STRUCTURE-ACTIVITY OF THE REGION OF THROMBOMODULIN THAT BINDS THROMBIN

Peptides corresponding to the thrombin-binding loop of the fifth EGF-l ike domain of thrombomodulin (TM) extending from C407 to E426 (CECPEGY ILDDGFICTDIDE) have been synthesized and their analysis has afforded a n understanding of the structure and function of this region of TM. Th e results have implications for the design of thrombin inhibitors and give insight into the energetic factors that drive protein-protein int eractions. Previous work had shown that the peptide corresponding to C 409-E426 but missing I420 binds to thrombin by an induced-fit mechanis m and that the ''tail'' residues C-terminal to the last cysteine are c ritical for thrombin binding. In this study, we probe the requirements for the C409-C421 disulfide bond and for each of the tail amino acids . Thrombin binding was assayed as inhibition of fibrinogen clotting an d as inhibition of the thrombin-TM interaction that results in activat ion of protein C. Peptides with C407-C421 disulfide bond and C409 chan ged to A were inhibitors of thrombin, but were weaker than peptides wi th the C409-C421 disulfide bond. Peptides from the des-I420 series sho wed an absolute requirement for I424 while the native sequence peptide s did not, and the peptides from the des-I420 series that contain I424 were more potent thrombin inhibitors than the native sequence peptide s. Analysis of the thrombin-bound structures suggests that deletion of I420 places I424 on the other side of the beta-pleated sheet where it undergoes a favorable intramolecular hydrophobic interaction with I41 4 stabilizing the bound conformation. (C) 1995 Academic Press, Inc.