1EOL

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

Summary for 1EOL

• Entry DOI: 10.2210/pdb1eol/pdb
• Related: 1EOJ 1IHS
• Descriptor: ALPHA THROMBIN, THROMBIN INHIBITOR P628 (3 entities in total)
• Functional Keywords: thrombin inhibitors, serine protease, hydrolase-hydrolase inhibitor complex, hydrolase/hydrolase inhibitor
• Biological source: Homo sapiens (human); More
• Cellular location: Secreted, extracellular space: P00734
• Total number of polymer chains: 2
• Total formula weight: 35116.33

Authors

Slon-Usakiewicz, J.J.,Sivaraman, J.,Li, Y.,Cygler, M.,Konishi, Y. (deposition date: 2000-03-23, release date: 2000-05-03, Last modification date: 2023-11-15)

Primary citation

Slon-Usakiewicz, J.J.,Sivaraman, J.,Li, Y.,Cygler, M.,Konishi, Y.
Design of P1' and P3' residues of trivalent thrombin inhibitors and their crystal structures.
Biochemistry, 39:2384-2391, 2000

PubMed 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.

PubMed: 10694407
DOI: 10.1021/bi992419b

Experimental method

X-RAY DIFFRACTION (2.1 Å)