms / _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Thrombin
B: Thrombin
D: Hirudin variant-1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)40,9658
Polymers40,5443
Non-polymers4215
Water1,76598
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area6020 Å2
ΔGint-30 kcal/mol
Surface area16330 Å2
MethodPISA
Unit cell
Length a, b, c (Å)89.506, 89.506, 133.148
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number96
Space group name H-MP43212

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Components

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Protein , 2 types, 2 molecules BD

#2: Protein Thrombin


Mass: 29779.234 Da / Num. of mol.: 1 / Fragment: Thrombin heavy chain (UNP residues 364-622) / Mutation: D102N
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: F2 / Cell (production host): Kidney Fibroblast Cells (BHK-21) / Production host: Cricetulus griseus (Chinese hamster) / References: UniProt: P00734, thrombin
#3: Protein Hirudin variant-1 / Hirudin-1 / Hirudin-I / Lepirudin


Mass: 6973.505 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Hirudo medicinalis (medicinal leech) / Production host: Escherichia coli (E. coli) / References: UniProt: P01050

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Protein/peptide / Sugars , 2 types, 2 molecules A

#1: Protein/peptide Thrombin


Mass: 3791.204 Da / Num. of mol.: 1 / Fragment: Thrombin light chain (UNP residues 331-363)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: F2 / Cell (production host): Kidney Fibroblast Cells (BHK-21) / Production host: Cricetulus griseus (Chinese hamster) / References: UniProt: P00734, thrombin
#5: 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.: 1
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

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Non-polymers , 3 types, 102 molecules

#4: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Na
#6: Chemical ChemComp-ACT / ACETATE ION


Mass: 59.044 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C2H3O2
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 98 / 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: 3.29 Å3/Da / Density % sol: 62.6 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 7.3
Details: 20% PEG 3350, 0.2M Ca acetate, pH 7.3, VAPOR DIFFUSION, HANGING DROP, temperature 293K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU MICROMAX-007 HF / Wavelength: 1.5418 Å
DetectorType: RIGAKU RAXIS IV++ / Detector: IMAGE PLATE / Date: Aug 11, 2013
RadiationMonochromator: Mirror / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
ReflectionResolution: 2.2→74.28 Å / Num. all: 28265 / Num. obs: 26993 / % possible obs: 95.5 % / Observed criterion σ(F): -0.5 / Observed criterion σ(I): -0.5 / Redundancy: 11.5 % / Biso Wilson estimate: 39.5 Å2 / Rmerge(I) obs: 0.07 / Rsym value: 0.07 / Net I/σ(I): 23.8
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. unique allRsym value% possible all
2.2-2.247.10.4813.313520.48197.6
2.24-2.287.50.4633.513220.46396.5
2.28-2.327.50.4353.713320.43596.2
2.32-2.377.70.414413370.41496.8
2.37-2.427.70.3814.313510.38196.2
2.42-2.4880.3694.713320.36996.8

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Processing

Software
NameVersionClassification
CrystalCleardata collection
MOLREPfrom ccp4phasing
REFMAC5.5.0109refinement
HKL-2000data reduction
HKL-2000data scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 1SHH

