PDBsum entry 3hk3

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Hydrolase PDB id
Protein chains
30 a.a. *
252 a.a. *
Waters ×240
* Residue conservation analysis
PDB id:
Name: Hydrolase
Title: Crystal structure of murine thrombin mutant w215a/e217a (one molecule in the asymmetric unit)
Structure: Thrombin light chain. Chain: a. Fragment: light chain: unp residues 317-360. Synonym: coagulation factor ii. Engineered: yes. Thrombin heavy chain. Chain: b. Fragment: heavy chain: unp residues 361-618. Synonym: coagulation factor ii.
Source: Mus musculus. Mouse. Organism_taxid: 10090. Gene: f2, cf2. Expressed in: cricetulus griseus. Expression_system_taxid: 10029. Expression_system_organ: baby hamster kidney. Expression_system_cell: bhk cells.
1.94Å     R-factor:   0.183     R-free:   0.235
Authors: P.S.Gandhi,M.J.Page,Z.Chen,L.Bush-Pelc,E.Di Cera
Key ref:
P.S.Gandhi et al. (2009). Mechanism of the anticoagulant activity of thrombin mutant W215A/E217A. J Biol Chem, 284, 24098-24105. PubMed id: 19586901 DOI: 10.1074/jbc.M109.025403
22-May-09     Release date:   07-Jul-09    
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Protein chain
Pfam   ArchSchema ?
P19221  (THRB_MOUSE) -  Prothrombin
618 a.a.
30 a.a.
Protein chain
Pfam   ArchSchema ?
P19221  (THRB_MOUSE) -  Prothrombin
618 a.a.
252 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 2 residue positions (black crosses)

 Enzyme reactions 
   Enzyme class: Chains A, B: E.C.  - Thrombin.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: Preferential cleavage: Arg-|-Gly; activates fibrinogen to fibrin and releases fibrinopeptide A and B.
 Gene Ontology (GO) functional annotation 
  GO annot!
  Cellular component     extracellular region   1 term 
  Biological process     blood coagulation   2 terms 
  Biochemical function     catalytic activity     3 terms  


DOI no: 10.1074/jbc.M109.025403 J Biol Chem 284:24098-24105 (2009)
PubMed id: 19586901  
Mechanism of the anticoagulant activity of thrombin mutant W215A/E217A.
P.S.Gandhi, M.J.Page, Z.Chen, L.Bush-Pelc, E.Di Cera.
The thrombin mutant W215A/E217A (WE) is a potent anticoagulant both in vitro and in vivo. Previous x-ray structural studies have shown that WE assumes a partially collapsed conformation that is similar to the inactive E* form, which explains its drastically reduced activity toward substrate. Whether this collapsed conformation is genuine, rather than the result of crystal packing or the mutation introduced in the critical 215-217 beta-strand, and whether binding of thrombomodulin to exosite I can allosterically shift the E* form to the active E form to restore activity toward protein C are issues of considerable mechanistic importance to improve the design of an anticoagulant thrombin mutant for therapeutic applications. Here we present four crystal structures of WE in the human and murine forms that confirm the collapsed conformation reported previously under different experimental conditions and crystal packing. We also present structures of human and murine WE bound to exosite I with a fragment of the platelet receptor PAR1, which is unable to shift WE to the E form. These structural findings, along with kinetic and calorimetry data, indicate that WE is strongly stabilized in the E* form and explain why binding of ligands to exosite I has only a modest effect on the E*-E equilibrium for this mutant. The E* --> E transition requires the combined binding of thrombomodulin and protein C and restores activity of the mutant WE in the anticoagulant pathway.
  Selected figure(s)  
Figure 1.
Cα traces of engineered proteases hWE and mWE compared with native thrombin. Left, new hWE structure with only one molecule in the asymmetric unit (wheat) is nearly identical to the previous structure, 1TQ0 (light green), and differs from the active E form (1SGT (red)) (48) for the collapse of the 215–217 β-strand (arrow) into the active site. Right, mWE-1 (light blue), mWE-2 (wheat), and mWE-3 (light green) differ from wild-type murine thrombin (2OCV (red)) (51) at both the 215–217 β-strand (arrow) and the oxyanion hole.
Figure 4.
PAR1 binding to hWE and mWE does not restore active site architecture. Left, hWE free (wheat) and hWE bound (light green) to the PAR1 peptide (gold) are nearly identical with the exception of the oxyanion hole (see legend for Fig. 3). Right, mWE free (wheat) is nearly identical to the PAR1 (gold) bound state (light green).
  The above figures are reprinted by permission from the ASBMB: J Biol Chem (2009, 284, 24098-24105) copyright 2009.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
20809655 A.D.Vogt, A.Bah, and E.Di Cera (2010).
Evidence of the E*-E equilibrium from rapid kinetics of Na+ binding to activated protein C and factor Xa.
  J Phys Chem B, 114, 16125-16130.  
19846563 W.Niu, Z.Chen, L.A.Bush-Pelc, A.Bah, P.S.Gandhi, and E.Di Cera (2009).
Mutant N143P reveals how Na+ activates thrombin.
  J Biol Chem, 284, 36175-36185.
PDB codes: 3jz1 3jz2
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