Structure/Activity of the Region of Thrombomodulin That Binds Thrombin

Peptides corresponding to the thrombin-binding loop of the fifth EGF-like domain of thrombomodulin (TM) extending from C407 to E426 (CECPEGYILDDGFICTDIDE) have been synthesized and their analysis has afforded an understanding of the structure and function of this region of TM. The results have implications for the design of thrombin inhibitors and give insight into the energetic factors that drive protein-protein interactions. Previous work had shown that the peptide corresponding to C409-E426 but missing I420 binds to thrombin by an induced-fit mechanism and that the "tail" residues C-terminal to the last cysteine are critical 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 and as inhibition of the thrombin-TM interaction that results in activation of protein C. Peptides with C407-C421 disulfide bond and C409 changed to A were inhibitors of thrombin, but were weaker than peptides with the C409-C421 disulfide bond. Peptides from the des-I420 series showed an absolute requirement for I424 while the native sequence peptides did not, and the peptides from the des-I420 series that contain I424 were more potent thrombin inhibitors than the native sequence peptides. Analysis of the thrombin-bound structures suggests that deletion of I420 places I424 on the other side of the β-pleated sheet where it undergoes a favorable intramolecular hydrophobic interaction with I414 stabilizing the bound conformation.