1shh


Resolution: 2.2→40 Å / Cor.coef. Fo:Fc: 0.957 / Cor.coef. Fo:Fc free: 0.936 / SU B: 13.225 / SU ML: 0.145 / Isotropic thermal model: Isotropic / Cross valid method: THROUGHOUT / σ(F): -0.5 / σ(I): -0.5 / ESU R: 0.208 / ESU R Free: 0.18 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.2335 1348 5 %RANDOM
Rwork0.19818 ---
obs0.19996 25485 95.16 %-
all-26781 --
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.4 Å / Solvent model: MASK
Displacement parametersBiso mean: 52.611 Å2
Baniso -1Baniso -2Baniso -3
1--0.47 Å20 Å20 Å2
2---0.47 Å20 Å2
3---0.94 Å2
Refinement stepCycle: LAST / Resolution: 2.2→40 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2802 0 27 98 2927
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0110.0222905
X-RAY DIFFRACTIONr_angle_refined_deg1.4631.9673925
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.7985350
X-RAY DIFFRACTIONr_dihedral_angle_2_deg36.18224.317139
X-RAY DIFFRACTIONr_dihedral_angle_3_deg18.53115508
X-RAY DIFFRACTIONr_dihedral_angle_4_deg18.2061520
X-RAY DIFFRACTIONr_chiral_restr0.1050.2408
X-RAY DIFFRACTIONr_gen_planes_refined0.0050.0212229
X-RAY DIFFRACTIONr_mcbond_it0.4871.51758
X-RAY DIFFRACTIONr_mcangle_it0.92322822
X-RAY DIFFRACTIONr_scbond_it1.77131147
X-RAY DIFFRACTIONr_scangle_it2.8774.51103
LS refinement shellResolution: 2.2→2.257 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.37 113 -
Rwork0.322 1861 -
obs-1861 96.72 %
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)
18.6794-2.167910.38462.9416-2.050912.8879-0.55460.40640.8085-0.2648-0.0236-0.2766-1.09380.38860.57820.4210.08960.31170.11490.12410.538411.41159.3156-17.7248
24.12911.25883.99145.8194-1.043110.04380.2568-0.1775-0.02480.52570.12760.56940.5523-1.3328-0.38440.1452-0.05320.04120.29580.00960.15995.07270.2189-7.7485
31.6161-0.72970.46615.0804-0.49992.84390.16780.2523-0.3373-0.1326-0.03880.03070.00840.012-0.12890.0660.0151-0.03140.0706-0.0750.091319.1667-7.3595-20.8459
46.634-0.65330.70643.51440.35142.81450.04850.2757-0.2114-0.45070.179-0.50270.13720.1194-0.22750.13480.00910.06610.071-0.05150.099928.3249-0.8894-20.7072
56.07981.17722.31443.26760.67523.30770.18020.2451-0.166-0.3382-0.0244-0.64160.21090.5305-0.15580.16910.09110.09520.2145-0.0540.175928.2642-7.4068-25.7849
64.1287-0.20860.8392.64350.0883.76670.1330.25990.1604-0.1333-0.0014-0.5159-0.10920.4586-0.13160.08690.0030.06490.0713-0.02670.190629.19032.9672-17.9719
71.3196-0.1929-0.03663.71872.88074.0267-0.0296-0.1890.04840.21580.0365-0.04370.0885-0.0127-0.00690.08990.03780.01170.0689-0.01020.027817.0338-2.6679-6.3977
80.0027-0.2186-0.191323.093716.238915.8258-0.03080.0093-0.011-0.0998-0.21791.1993-0.3207-0.62880.24870.23650.05420.03240.1876-0.03030.308420.9587-25.883-16.2078
93.98670.23110.96352.5244-0.23454.17240.1542-0.5339-0.09020.5632-0.0738-0.26820.23810.2657-0.08030.22770.0136-0.02460.1180.00640.087423.6826-8.1461-2.3914
102.71961.3308-0.56116.2731-1.46937.9989-0.0829-0.3207-0.30790.59810.15930.28160.81880.097-0.07630.25540.0680.01030.09720.01080.142519.0585-13.4931-5.239
111.961-0.8822-0.23424.0795-0.44451.72530.0047-0.2215-0.00770.37240.0815-0.16830.13740.0545-0.08610.10290.0055-0.02430.059-0.02070.030921.2827-1.9835-6.0958
129.93070.7205-12.97520.1331-14.495634.93790.47850.68571.3084-1.2624-0.2353-0.5454-1.34090.3222-0.24330.3946-0.1453-0.06160.26430.06350.574233.425212.6541-15.1334
136.73641.6260.19860.6908-0.92273.99390.085-0.1249-0.85810.057-0.1298-0.4080.3980.52280.04480.47370.214-0.27290.1604-0.07340.685235.4831-22.0906-5.952
1413.51172.1156-0.15456.56471.633410.9424-0.07770.2403-0.45060.3514-0.0095-0.66040.07670.5110.08720.35810.187-0.07230.3046-0.01140.668640.091-22.9621-6.5573
1520.320619.0214-9.324518.1143-6.289424.4879-0.6046-0.3246-1.8557-0.5465-0.2278-1.77170.35541.90210.83240.23670.32140.14070.67370.05170.478726.0952-10.349-35.5604
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A1 - 8
2X-RAY DIFFRACTION2A9 - 14
3X-RAY DIFFRACTION3B16 - 37
4X-RAY DIFFRACTION4B38 - 59
5X-RAY DIFFRACTION5B60 - 78
6X-RAY DIFFRACTION6B79 - 127
7X-RAY DIFFRACTION7B128 - 144
8X-RAY DIFFRACTION8B145 - 149
9X-RAY DIFFRACTION9B150 - 184
10X-RAY DIFFRACTION10B185 - 197
11X-RAY DIFFRACTION11B198 - 234
12X-RAY DIFFRACTION12B235 - 247
13X-RAY DIFFRACTION13D301 - 320
14X-RAY DIFFRACTION14D321 - 352
15X-RAY DIFFRACTION15D353 - 365

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