The crystallographic structure has been determined of a complex between a nonadecapeptide from the fifth epidermal growth factor (EGF5) domain of human thrombomodulin and human D-PheProArg-alpha-thrombin. The peptide corresponds to amino acid residues Glu408-Glu426 of thrombomodulin and contains the third disulfide loop of EGF5 and its linker to EGF6. The structure was refined at 3.0-A resolution to an R-value of 0.146. There are two thrombin molecules in the asymmetric unit, and the structure in the crystal is a 2:1 thrombin complex. The folding of the peptide corresponds closely to the third disulfide loop of EGF2 of factor Xa (rms delta = 1.0 A). The peptide is squeezed between cofacial electropositive fibrinogen recognition exo sites of the two thrombin molecules. Since the peptide has a total of seven aspartic and glutamic acid residues, the principal binding interaction with thrombin is electrostatic. A major hydrophobic association, which is highly directional in such a pronounced electrostatic environment, involves a TyrIleLeu triplet of the peptide and Phe34, Leu65, Tyr76, and Ile82 (chymotrypsinogen numbering) of one thrombin molecule. The tyrosine of the peptide is sandwiched between the thrombin aromatic rings and is most likely the prime source of the specificity of the thrombomodulin-thrombin interaction.

Thrombin displays remarkable specificity, effecting the removal of fibrinopeptides A and B of fibrinogen through the selective cleavage of two Arg-Gly bonds between the 181 Arg/Lys-Xaa bonds in fibrinogen. Significant advances have been made in recent years towards understanding the origin of the specificity of cleavage of the Arg16-Gly17 bond of the A alpha-chain of human fibrinogen. We have previously proposed a model for the bound structure of fibrinopeptide A7-16 (FPA), based upon NMR data, computer-assisted molecular modeling and the synthesis and study of peptidomimetic substrates and inhibitors of thrombin. We now report the structure of the ternary complex of an FPA mimetic (FPAM), hirugen and thrombin at 2.5 A resolution (R-factor = 0.138) and specificity data for the inhibition of thrombin and related trypsin-like proteinases by FPAM. The crystallographic structures of FPA and its chloromethyl ketone derivative bound to thrombin were determined. Although there are differences between these structures in the above modeled FPA structure and that of the crystal structure of FPAM bound to thrombin, the phi, psi angles in the critical region of P1-P2-P3 in all of the structures are similar to those of bovine pancreatic trypsin inhibitor (BPTI) in the BPTI-trypsin complex and D-Phe-Pro-Arg (PPACK) in the PPACK-thrombin structure. A comparison between these and an NMR-derived structure is carried out and discussed.

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