THROMBIN-BOUND STRUCTURE OF AN EGF SUBDOMAIN FROM HUMAN THROMBOMODULIN DETERMINED BY TRANSFERRED NUCLEAR OVERHAUSER EFFECTS

The EGF-like domains in human thrombomodulin interact with and change the specificity of thrombin from a procoagulant enzyme to an anticoagu lant enzyme. Recent experiments identified the minimal thrombin-bindin g region of thrombomodulin as the most acidic loop of the fifth EGF-li ke domain with a sequence of E(408)CPEGYILDDGFI(420)CTDIDE. High-resol ution NMR spectroscopy was employed to characterize the interaction of a des-Ile420 thrombomodulin peptide, Cys(1(409))Pro(2)Glu(3)- Gly(4)T yr(5)Ile(6)Leu(7)Asp(8)Asp(9)Gly(10)- 1)Cys(12)Thr(13)Asp(14)Ile(15)As p(16)Glu(17(426)), With its target coagulation protein, thrombin. The disulfide-bonded peptide was found to be structured only upon binding, while neither the linear nor the cyclized peptide exhibited any struc tural preference free in solution. The thrombin-bound structure of the cyclic thrombomodulin peptide was determined by transferred nuclear O verhauser effects (transferred NOEs) and by distance geometry and Mont e Carlo calculations. The thrombin-bound cyclic peptide assumes an ove rall conformation similar to those observed in the free but intact EGF molecules. There is a type II. beta-turn involving residues Pro2-Tyr5 , followed by an optimized antiparallel beta-sheet involving residues Gly4-Asp8 and residues Phe11-Ile15. The thrombomodulin peptide provide s a potential thrombin-binding surface between residues Tyr5 and Phe11 , which are brought close by a chain reversal within the central beta- sheet. Comparison of the thrombin-bound structure of the EGF-like subd omain with other thrombin-peptide complexes revealed that a common thr ombin-binding surface can be organized by different secondary structur e elements with entirely different peptide sequences. The thrombin-bou nd structure of the thrombomodulin peptide may serve as a basis to und erstand the regulatory functions of thrombomodulin and as a guide for the design of specific inhibitors for thrombin.