Structure around the cleavage site in the thrombin receptor determined by NMR spectroscopy

NMR spectroscopic experiments were performed to study the structure of synthetic peptides identical to two extracellular regions of the human thrombin receptor. The smaller molecule, comprising 14 amino acids, was biologically active and was equivalent to the "tethered ligand" exposed after cleavage of the receptor by thrombin. The principal structural elements were two overlapping turns (amino acids 5-8 and 6-9), the second of which was stabilized by a hydrogen bond, 6CO-9NH. The five N-terminal residues, considered to be responsible for biological activity, were essentially unstructured. A second version of this peptide, biologically inactive due to the reversal of the two N-terminal amino acids, had a very similar structure. A longer peptide (23 amino acids) covering the proposed thrombin cleavage site was found to be more highly structured. The seven residues from Pro-2 to Arg5 (the N-terminal amino acid exposed after cleavage is taken as residue 1) formed a 3(10) helix which is not present in the shorter tethered ligand peptide. The structure is partially stabilized by a charged hydrogen bond between the side chains of Arg-1 and Asp-3. The overlapping turns observed in the shorter peptides could also be distinguished in the longer molecule. On the basis of the structure determined for the peptide which encompasses the cleavage site and the determined structure of thrombin, a model is postulated for the interaction of the thrombin receptor and the protease during activation.