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 p lus the junction between the fifth and the sixth EGF-like domains was chara cterized in solution by use of NMR spectroscopy, particularly differential resonance perturbations and transferred nuclear Overhauser effects (transfe rred NOEs). The EGF-like thrombomodulin fragment, or hTM422-449, is conform ationally flexible in the absence of thrombin. Upon addition of thrombin, d ifferential resonance perturbations and transferred NOEs are observed for t he thrombomodulin peptide, suggesting specific and rapidly reversible bindi ng and structuring of hTM422-449 in complex with thrombin. Residue-specific analysis of the differential line broadening, resonance shifts, and transf erred NOEs identified regions of hTM422-449 responding to thrombin binding as the N-terminal residues Thr422-Ile424 and residues His438-Ile447 corresp onding to the central beta-hairpin, or beta-loop, of the consensus EGF-like repeat. The formation of the beta-hairpin is supported by the pattern of t ransferred NOEs bringing the two beta-strands together and characterizing a type I beta-turn. Docking of the thrombomodulin peptide to the anion-bindi ng exosite I of thrombin revealed structural details capturing binding cont acts identified so far as essential for the thrombin-thrombomodulin interac tion. Definition of specific interactions between thrombin and a minimal fr agment of the sixth EGF-like domain of human TM may lead to the discovery o f new peptidomimetic molecules as modulators of blood coagulation.