PDBsum entry 1eol

Hydrolase/hydrolase inhibitor

PDB id: 1eol

Contents

Protein chains

278 a.a. *

15 a.a. *

Waters ×142

* Residue conservation analysis

PDB id:

1eol

Name:

Hydrolase/hydrolase inhibitor

Title:

Design of p1' and p3' residues of trivalent thrombin inhibitors and their crystal structures

Structure:

Alpha thrombin. Chain: a. Thrombin inhibitor p628. Chain: b. Engineered: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Synthetic: yes. Other_details: this sequence was chemically synthesized

Biol. unit:

Dimer (from PQS)

Resolution:

2.10Å R-factor: 0.193 R-free: 0.240

Authors:

J.J.Slon-Usakiewicz,J.Sivaraman,Y.Li,M.Cygler,Y.Konishi

Key ref:

J.J.Slon-Usakiewicz et al. (2000). Design of P1' and P3' residues of trivalent thrombin inhibitors and their crystal structures. Biochemistry, 39, 2384-2391. PubMed id: 10694407 DOI: 10.1021/bi992419b

Date:

23-Mar-00 Release date: 03-May-00

Protein chain

P00734  (THRB_HUMAN) -  Prothrombin from Homo sapiens

Seq: 622 a.a.

Struc: 278 a.a.*

Protein chain

No UniProt id for this chain

Struc: 15 a.a.

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

Enzyme reactions

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

DOI no: 10.1021/bi992419b

Biochemistry 39:2384-2391 (2000)

PubMed id: 10694407

Design of P1' and P3' residues of trivalent thrombin inhibitors and their crystal structures.

J.J.Slon-Usakiewicz, J.Sivaraman, Y.Li, M.Cygler, Y.Konishi.

ABSTRACT

Synthetic bivalent thrombin inhibitors comprise an active site blocking segment, a fibrinogen recognition exosite blocking segment, and a linker connecting these segments. Possible nonpolar interactions of the P1' and P3' residues of the linker with thrombin S1' and S3' subsites, respectively, were identified using the "Methyl Scan" method [Slon-Usakiewicz et al. (1997) Biochemistry 36, 13494-13502]. A series of inhibitors (4-tert-butylbenzenesulfonyl)-Arg-(D-pipecolic acid)-Xaa-Gly-Yaa-Gly-betaAla-Asp-Tyr-Glu-Pro-Ile-Pro-Glu-Glu-Ala- (be ta-cyclohexylalanine)-(D-Glu)-OH, in which nonpolar P1' residue Xaa or P3' residue Yaa was incorporated, were designed and improved the affinity to thrombin. Substitution of the P3' residue with D-phenylglycine or D-Phe improved the K(i) value to (9.5 +/- 0.6) x 10(-14) or 1.3 +/- 0.5 x 10(-13) M, respectively, compared to that of a reference inhibitor with Gly residues at Xaa and Yaa residues (K(i) = (2.4 +/- 0.5) x 10(-11) M). Similarly, substitution of the P1' residue with L-norleucine or L-beta-(2-thienyl)alanine lowered the K(i) values to (8.2 +/- 0.6) x 10(-14) or (5.1 +/- 0.4) x 10(-14) M, respectively. The linker Gly-Gly-Gly-betaAla of the inhibitors in the previous sentence was simplified with 12-aminododecanoic acid, resulting in further improvement of the K(i) values to (3.8 +/- 0.6) x 10(-14) or (1.7 +/- 0.4) x 10(-14) M, respectively. These K(i) values are equivalent to that of natural hirudin (2.2 x 10(-14) M), yet the size of the synthetic inhibitors (2 kD) is only one-third that of hirudin (7 kD). Two inhibitors, with L-norleucine or L-beta-(2-thienyl)alanine at the P1' residue and the improved linker of 12-aminododecanoic acid, were crystallized in complex with human alpha-thrombin. The crystal structures of these complexes were solved and refined to 2.1 A resolution. The Lys(60F) side chain of thrombin moved significantly and formed a large nonpolar S1' subsite to accommodate the bulky P1' residue.