GAMMA-FIBRINOGEN: CRYSTAL STRUCTURE OF A 30 KDA C-TERMINUS FRAGMENT AT 2.1 Å RESOLUTION. V.C. Yee, K.P. Pratt, H.C. Cote, I. Le Trong, D.W. Chung, R.E. Stenkamp, and D.C. Teller, Departments of Biochemistry and Biological Structure, University of Washington, Seattle, WA, 98195

The crystal structure of a 30 kDa carboxyl terminus fragment of the fibrinogen gamma chain has been determined using MIR phases to 2.5 è resolution, and refined against diffraction data to 2.1 è resolution. Fibrinogen is the central structural protein in the blood coagulation process. Cleavage of fibrinogen by the serine protease thrombin yields fibrin, which spontaneously polymerizes to form a clot matrix. Subsequent covalent crosslinking by factor XIIIa, a transglutaminase, produces a mechanically and proteolytically stable blood clot.

Fibrinogen is a disulfide-crosslinked dimer of heterotrimers, and contains two copies each of the alpha, beta, and gamma chains. A recombinant 30 kDa C-terminus fragment of the gamma chain containing the principle factor XIIIa crosslinking site, the calcium-binding site, the platelet receptor recognition domain, part of the polymerization surface, and one of the sites which accelerate t-PA activation of plasminogen, has been the focus of our crystallographic studies. This fragment crystallizes in space group P21 and its structure has been determined using MIR phases to 2.5è resolution. The model has been refined against 2.1è resolution diffraction data to give Rcryst=15.5% and Rfree=22.7%.

This first structure of a large fibrinogen fragment provides fresh insight into the polymerization and crosslinking events important during blood clot formation. In other work, we have determined several crystal structures of factor XIII in various forms. Our structural characterization of both enzyme and substrate extends our understanding of the transglutaminase reaction, and provides information that will be helpful in continuing studies toward obtaining the structure of an enzyme-substrate complex.

This work has been funded by NIH grants HL-50355 and HL-16919.