PDBsum entry 2pgq

Hydrolase/hydrolase inhibitor

PDB id: 2pgq

Contents

Protein chains

43 a.a. *

257 a.a. *

Ligands

0G6

NAG

Metals

_ZN ×3

Waters ×304

* Residue conservation analysis

PDB id:

2pgq

Name:

Hydrolase/hydrolase inhibitor

Title:

Human thrombin mutant c191a-c220a in complex with the inhibitor ppack

Structure:

Thrombin light chain. Chain: a. Fragment: residues 319-363. Synonym: coagulation factor ii. Engineered: yes. Thrombin heavy chain. Chain: b. Fragment: residues 364-622. Synonym: coagulation factor ii.

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: f2. Expressed in: cricetulus griseus. Expression_system_taxid: 10029. Expression_system_cell_line: bhk-21. Expression_system_organ: kidney.

Resolution:

1.80Å R-factor: 0.198 R-free: 0.222

Authors:

L.A.Bush-Pelc,F.Marino,Z.Chen,A.O.Pineda,F.S.Mathews,E.Di Cera

Key ref:

L.A.Bush-Pelc et al. (2007). Important role of the cys-191 cys-220 disulfide bond in thrombin function and allostery. J Biol Chem, 282, 27165-27170. PubMed id: 17636263 DOI: 10.1074/jbc.M703202200

Date:

10-Apr-07 Release date: 17-Jul-07

Protein chain

P00734  (THRB_HUMAN) -  Prothrombin from Homo sapiens

Seq: 622 a.a.

Struc: 43 a.a.

Protein chain

P00734  (THRB_HUMAN) -  Prothrombin from Homo sapiens

Seq: 622 a.a.

Struc: 257 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.M703202200

J Biol Chem 282:27165-27170 (2007)

PubMed id: 17636263

Important role of the cys-191 cys-220 disulfide bond in thrombin function and allostery.

L.A.Bush-Pelc, F.Marino, Z.Chen, A.O.Pineda, F.S.Mathews, E.Di Cera.

ABSTRACT

Little is known on the role of disulfide bonds in the catalytic domain of serine proteases. The Cys-191-Cys-220 disulfide bond is located between the 190 strand leading to the oxyanion hole and the 220-loop that contributes to the architecture of the primary specificity pocket and the Na+ binding site in allosteric proteases. Removal of this bond in thrombin produces an approximately 100-fold loss of activity toward several chromogenic and natural substrates carrying Arg or Lys at P1. Na+ activation is compromised, and no fluorescence change can be detected in response to Na+ binding. A 1.54-A resolution structure of the C191A/C220A mutant in the free form reveals a conformation similar to the Na+-free slow form of wild type. The lack of disulfide bond exposes the side chain of Asp-189 to solvent, flips the backbone O atom of Gly-219, and generates disorder in portions of the 186 and 220 loops defining the Na+ site. This conformation, featuring perturbation of the Na+ site but with the active site accessible to substrate, offers a possible representation of the recently identified E* form of thrombin. Disorder in the 186 and 220 loops and the flip of Gly-219 are corrected by the active site inhibitor H-D-Phe-Pro-Arg-CH(2)Cl, as revealed by the 1.8-A resolution structure of the complex. We conclude that the Cys-191-Cys-220 disulfide bond confers stability to the primary specificity pocket by shielding Asp-189 from the solvent and orients the backbone O atom of Gly-219 for optimal substrate binding. In addition, the disulfide bond stabilizes the 186 and 220 loops that are critical for Na+ binding and activation.

Selected figure(s)

Figure 1.
FIGURE 1. Overlay of the ribbon plots of the structures of the thrombin mutant C191A/C220A in the free (wheat) and PPACK-inhibited (cyan) forms. The r.m.s.d. between the two structures is 0.42 Å. Structures are displayed in the standard Bode orientation (3) with the active site in the middle. The catalytic residues His-57, Asp-102, and Ser-195 are rendered as sticks as is Asp-189 in the primary specificity pocket and the inhibitor PPACK (green). Relevant regions of the enzyme are noted. The C atoms of Ala-191 and Ala-220 at the sites of mutation are indicated by arrows (black for Ala-220, red for Ala-191). Note the autolysis loop that is completely ordered only in the PPACK-bound form.

Figure 2.
FIGURE 2. Electron density maps 2F[0] - F[C] contoured at 0. 7 for the thrombin mutant C191A/C220A in its free CCF (A) and PPACK-bound CCB (B) forms. Shown is the region around the mutations (arrows) with the adjacent 186 loop, the 217–220 strand, the primary specificity pocket up to the catalytic Ser-195 and His-57. Removal of he Cys-191–Cys-220 disulfide bond increases exposure of Asp-189 to solvent. Note the flip of the backbone O atom of Gly-219 in the CCF structure. The O atom of Ser-195 is oriented away from His-57 in CCF, as seen in the slow form of wild type (22). Disorder in the side chains of residues in the 186-loop and around Glu-217 and Gly-219 in the CCF structure (A) is corrected by the presence of PPACK (stick model in green) in the CCB structure (B). Disorder in the Na^+ binding site (186 and 220 loops) suggests that the conformation of CCF is unable to bind Na^+, in agreement with functional data on the mutant.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2007, 282, 27165-27170) copyright 2007.