Structure of Dansyl-EGR-Chloromethylketone Inhibited Factor VIIa Complex with Soluble Tissue Factor. S. Paul Bajaj, Ravi G. Kurumbail, Roderick A. Stegeman, Anna M. Stevens, Huey-Sheng Shieh, Thomas J. Girard, and William C. Stallings, Saint Louis University Health Sciences Center and Searle/Monsanto, St. Louis, Missouri

Physiologic blood coagulation is initiated when factor VIIa (fVIIa) binds to its high-affinity cofactor, tissue factor (TF) exposed at the site of vascular injury. FVIIa consists of two polypeptide chains linked by a disulfide bond. The light chain consists of an N-terminal gamma-carboxyglutamic acid (Gla) domain followed by two EGF domains while the serine protease domain constitutes the heavy chain. Recombinant fVIIa produced in 293 kidney cells was inhibited by the active site inhibitor dansyl-EGR chloromethylketone, complexed with soluble TF (sTF) and crystallized using hanging drop vapor diffusion methods. Crystals grew in two to three days and were orthorhombic, space group P2₁2₁2₁, with unit cell constants a=69.8 A, b=81.4 A, c=125.9 A. These crystals are similar to those reported by Banner et al. (Nature 380, 41-46, 1996) for (D)Phe-(L)Phe-(L)Arg-chloromethylketone inhibited fVIIa/sTF complex. Diffraction data have been measured to a resolution of 1.9 A from crystals cooled to liquid nitrogen temperatures.

The structure of dansyl-EGR/fVIIa/sTF complex was solved by isomorphous replacement methods using the coordinates of (D)Phe-(L)Phe-(L)Arg-chloromethylketone inhibited fVIIa/sTF complex (Kurumbail et al., unpublished data). Formation of a tetrahedral hemiketal with the active site Ser 195 and covalent bond with His 57 is readily evident. The arginine side chain of the inhibitor forms an ion-pair with Asp 189 of fVIIa in the S1 site, as observed in the structures of other coagulation proteases. Dansyl group of the inhibitor packs tightly against Pro 174 of fVIIa. Both EGF domains have been modeled into electron density. Efforts are underway to locate the Gla domain of fVIIa. Further refinement of the structure is in progress.