THE CRYSTAL-STRUCTURE OF THE CATALYTIC DOMAIN OF HUMAN UROKINASE-TYPEPLASMINOGEN-ACTIVATOR

Background: Urokinase-type plasminogen activator (u-PA) promotes fibri nolysis by catalyzing the conversion of plasminogen to the active prot ease plasmin via the cleavage of a peptide bond. When localized to the external cell surface it contributes to tissue remodelling and cellul ar migration; inhibition of its activity impedes the spread of cancer. u-PA has three domains: an N-terminal receptor-binding growth factor domain, a central kringle domain and a C-terminal catalytic protease d omain. The biological roles of the fibrinolytic enzymes render them th erapeutic targets, however, until now no structure of the protease dom ain has been available. Solution of the structure of the u-PA serine p rotease was undertaken to provide such data. Results: The crystal stru cture of the catalytic domain of recombinant, non-glycosylated human u -PA, complexed with the inhibitor Glu-Gly-Arg chloromethyl ketone (EGR cmk), has been determined at a nominal resolution of 2.5 Angstrom and refined to a crystallographic R-factor of 22.4% on all data (20.4% on data >3 sigma). The enzyme has the expected topology of a trypsin-like serine protease. Conclusions: The enzyme has an S1 specificity pocket similar to that of trypsin, a restricted, less accessible, hydrophobi c S2 pocket and a solvent-accessible S3 pocket which is capable of acc ommodating a wide range of residues. The EGRcmk inhibitor binds covale ntly at the active site to form a tetrahedral hemiketal structure. Alt hough the overall structure is similar to that of homologous serine pr oteases, at six positions insertions of extra residues in loop regions create unique surface areas. One of these loop regions is highly mobi le despite being anchored by the disulphide bridge which is characteri stic of a small subset of serine proteases namely tissuetype plasminog en activator, Factor XII and Complement Factor I.