Identification of a thrombin-binding region in the sixth epidermal growth factor-like repeat of human thrombomodulin.
The interaction of thrombin with a 28-residue peptide corresponding to the N-terminal subdomain of the sixth EGF-like repeat of human thrombomodulin plus the junction between the fifth and the sixth EGF-like domains was characterized in solution by use of NMR spectroscopy, particularly differential resonance perturbations and transferred nuclear Overhauser effects (transferred NOEs). The EGF-like thrombomodulin fragment, or hTM422-449, is conformationally flexible in the absence of thrombin. Upon addition of thrombin, differential resonance perturbations and transferred NOEs are observed for the thrombomodulin peptide, suggesting specific and rapidly reversible binding and structuring of hTM422-449 in complex with thrombin. Residue-specific analysis of the differential line broadening, resonance shifts, and transferred NOEs identified regions of hTM422-449 responding to thrombin binding as the N-terminal residues Thr422-Ile424 and residues His438-Ile447 corresponding to the central beta-hairpin, or B-loop, of the consensus EGF-like repeat. The formation of the beta-hairpin is supported by the pattern of transferred NOEs bringing the two beta-strands together and characterizing a type I beta-turn. Docking of the thrombomodulin peptide to the anion-binding exosite I of thrombin revealed structural details capturing binding contacts identified so far as essential for the thrombin-thrombomodulin interaction. Definition of specific interactions between thrombin and a minimal fragment of the sixth EGF-like domain of human TM may lead to the discovery of new peptidomimetic molecules as modulators of blood coagulation.
Tolkatchev
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Biomolecular NMR Laboratory and Montreal Joint Centre for Structural Biology, Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Avenue, Montreal, Quebec, Canada H4P 2R2.
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A
Zhu
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B
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F
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D004815:Epidermal Growth Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D020449:Repetitive Sequences, Amino Acid; D013917:Thrombin / Q000737:chemistry / Q000378:metabolism*; D018180:Thrombomodulin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D010446:Peptide Fragments; D018180:Thrombomodulin; D004815:Epidermal Growth Factor; D013917:Thrombin