1OYH

Crystal Structure of P13 Alanine Variant of Antithrombin


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.62 Å
  • R-Value Free: 0.258 
  • R-Value Work: 0.221 

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Literature

The influence of hinge region residue Glu-381 on antithrombin allostery and metastability

Johnson, D.J.D.Huntington, J.A.

(2004) J Biol Chem 279: 4913-4921

  • DOI: https://doi.org/10.1074/jbc.M311644200
  • Primary Citation of Related Structures:  
    1OYH

  • PubMed Abstract: 

    Antithrombin becomes an efficient inhibitor of factor Xa and thrombin by binding a specific pentasaccharide sequence found on a small fraction of the heparan sulfate proteoglycans lining the microvaculature. In the structure of native antithrombin, the reactive center loop is restrained due to the insertion of its hinge region into the main beta-sheet A, whereas in the heparin-activated state the reactive center loop is freed from beta-sheet A. In both structures, hinge region residue Glu-381 makes several stabilizing contacts. To determine the role of these contacts in the allosteric mechanism of antithrombin activation, we replaced Glu-381 with an alanine. This variant is less active toward its target proteases than control antithrombin, due to a perturbation of the equilibrium between the two forms, and to an increase in stoichiometry of inhibition. Pentasaccharide binding affinity is reduced 4-fold due to an increase in the off-rate. These data suggest that the main role of Glu-381 is to stabilize the activated conformation. Stability studies also showed that the E381A variant is resistant to continued insertion of its reactive center loop upon incubation at 50 degrees C, suggesting new stabilizing interactions in the native structure. To test this hypothesis, and to aid in the interpretation of the kinetic data we solved to 2.6 A the structure of the variant. We conclude that wild-type Glu-381 interactions stabilize the activated state and decreases the energy barrier to full loop insertion.


  • Organizational Affiliation

    University of Cambridge, Department of Haematology, Cambridge Institute for Medical Research, Division of Structural Medicine, Thrombosis Research Unit, Wellcome Trust MRC Building, Hills Road, Cambridge CB2 2XY, United Kingdom.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Antithrombin-IIIA [auth I]432Homo sapiensMutation(s): 2 
UniProt & NIH Common Fund Data Resources
Find proteins for P01008 (Homo sapiens)
Explore P01008 
Go to UniProtKB:  P01008
PHAROS:  P01008
GTEx:  ENSG00000117601 
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP01008
Sequence Annotations
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  • Reference Sequence
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 2
MoleculeChains Sequence LengthOrganismDetailsImage
Antithrombin-IIIB [auth L]432Homo sapiensMutation(s): 0 
UniProt & NIH Common Fund Data Resources
Find proteins for P01008 (Homo sapiens)
Explore P01008 
Go to UniProtKB:  P01008
PHAROS:  P01008
GTEx:  ENSG00000117601 
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP01008
Sequence Annotations
Expand
  • Reference Sequence
Oligosaccharides

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Entity ID: 3
MoleculeChains Length2D Diagram Glycosylation3D Interactions
2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranoseC [auth A],
D [auth B]
2N-Glycosylation
Glycosylation Resources
GlyTouCan:  G42666HT
GlyCosmos:  G42666HT
GlyGen:  G42666HT
Entity ID: 4
MoleculeChains Length2D Diagram Glycosylation3D Interactions
alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranoseE [auth C]5N-Glycosylation
Glycosylation Resources
GlyTouCan:  G22768VO
GlyCosmos:  G22768VO
GlyGen:  G22768VO
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.62 Å
  • R-Value Free: 0.258 
  • R-Value Work: 0.221 
  • Space Group: P 1 21 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 68.998α = 90
b = 98.573β = 105.53
c = 89.35γ = 90
Software Package:
Software NamePurpose
MAR345data collection
SCALAdata scaling
MOLREPphasing
CNSrefinement
CCP4data scaling

Structure Validation

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Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2004-04-13
    Type: Initial release
  • Version 1.1: 2008-04-29
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Non-polymer description, Version format compliance
  • Version 1.3: 2017-10-11
    Changes: Refinement description
  • Version 2.0: 2020-07-29
    Type: Remediation
    Reason: Carbohydrate remediation
    Changes: Advisory, Atomic model, Data collection, Derived calculations, Non-polymer description, Structure summary
  • Version 2.1: 2021-10-27
    Changes: Database references, Structure summary
  • Version 2.2: 2023-08-16
    Changes: Data collection, Refinement description