PDBsum entry 2gp9

Hydrolase

PDB id: 2gp9

Contents

Protein chains

32 a.a. *

247 a.a. *

Ligands

EPE

Waters ×150

* Residue conservation analysis

PDB id:

2gp9

Name:

Hydrolase

Title:

Crystal structure of the slow form of thrombin in a self-inhibited conformation

Structure:

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

Dimer (from PQS)

Resolution:

1.87Å R-factor: 0.195 R-free: 0.218

Authors:

A.Pineda,Z.Chen,F.S.Mathews,E.Di Cera

Key ref:

A.O.Pineda et al. (2006). Crystal structure of thrombin in a self-inhibited conformation. J Biol Chem, 281, 32922-32928. PubMed id: 16954215 DOI: 10.1074/jbc.M605530200

Date:

17-Apr-06 Release date: 12-Sep-06

Protein chain

P00734  (THRB_HUMAN) -  Prothrombin from Homo sapiens

Seq: 622 a.a.

Struc: 32 a.a.

Protein chain

P00734  (THRB_HUMAN) -  Prothrombin from Homo sapiens

Seq: 622 a.a.

Struc: 247 a.a.*

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

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.M605530200

J Biol Chem 281:32922-32928 (2006)

PubMed id: 16954215

Crystal structure of thrombin in a self-inhibited conformation.

A.O.Pineda, Z.W.Chen, A.Bah, L.C.Garvey, F.S.Mathews, E.Di Cera.

ABSTRACT

The activating effect of Na(+) on thrombin is allosteric and depends on the conformational transition from a low activity Na(+)-free (slow) form to a high activity Na(+)-bound (fast) form. The structures of these active forms have been solved. Recent structures of thrombin obtained in the absence of Na(+) have also documented inactive conformations that presumably exist in equilibrium with the active slow form. The validity of these inactive slow form structures, however, is called into question by the presence of packing interactions involving the Na(+) site and the active site regions. Here, we report a 1.87A resolution structure of thrombin in the absence of inhibitors and salts with a single molecule in the asymmetric unit and devoid of significant packing interactions in regions involved in the allosteric slow --> fast transition. The structure shows an unprecedented self-inhibited conformation where Trp-215 and Arg-221a relocate >10A to occlude the active site and the primary specificity pocket, and the guanidinium group of Arg-187 penetrates the protein core to fill the empty Na(+)-binding site. The extreme mobility of Trp-215 was investigated further with the W215P mutation. Remarkably, the mutation significantly compromises cleavage of the anticoagulant protein C but has no effect on the hydrolysis of fibrinogen and PAR1. These findings demonstrate that thrombin may assume an inactive conformation in the absence of Na(+) and that its procoagulant and anticoagulant activities are closely linked to the mobility of residue 215.

Selected figure(s)

Figure 1.
FIGURE 1. Surface rendering of the structures of inactive thrombin in the absence of Na^+, labeled with their corresponding Protein Data Bank accession codes. Except for the structure of the W215A/E217A mutant (14) (middle right, 1TQ0), all of the molecules are in the standard Bode orientation (27), with the active site at the center and the Na^+ site in the southwest quadrant. The structure of D102N (top left, 2GP9) is used as reference, with key residues labeled. Also shown for reference is the structure of the active slow form (9) (top right, 1SGI). The areas in cyan correspond to the intermolecular contacts <4Å of the two molecules in the asymmetric unit, related by noncrystallographic 2-fold symmetry (see also Table 2). Only one representative monomer in the asymmetric unit is shown for clarity. The structure of the W215A/E217A mutant (middle right, 1TQ0) is rotated 120° about the y axis relative to the standard orientation to show the contact areas. The other structures refer to the E217K mutant (13) (middle left, 1RD3), wild type in the presence of Li^+ (17) (bottom left, 2AFQ), and molecule 2 of the R77aA mutant in the presence of K^+ (15, 16) (bottom right, 2A0Q).

Figure 3.
FIGURE 3. Stereo view of the overlay of the structures of D102N (CPK, with C in yellow) and the PPACK-inhibited Na^+-bound form (CPK, with C in cyan) (9) reveals the molecular basis of self-inhibition in the D102N structure. Trp-215 and Arg-221a of D102N produce a self-inhibited conformation of the enzyme by occupying positions analogous to Pro and Arg of PPACK (stick model, green) in the fast form. Also shown is the bound Na^+, with the coordinating water molecules and the H-bonding network (dashed lines). Note the significant shift of the 220 loop with disruption of the ionic interactions with the 186 loop, causing Arg-187 to position its guanidinium group within 1 Å from where Na^+ binds in the fast form. The arrows point to the position of residue Asp-189 and the flip of the nitrogen atom of Gly-193 in the oxyanion hole. The structure is a remarkable example of molecular mimicry of bound substrate/inhibitor (Trp-215 and Arg-221a) and Na^+ (Arg-187) made possible by the flexibility of the thrombin fold in the free form.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2006, 281, 32922-32928) copyright 2006.

Figures were selected by an automated process.