PDBsum entry 1tq0

Blood clotting

PDB id: 1tq0

Contents

Protein chains

31 a.a. *

247 a.a. *

28 a.a. *

Waters ×176

* Residue conservation analysis

PDB id:

1tq0

Name:

Blood clotting

Title:

Crystal structure of the potent anticoagulant thrombin mutant w215a/e217a in free form

Structure:

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

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: f2. Expressed in: cricetulus griseus. Expression_system_taxid: 10029. Expression_system_cell_line: kidney.

Biol. unit:

Dimer (from PQS)

Resolution:

2.80Å R-factor: 0.234 R-free: 0.292

Authors:

A.O.Pineda,Z.-W.Chen,S.Caccia,S.N.Savvides,G.Waksman,F.S.Mathews,E.Di Cera

Key ref:

A.O.Pineda et al. (2004). The anticoagulant thrombin mutant W215A/E217A has a collapsed primary specificity pocket. J Biol Chem, 279, 39824-39828. PubMed id: 15252033 DOI: 10.1074/jbc.M407272200

Date:

16-Jun-04 Release date: 03-Aug-04

Protein chain

P00734  (THRB_HUMAN) -  Prothrombin from Homo sapiens

Seq: 622 a.a.

Struc: 31 a.a.

Protein chains

P00734  (THRB_HUMAN) -  Prothrombin from Homo sapiens

Seq: 622 a.a.

Struc: 247 a.a.*

Protein chain

P00734  (THRB_HUMAN) -  Prothrombin from Homo sapiens

Seq: 622 a.a.

Struc: 28 a.a.

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

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.M407272200

J Biol Chem 279:39824-39828 (2004)

PubMed id: 15252033

The anticoagulant thrombin mutant W215A/E217A has a collapsed primary specificity pocket.

A.O.Pineda, Z.W.Chen, S.Caccia, A.M.Cantwell, S.N.Savvides, G.Waksman, F.S.Mathews, E.Di Cera.

ABSTRACT

The thrombin mutant W215A/E217A features a drastically impaired catalytic activity toward chromogenic and natural substrates but efficiently activates the anticoagulant protein C in the presence of thrombomodulin. As the remarkable anticoagulant properties of this mutant continue to be unraveled in preclinical studies, we solved the x-ray crystal structures of its free form and its complex with the active site inhibitor H-d-Phe-Pro-Arg-CH(2)Cl (PPACK). The PPACK-bound structure of W215A/E217A is identical to the structure of the PPACK-bound slow form of thrombin. On the other hand, the structure of the free form reveals a collapse of the 215-217 strand that crushes the primary specificity pocket. The collapse results from abrogation of the stacking interaction between Phe-227 and Trp-215 and the polar interactions of Glu-217 with Thr-172 and Lys-224. Other notable changes are a rotation of the carboxylate group of Asp-189, breakage of the H-bond between the catalytic residues Ser-195 and His-57, breakage of the ion pair between Asp-222 and Arg-187, and significant disorder in the 186- and 220-loops that define the Na(+) site. These findings explain the impaired catalytic activity of W215A/E217A and demonstrate that the analysis of the molecular basis of substrate recognition by thrombin and other proteases requires crystallization of both the free and bound forms of the enzyme.

Selected figure(s)

Figure 2.
FIG. 2. Stereo view of the active site, primary specificity pocket, and Na^+-binding site of the thrombin mutant WE. The PPACK-inhibited WE structure (blue) is superimposed to the SL structure (red) of the wild type (15). Notwithstanding the drastic difference in atomic resolution (2.4 Å for WE-PPACK and 1.55 Å for SL), the two structures are remarkably similar overall (r.m.s. deviation = 0.4 Å). There is no evidence of bound Na^+ in the WE-PPACK structure, and there is a notable 1:1 correspondence for the water molecules in the Na^+ site between the two structures. Relevant side chains are labeled. In the WE structure, the side chain of Lys-224 moves away from residue 217 because of the E217A mutation.

Figure 3.
FIG. 3. Stereo view of the active site and primary specificity pocket of the thrombin mutant WE. The free form of WE (red), shown with the 2F[o] - F[c] electron density map contoured at 0.7 level (orange), is superimposed to the PPACK-inhibited form (blue). The 215-217 strand in the free form collapses into the primary specificity pocket and clashes with the Arg residue at the P1 position of PPACK (green). The r.m.s. deviation between free WE and WE-PPACK in the 215-221 segment is 2.5 Å. The r.m.s. deviation between the two monomers in the asymmetric unit of the free WE structure in the same segment is 0.5 Å. Also notable is the rotation of the side chain of Asp-189 in the free form that aligns almost parallel to the backbone as well as the shift in the side chain of Ser-195 that moves away from its H-bonding partner His-57.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2004, 279, 39824-39828) copyright 2004.

Figures were selected by an automated process.