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'''DESIGN OF P1' AND P3' RESIDUES OF TRIVALENT THROMBIN INHIBITORS AND THEIR CRYSTAL STRUCTURES''' | '''DESIGN OF P1' AND P3' RESIDUES OF TRIVALENT THROMBIN INHIBITORS AND THEIR CRYSTAL STRUCTURES''' | ||
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[[Category: Sivaraman, J.]] | [[Category: Sivaraman, J.]] | ||
[[Category: Slon-Usakiewicz, J J.]] | [[Category: Slon-Usakiewicz, J J.]] | ||
[[Category: | [[Category: Crystal structure]] | ||
[[Category: | [[Category: Serine protease]] | ||
[[Category: | [[Category: Thrombin inhibitor]] | ||
''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Fri May 2 15:21:03 2008'' | |||
''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on | |||
Revision as of 15:21, 2 May 2008
DESIGN OF P1' AND P3' RESIDUES OF TRIVALENT THROMBIN INHIBITORS AND THEIR CRYSTAL STRUCTURES
OverviewOverview
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.
About this StructureAbout this Structure
1EOL is a Single protein structure of sequence from Homo sapiens. Full crystallographic information is available from OCA.
ReferenceReference
Design of P1' and P3' residues of trivalent thrombin inhibitors and their crystal structures., Slon-Usakiewicz JJ, Sivaraman J, Li Y, Cygler M, Konishi Y, Biochemistry. 2000 Mar 7;39(9):2384-91. PMID:10694407 Page seeded by OCA on Fri May 2 15:21:03 2008