Human
tissue kallikrein, a
trypsin-like serine protease involved in blood 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 A resolution is described and compared with that of porcine
kallikrein and of other
trypsin-like
proteases. The active and S1 sites (nomenclature of Schechter I, Berger A, 1967, Biochem Biophys Res Commun 27:157-162) are similar to those of porcine
kallikrein. Compared to
trypsin, the S1 site is enlarged owing to the insertion of an additional residue, cis-Pro 219. The replacement Tyr 228 --> Ala further enlarges the S1 pocket. However, the replacement of Gly 226 in
trypsin with Ser in human
tissue kallikrein restricts accessibility of substrates and inhibitors to Asp 189 at the base of the S1 pocket; there is a hydrogen bond between O delta1Asp189 and O gammaSer226. These changes in the architecture of the S1 site perturb the binding of inhibitors or substrates from the modes determined or inferred for
trypsin. The crystal structure gives insight into the structural differences responsible for changes in specificity in human
tissue kallikrein compared with other
trypsin-like
proteases, and into the structural basis for the unusual specificity of human
tissue kallikrein in cleaving both an Arg-Ser and a Met-Lys
peptide bond in its natural
protein substrate,
kininogen. A Zn+2-dependent, small-molecule competitive inhibitor of
kallikrein (Ki = 3.3 microM) has been identified and the bound structure modeled to guide
drug design.