PDBsum entry 1shh

Hydrolase/hydrolase inhibitor

PDB id: 1shh

Contents

Protein chains

29 a.a. *

252 a.a. *

29 a.a. *

Ligands

0G6 ×2

NAG

Waters ×483

* Residue conservation analysis

PDB id:

1shh

Name:

Hydrolase/hydrolase inhibitor

Title:

Slow form of thrombin bound with ppack

Structure:

Thrombin. Chain: a, d. Fragment: thrombin light chain (a). Synonym: coagulation factor ii. Engineered: yes. Thrombin. Chain: b, e. Fragment: thrombin heavy chain (b). Synonym: coagulation factor ii.

Source:

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

Biol. unit:

Trimer (from PQS)

Resolution:

1.55Å R-factor: 0.195 R-free: 0.216

Authors:

A.O.Pineda,C.J.Carrell,L.A.Bush,S.Prasad,S.Caccia,Z.W.Chen, F.S.Mathews,E.Di Cera

Key ref:

A.O.Pineda et al. (2004). Molecular dissection of Na+ binding to thrombin. J Biol Chem, 279, 31842-31853. PubMed id: 15152000 DOI: 10.1074/jbc.M401756200

Date:

25-Feb-04 Release date: 08-Jun-04

Protein chain

P00734  (THRB_HUMAN) -  Prothrombin from Homo sapiens

Seq: 622 a.a.

Struc: 29 a.a.

Protein chains

P00734  (THRB_HUMAN) -  Prothrombin from Homo sapiens

Seq: 622 a.a.

Struc: 252 a.a.*

Protein chain

P00734  (THRB_HUMAN) -  Prothrombin from Homo sapiens

Seq: 622 a.a.

Struc: 29 a.a.

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

Enzyme reactions

• Enzyme class: Chains A, B, D, E: 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.M401756200

J Biol Chem 279:31842-31853 (2004)

PubMed id: 15152000

Molecular dissection of Na+ binding to thrombin.

A.O.Pineda, C.J.Carrell, L.A.Bush, S.Prasad, S.Caccia, Z.W.Chen, F.S.Mathews, E.Di Cera.

ABSTRACT

Na(+) binding near the primary specificity pocket of thrombin promotes the procoagulant, prothrombotic, and signaling functions of the enzyme. The effect is mediated allosterically by a communication between the Na(+) site and regions involved in substrate recognition. Using a panel of 78 Ala mutants of thrombin, we have mapped the allosteric core of residues that are energetically linked to Na(+) binding. These residues are Asp-189, Glu-217, Asp-222, and Tyr-225, all in close proximity to the bound Na(+). Among these residues, Asp-189 shares with Asp-221 the important function of transducing Na(+) binding into enhanced catalytic activity. None of the residues of exosite I, exosite II, or the 60-loop plays a significant role in Na(+) binding and allosteric transduction. X-ray crystal structures of the Na(+)-free (slow) and Na(+)-bound (fast) forms of thrombin, free or bound to the active site inhibitor H-d-Phe-Pro-Arg-chloromethyl-ketone, document the conformational changes induced by Na(+) binding. The slow --> fast transition results in formation of the Arg-187:Asp-222 ion pair, optimal orientation of Asp-189 and Ser-195 for substrate binding, and a significant shift of the side chain of Glu-192 linked to a rearrangement of the network of water molecules that connect the bound Na(+) to Ser-195 in the active site. The changes in the water network and the allosteric core explain the thermodynamic signatures linked to Na(+) binding and the mechanism of thrombin activation by Na(+). The role of the water network uncovered in this study establishes a new paradigm for the allosteric regulation of thrombin and other Na(+)-activated enzymes involved in blood coagulation and the immune response.

Selected figure(s)

Figure 7.
FIG. 7. Stereo view of the Na^+ binding environment in the structures of F (free fast form, gold), S (free slow form, red), FL (PPACK-bound fast form, blue), and SL (PPACK-bound slow form, green). Shown are all atoms within 3 Å of the bound Na^+ in the F structure, in addition to the side chains of Asp-189 and Asp-221. Note the similarity of the Na^+ coordination shell between F and FL; the bound Na^+ is coordinated octahedrally by the backbone O atoms of Lys-224 and Arg-221a and by four buried water molecules that H-bond to (clockwise) Asp-189, Asp-221, Gly-223, and Tyr-184a. Only some of these water molecules are replaced in the absence of Na^+ (S and SL). Note the rearrangement of the side chain of Asp-189 in the S structure and the significant shift in the backbone O atom of Arg-221a that assumes a position incompatible with Na^+ coordination. H-bonds are shown by broken lines and refer to the F structure.

Figure 8.
FIG. 8. Stereo view of the electron density maps of the S (A), F (B), SL (C), and FL (D) intermediates of thrombin in the regions bearing the most significant structural transitions. Residues are rendered in CPK. The bound Na^+ is rendered as a cyan ball. Shown are the 221–224 loop region and the 187–195 domain. Note how Asp-222 and Arg-187 have joined densities in the F form, indicative of ion pair interaction, but not in the S form. Also notable are the reorientation of Asp-189 and Glu-192 in the S form, as well as the shift in the position of Ser-195. Other changes observed in the slow fast transition involve the network of water molecules (red balls) embedding the Na^+ site, the S1 pocket, and the active site region. In the fast form, this network is well organized and contains 11 water molecules. In the slow form, the water molecules are reduced to seven, and the long range connectivity of the network is lost (see also Fig. 9). The 2F[o] - F[c] electron density maps are contoured at 0.7 for S and F and at 1.0 for SL and FL.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2004, 279, 31842-31853) copyright 2004.

Figures were selected by an automated process.