Interaction of hirudin with thrombin: identification of a minimal binding domain of hirudin that inhibits clotting activity.
Hirudin, isolated from the European leech Hirudo medicinalis, is a potent inhibitor of thrombin, forming an almost irreversible thrombin-hirudin complex. Previously, we have shown that the carboxyl terminus of hirudin (residues 45-65) inhibits clotting activity and without binding to the catalytic site of thrombin. In the present study, a series of peptides corresponding to this carboxyl-terminal region of hirudin have been synthesized, and their anticoagulant activity and binding properties to thrombin were examined. Binding was assessed by their ability to displace 125I-hirudin 45-65 from Sepharose-immobilized thrombin and by isolation of peptide-thrombin complexes. We show that the carboxyl-terminal 10 amino acid residues 56-65 (Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-Gln) are minimally required for binding to thrombin and inhibition of clotting. Phe-56 was critical for maintaining anticoagulant activity as demonstrated by the loss of activity when Phe-56 was substituted with D-Phe, Glu, or Leu. In addition, we found that the binding of the carboxyl-terminal peptide of hirudin with thrombin was associated with a significant conformational change of thrombin as judged by circular dichroism. This conformational change might be responsible for the loss of clotting activity of thrombin.
Mao
S J
SJ
Merrell Dow Research Institute, Cincinnati, Ohio 45215.
Yates
M T
MT
Owen
T J
TJ
Krstenansky
J L
JL
D001665:Binding Sites; D002942:Circular Dichroism; D005260:Female; D006629:Hirudins / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000378:metabolism*
D006629:Hirudins; D009842:Oligopeptides; D013917:Thrombin