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 anticoagulant enzyme. Recent experiments identified the minimal thrombin-binding region of thrombomodulin as the most acidic loop of the fifth EGF-like domain with a sequence of E(408)CPEGYILDDGFI(420)CTDIDE. High-resolution NMR spectroscopy was employed to characterize the interaction of a des-Ile420 thrombomodulin peptide, Cys(1(409))Pro(2)Glu(3)- Gly(4)Tyr(5)Ile(6)Leu(7)Asp(8)Asp(9)Gly(10)- Phe(11)Cys(12)Thr(13)Asp(14)Ile(15)Asp(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 structural preference free in solution. The thrombin-bound structure of the cyclic thrombomodulin peptide was determined by transferred nuclear Overhauser effects (transferred NOEs) and by distance geometry and Monte Carlo calculations. The thrombin-bound cyclic peptide assumes an overall 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 provides 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 subdomain with other thrombin-peptide complexes revealed that a common thrombin-binding surface can be organized by different secondary structure elements with entirely different peptide sequences. The thrombin-bound structure of the thrombomodulin peptide may serve as a basis to understand the regulatory functions of thrombomodulin and as a guide for the design of specific inhibitors for thrombin.