PDBsum entry 4hzh

Hydrolase

PDB id: 4hzh

Contents

Protein chains

392 a.a.

468 a.a.

Ligands

NAG ×4

PDB id:

4hzh

Name:

Hydrolase

Title:

Structure of recombinant gla-domainless prothrombin mutant s525a

Structure:

Prothrombin. Chain: a, b. Fragment: unp residues 90-622. Synonym: coagulation factor ii, activation peptide fragment 1, activation peptide fragment 2, thrombin light chain, thrombin heavy chain. Engineered: yes. Mutation: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: f2. Expressed in: homo sapiens. Expression_system_taxid: 9606. Expression_system_cell_line: bhk cell

Resolution:

3.30Å R-factor: 0.296 R-free: 0.329

Authors:

N.Pozzi,W.Niu,D.W.Gohara,Z.Chen,E.Di Cera

Key ref:

N.Pozzi et al. (2013). Crystal structure of prothrombin reveals conformational flexibility and mechanism of activation. J Biol Chem, 288, 22734-22744. PubMed id: 23775088 DOI: 10.1074/jbc.M113.466946

Date:

15-Nov-12 Release date: 26-Jun-13

Protein chain

P00734  (THRB_HUMAN) -  Prothrombin from Homo sapiens

Seq: 622 a.a.

Struc: 392 a.a.*

Protein chain

P00734  (THRB_HUMAN) -  Prothrombin from Homo sapiens

Seq: 622 a.a.

Struc: 468 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

Enzyme reactions

• Enzyme class: Chains A, B: 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.M113.466946

J Biol Chem 288:22734-22744 (2013)

PubMed id: 23775088

Crystal structure of prothrombin reveals conformational flexibility and mechanism of activation.

N.Pozzi, Z.Chen, D.W.Gohara, W.Niu, T.Heyduk, E.Di Cera.

ABSTRACT

The zymogen prothrombin is composed of fragment 1 containing a Gla domain and kringle-1, fragment 2 containing kringle-2, and a protease domain containing A and B chains. The prothrombinase complex assembled on the surface of platelets converts prothrombin to thrombin by cleaving at Arg-271 and Arg-320. The three-dimensional architecture of prothrombin and the molecular basis of its activation remain elusive. Here we report the first x-ray crystal structure of prothrombin as a Gla-domainless construct carrying an Ala replacement of the catalytic Ser-525. Prothrombin features a conformation 80 Å long, with fragment 1 positioned at a 36° angle relative to the main axis of fragment 2 coaxial to the protease domain. High flexibility of the linker connecting the two kringles suggests multiple arrangements for kringle-1 relative to the rest of the prothrombin molecule. Luminescence resonance energy transfer measurements detect two distinct conformations of prothrombin in solution, in a 3:2 ratio, with the distance between the two kringles either fully extended (54 ± 2 Å) or partially collapsed (≤34 Å) as seen in the crystal structure. A molecular mechanism of prothrombin activation emerges from the structure. Of the two sites of cleavage, Arg-271 is located in a disordered region connecting kringle-2 to the A chain, but Arg-320 is well defined within the activation domain and is not accessible to proteolysis in solution. Burial of Arg-320 prevents prothrombin autoactivation and directs prothrombinase to cleave at Arg-271 first. Reversal of the local electrostatic potential then redirects prothrombinase toward Arg-320, leading to thrombin generation via the prethrombin-2 intermediate.