PDBsum entry 1eb1

Hydrolase/hydrolase inhibitor

PDB id: 1eb1

Contents

Protein chains

257 a.a. *

27 a.a. *

Ligands

ASP-TYR-GLU-PRO-ILE-PRO-GLU-GLU-ALA-PHE

ZAL-PRO-MMO

Waters ×320

* Residue conservation analysis

PDB id:

1eb1

Name:

Hydrolase/hydrolase inhibitor

Title:

Complex structure of human thrombin with n-methyl-arginine inhibitor

Structure:

Peptide inhibitor. Chain: a. Engineered: yes. Thrombin heavy chain. Chain: h. Thrombin light chain. Chain: l. Fragment: catalytic domain residues 364-620. 3-cyclohexyl-d-alanyl-l-prolyl-n~2~-methyl-l-arginine.

Source:

Synthetic: yes. Synthetic construct. Organism_taxid: 32630. Homo sapiens. Human. Organism_taxid: 9606. Organ: blood. Organism_taxid: 32630

Biol. unit:

Hetero-Trimer (from PDB file)

Resolution:

1.80Å R-factor: 0.191 R-free: 0.220

Authors:

R.Friedrich,T.Steinmetzer,W.Bode

Key ref:

R.Friedrich et al. (2002). The methyl group of N(alpha)(Me)Arg-containing peptides disturbs the active-site geometry of thrombin, impairing efficient cleavage. J Mol Biol, 316, 869-874. PubMed id: 11884127 DOI: 10.1006/jmbi.2001.5394

Date:

18-Jul-01 Release date: 28-Jan-02

Protein chain

P00734  (THRB_HUMAN) -  Prothrombin from Homo sapiens

Seq: 622 a.a.

Struc: 257 a.a.

Protein chain

P00734  (THRB_HUMAN) -  Prothrombin from Homo sapiens

Seq: 622 a.a.

Struc: 27 a.a.

Enzyme reactions

• Enzyme class: Chains H, L: E.C.3.4.21.5 - thrombin.
• Reaction: Preferential cleavage: Arg-|-Gly; activates fibrinogen to fibrin and releases fibrinopeptide A and B.

DOI no: 10.1006/jmbi.2001.5394

J Mol Biol 316:869-874 (2002)

PubMed id: 11884127

The methyl group of N(alpha)(Me)Arg-containing peptides disturbs the active-site geometry of thrombin, impairing efficient cleavage.

R.Friedrich, T.Steinmetzer, R.Huber, J.Stürzebecher, W.Bode.

ABSTRACT

Bivalent peptidic thrombin inhibitors consisting of an N-terminal d-cyclohexylalanine-Pro-N(alpha)(Me)Arg active-site fragment, a flexible polyglycine linker, and a C-terminal hirugen-like segment directed towards the fibrinogen recognition exosite inhibit thrombin with K(i) values in the picomolar range, remaining stable in buffered solution at pH 7.8 for at least 15 hours. In order to investigate the structural basis of this increased stability, the most potent of these inhibitors, I-11 (K(i)=37pM), containing an N(alpha)(Me)Arg-Thr bond, was crystallized in complex with human alpha-thrombin. X-ray data were collected to 1.8A resolution and the crystal structure of this complex was determined. The Fourier map displays clear electron density for the N-terminal fragment and for the exosite binding segment. It indicates, however, that in agreement with Edman sequencing, the peptide had been cleaved in the crystal, presumably due to the long incubation time of 14 days needed for crystallization and data collection. The N(alpha)(Me) group is directed toward the carbonyl oxygen atom of Ser214, pushing the Ser195 O(gamma) atom out of its normal site. This structure suggests that upon thrombin binding, the scissile peptide bond of the intact peptide and the Ser195 O(gamma) are separated from each other, impairing the nucleophilic attack of the Ser195 O(gamma) toward the N(alpha)(Me)Arg carbonyl group. In the time-scale of two weeks, however, cleavage geometries favoured by the crystal allow catalysis at a slow rate.

Selected figure(s)

Figure 1.
Figure 1. Stereo view of a section of the I-11-thrombin complex around the bound Pro-N a (Me)Arg segment of I-11 (orange), superimposed with the final 1.8 Å electron density. The picture is obtained from standard orientation by a 90 ° rotation about a vertical axis. The density is contoured at 1.2s. Relevant residues are labelled. The Figure was made with MAIN. 27

Figure 2.
Figure 2. (a) Stereo view of the active-site cleft of the thrombin component (white sticks) of I-11-thrombin shown in standard orientation. The inhibitors I-11 (orange), PPACK (green), and hirulog 3 (blue) are superimposed, and the active-site residues, Asp189, Trp215, Glu192 and Trp60D are labelled. The Glu192 carboxylate is not defined by elec- tron density. The Figure was made with MAIN. 27 (b) Stereo view into the non-primed region of I-11-thrombin, con- taining the bound inhibitors I-11 (orange) and PPACK (green). The thrombin surface is represented as a solid surface, with the colours showing the electrostatic surface potential from -15kT/e (intense red) to +15kT/e (intense blue). The Figure was made with WEBLABVIEWER (http://www.msi.com).

The above figures are reprinted by permission from Elsevier: J Mol Biol (2002, 316, 869-874) copyright 2002.