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 E408CPEGYILDDGFI420CTDIDE. High-resolution NMR spectroscopy was employed to characterize the interaction of a des-Ile420 thrombomodulin peptide, Cys1(409)Pro2Glu3Gly4Tyr5Ile6- Leu7Asp8Asp9Gly10Phe11Cys12Thr13Asp14Ile15Asp16Glu17(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.
Srinivasan
J
J
Biotechnology Research Institute, National Research Council Canada, Montreal, Quebec, Canada.
Hu
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Hrabal
R
R
Zhu
Y
Y
Komives
E A
EA
Ni
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F
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D004815:Epidermal Growth Factor / Q000737:chemistry*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D013917:Thrombin / Q000737:chemistry*; D018180:Thrombomodulin / Q000737:chemistry*
D010446:Peptide Fragments; D018180:Thrombomodulin; D004815:Epidermal Growth Factor; D013917:Thrombin