PDBsum entry 2od3

Hydrolase/hydrolase inhibitor

PDB id: 2od3

Contents

Protein chains

28 a.a. *

249 a.a. *

Ligands

0G6

NAG

Waters ×271

* Residue conservation analysis

PDB id:

2od3

Name:

Hydrolase/hydrolase inhibitor

Title:

Human thrombin chimera with human residues 184a, 186, 186a, 186b, 186c and 222 replaced by murine thrombin equivalents.

Structure:

Thrombin light chain. Chain: a. Engineered: yes. Mutation: yes. Thrombin heavy chain. Chain: b. Engineered: yes

Source:

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

Resolution:

1.75Å R-factor: 0.199 R-free: 0.230

Authors:

F.Marino,Z.Chen,C.E.Ergenekan,L.A.Bush,F.S.Mathews,E.Di Cera

Key ref:

F.Marino et al. (2007). Structural basis of na+ activation mimicry in murine thrombin. J Biol Chem, 282, 16355-16361. PubMed id: 17428793 DOI: 10.1074/jbc.M701323200

Date:

21-Dec-06 Release date: 10-Apr-07

Protein chain

P00734  (THRB_HUMAN) -  Prothrombin from Homo sapiens

Seq: 622 a.a.

Struc: 28 a.a.

Protein chain

P00734  (THRB_HUMAN) -  Prothrombin from Homo sapiens

Seq: 622 a.a.

Struc: 249 a.a.*

* PDB and UniProt seqs differ at 6 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.M701323200

J Biol Chem 282:16355-16361 (2007)

PubMed id: 17428793

Structural basis of na+ activation mimicry in murine thrombin.

F.Marino, Z.W.Chen, C.E.Ergenekan, L.A.Bush-Pelc, F.S.Mathews, E.Di Cera.

ABSTRACT

Unlike human thrombin, murine thrombin lacks Na(+) activation due to the charge reversal substitution D222K in the Na(+) binding loop. However, the enzyme is functionally stabilized in a Na(+)-bound form and is highly active toward physiologic substrates. The structural basis of this peculiar property is unknown. Here, we present the 2.2 A resolution x-ray crystal structure of murine thrombin in the absence of inhibitors and salts. The enzyme assumes an active conformation, with Ser-195, Glu-192, and Asp-189 oriented as in the Na(+)-bound fast form of human thrombin. Lys-222 completely occludes the pore of entry to the Na(+) binding site and positions its side chain inside the pore, with the Nzeta atom H-bonded to the backbone oxygen atoms of Lys-185, Asp-186b, and Lys-186d. The same architecture is observed in the 1.75 A resolution structure of a thrombin chimera in which the human enzyme carries all residues defining the Na(+) pore in the murine enzyme. These findings demonstrate that Na(+) activation in thrombin is linked to the architecture of the Na(+) pore. The molecular strategy of Na(+) activation mimicry unraveled for murine thrombin is relevant to serine proteases and enzymes activated by monovalent cations in general.

Selected figure(s)

Figure 1.
FIGURE 1. A-C, surface rendering of the pore of entry to the Na^+ binding site of human thrombin in the structure 1SG8 (15) (A) when compared with the same region in murine thrombin (B) and the thrombin chimera (C). Residues lining the pore are color-coded according to their physical properties (red = positively charged, blue = negatively charged, orange = hydrophobic, white = all others). In the human enzyme, the pore is defined by residue Asp-222 in the 220-loop and the sequence PDEGKR from Pro-186 to Arg-187 in the 186-loop (Table 1) (A). In murine thrombin (B), residue 222 is Lys, and the corresponding sequence in the 186-loop is VNDTKR (Table 1). The side chain of Lys-222 completely occludes the pore. The side chain of Asn-186a is glycosylated (NAG). Occlusionofthe pore is also seen in the thrombin chimera (C), in which the human enzyme carries all residues around the pore as in murine thrombin. There is no glycosylation of Asn-186a in the chimera. D-F, architecture of the pore of entry to the Na^+ binding site in the same orientation as shown in the surface rendering (A-C), with relevant residues rendered in Corey-Pauling-Koltun model (carbon in yellow) and the 2F[o] - F[c] electron density maps contoured at the 0.7 level for the structures presented in this study (E and F). The human enzyme (D) shows the pore wide open, whereas Lys-222 in murine thrombin (E) occludes the pore and positions the N atom within H-bonding distance from Lys-185, Asp-186b, and Lys-186d. The backbone oxygen atom of residue 186b is flipped relative to the position assumed in the fast form of the human enzyme. Also shown is the indole side chain of Trp-20, which is Ser in human thrombin, as a structural signature of the murine enzyme. Lys-222 in the thrombin chimera (F) is positioned as in the murine thrombin structure.

Figure 2.
FIGURE 2. Overlay of key residues in murine thrombin (Corey-Pauling-Koltun, with carbon in yellow) and in the fast form (Corey-Pauling-Koltun, with carbon in green) of human thrombin (15). H-bonds (broken lines) refer to the murine thrombin structure. The presence of Lys-222 in murine thrombin stabilizes the conformation in a fast-like form. The O atom of the catalytic Ser-195 is within H-bonding distance (3.05 Å) from His-57. This H-bond is present in the fast form of the human enzyme (3.09 Å) but is broken (3.70 Å) in the Na^+-free slow form (15). The side chain of Asp-189 in the primary specificity pocket is oriented optimally for coordination of Arg of substrate, as seen in the fast form. The conformations of Asp-189 and Ser-195 are maintained by H-bonding interactions mediated by water molecules, as in the fast form of the human enzyme. However, only seven water molecules (red balls) are present in this region of the murine thrombin structure, as opposed to Na^+ (green ball) and 11 water molecules (cyan balls) present in the fast form of the human enzyme (15). The presence of Lys-222 in murine thrombin pushes Arg-187 away and closer (2.55 Å) to Asp-221. The N atom of Lys-222 and the O 1 atom of Asp-221 H-bond to water w153, which in turn stabilizes water w51 in a position equivalent (<1 Å away) to the bound Na^+ in the fast form (green ball) and in contact with the backbone oxygen atoms of Arg-221a (2.77 Å) and Lys-224 (2.61 Å). The H-bonding network around water w51 mimics that seen around the bound Na^+ in the fast form of the human enzyme (15) and establishes a connection to the O 2 atom of Asp-189 via water w97. The O 1 atom of Asp-189 is held in place by an H-bond with water w55 (2.74 Å). Ser-195 is fixed in its orientation by a water-mediated contact with the O 1 atom of Glu-192, with water w63 positioned 3.19 Å away from the O atom of Ser-195 and 2.82 Å away from the O 1 atom of Glu-192. The only two water molecules, w141 and w142, between Asp-189 and Ser-195 are too far away from either residue. Thus, murine thrombin lacks the connectivity between the primary specificity pocket and the catalytic triad seen in the fast form of the human enzyme.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2007, 282, 16355-16361) copyright 2007.

Figures were selected by an automated process.