CRYSTAL-STRUCTURE OF RECOMBINANT HUMAN TISSUE KALLIKREIN AT 2.0 ANGSTROM RESOLUTION

Human tissue kallikrein, a trypsin-like serine protease involved in bl ood pressure regulation and inflammation processes, was expressed in a deglycosylated form at high levels in Pichia pastoris, purified, and crystallized. The crystal structure at 2.0 Angstrom resolution is desc ribed and compared with that of porcine kallikrein and of other trypsi n-like proteases. The active and S1 sites (nomenclature of Schechter I , Berger A, 1967, Biochem Biophys Res Commun 27:157-162) are similar t o those of porcine kallikrein. Compared to trypsin, the S1 site is enl arged owing to the insertion of an additional residue, cis-Pro 219. Th e replacement Tyr 228 --> Ala further enlarges the S1 pocket. However, the replacement of Gly 226 in trypsin with Ser in human tissue kallik rein restricts accessibility of substrates and inhibitors to Asp 189 a t the base of the S1 pocket; there is a hydrogen bond between O delta 1(Asp189) and O gamma(Ser226). These changes in the architecture of th e S1 site perturb the binding of inhibitors or substrates from the mod es determined or inferred for trypsin. The crystal structure gives ins ight into the structural differences responsible for changes in specif icity in human tissue kallikrein compared with other trypsin-like prot eases, and into the structural basis for the unusual specificity of hu man tissue kallikrein in cleaving both an Arg-Ser and a Met-Lys peptid e bond in its natural protein substrate, kininogen. A Zn+2-dependent, small-molecule competitive inhibitor of kallikrein (K-i = 3.3 mu M) ha s been identified and the bound structure modeled to guide drug design .