PDBsum entry 1rd3

Hydrolase

PDB id: 1rd3

Contents

Protein chains

33 a.a. *

259 a.a. *

31 a.a. *

Ligands

NAG-NDG-BMA-MAN-FUC

NAG-NDG-FUC

PO4 ×3

GOL ×6

Waters ×44

* Residue conservation analysis

PDB id:

1rd3

Name:

Hydrolase

Title:

2.5a structure of anticoagulant thrombin variant e217k

Structure:

Prothrombin. Chain: a, c. Fragment: thrombin light chain. Synonym: coagulation factor ii. Engineered: yes. Prothrombin. Chain: b, d. Fragment: thrombin heavy chain. Synonym: coagulation factor ii.

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: f2. Expressed in: cricetinae gen. Sp.. Expression_system_taxid: 36483.

Biol. unit:

Dimer (from PQS)

Resolution:

2.50Å R-factor: 0.229 R-free: 0.259

Authors:

W.J.Carter,T.Myles,L.L.Leung,J.A.Huntington

Key ref:

W.J.Carter et al. (2004). Crystal structure of anticoagulant thrombin variant E217K provides insights into thrombin allostery. J Biol Chem, 279, 26387-26394. PubMed id: 15075325 DOI: 10.1074/jbc.M402364200

Date:

05-Nov-03 Release date: 04-May-04

Protein chain

P00734  (THRB_HUMAN) -  Prothrombin from Homo sapiens

Seq: 622 a.a.

Struc: 33 a.a.

Protein chains

P00734  (THRB_HUMAN) -  Prothrombin from Homo sapiens

Seq: 622 a.a.

Struc: 259 a.a.*

Protein chain

P00734  (THRB_HUMAN) -  Prothrombin from Homo sapiens

Seq: 622 a.a.

Struc: 31 a.a.

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: Chains A, B, C, D: E.C.3.4.21.5 - thrombin.
• Reaction: Preferential cleavage: Arg-|-Gly; activates fibrinogen to fibrin and releases fibrinopeptide A and B.

DOI no: 10.1074/jbc.M402364200

J Biol Chem 279:26387-26394 (2004)

PubMed id: 15075325

Crystal structure of anticoagulant thrombin variant E217K provides insights into thrombin allostery.

W.J.Carter, T.Myles, C.S.Gibbs, L.L.Leung, J.A.Huntington.

ABSTRACT

Thrombin is the ultimate protease of the blood clotting cascade and plays a major role in its own regulation. The ability of thrombin to exhibit both pro- and anti-coagulant properties has spawned efforts to turn thrombin into an anticoagulant for therapeutic purposes. This quest culminated in the identification of the E217K variant through scanning and saturation mutagenesis. The antithrombotic properties of E217K thrombin are derived from its inability to convert fibrinogen to a fibrin clot while maintaining its thrombomodulin-dependent ability to activate the anticoagulant protein C pathway. Here we describe the 2.5-A crystal structure of human E217K thrombin, which displays a dramatic restructuring of the geometry of the active site. Of particular interest is the repositioning of Glu-192, which hydrogen bonds to the catalytic Ser-195 and which results in the complete occlusion of the active site and the destruction of the oxyanion hole. Substrate binding pockets are further blocked by residues previously implicated in thrombin allostery. We have concluded that the E217K mutation causes the allosteric inactivation of thrombin by destabilizing the Na(+) binding site and that the structure thus may represent the Na(+)-free, catalytically inert "slow" form.

Selected figure(s)

Figure 2.
FIG. 2. E217K thrombin is inactive because of the altered hydrogen bonding of the active site residues. a, stereo representation of the electron density surrounding residues His-57, Trp-60d, 191-196 and 215-220. The conformation of this region is significantly altered for the E217K variant, and as a result of the movement of Glu-192, the hydrogen bonding of the catalytic residues has become non-catalytic. b, superposition of the catalytic residues (His-57 and residues 192-195) of E217K (yellow) and wild-type, active thrombin (1HAH [PDB] , magenta) illustrates the new hydrogen bonding pattern (green broken lines) and the consequent loss of the oxyanion hole, normally formed by the amide hydrogens of Gly-193 and Ser-195 (arrows). The amide hydrogen of Gly-193 is oriented away from the oxyanion hole, and the amide hydrogen of Ser-195 is hydrogen-bonded to the main chain oxygen of Glu-192. The hydrogens of Ser-195 are shown for clarity (white).

Figure 3.
FIG. 3. Surface representations of thrombin reveal the occlusion of the active site cleft caused by the E217K mutation. a, the surface of active thrombin (1HAH [PDB] , colored green for hydrophobicity) in the standard orientation demonstrates the open active site cleft of thrombin and the accessibility of the catalytic O of Ser-195 (red). This conformation represents an allosterically activated thrombin with nothing bound in the active site cleft. However, the structure can easily accommodate a natural thrombin substrate derived from the reactive center loop of heparin cofactor II (P4-P4', shown as rods). b, in contrast, the active site cleft of E217K is in a closed conformation, and although the catalytic O is colored as above, it is fully blocked by the conformational changes in the active site. In addition, it is clear that overlaps and steric clashes from P4 to P1 would prevent substrate binding to E217K. A video depiction of the structural transition from a closed (E217K) to an open (1JOU [PDB] , monomer AB) active site is given as supplementary material.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2004, 279, 26387-26394) copyright 2004.

Figures were selected by the author.