Crystal structure of recombinant human tissue kallikrein at 2.0 Å resolution

Abstract

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 8, 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 SI pocket; there is a hydrogen bond between and 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*'-dependent, small-molecule competitive inhibitor of kallikrein ( K , = 3.3 p M ) has been identified and the bound structure modeled to guide drug design.

Roles of the P1, P2, and P3 Residues in Determining Inhibitory Specificity of Kallistatin toward Human Tissue Kallikrein

“…In agreement with our results, other studies (16,17) using synthetic peptides showed that human tissue kallikrein displays substrate specificity for Arg, Met, and Phe with lesser specificity for Leu and Lys at the P1 position. The low selectivity of human tissue kallikrein for the P1 residue could be explained by an enlarged S1 site of tissue kallikrein, which may accommodate various amino acids as revealed by x-ray crystallography (25,26). The limited proteolytic nature of tissue kallikrein to its physiological substrates would, therefore, require other subsites in the reactive crevice to regulate the enzymatic specificity.…”

“…Molecular Modeling of Human Tissue Kallikrein-The atomic coordinates of the x-ray structure of human tissue kallikrein were generously provided by Dr. Katz (26). The coordinates of the "kallikrein loop," consisting of 14 residues from 95 [102] to 98K [115], are not available in this molecular model.…”

“…Modeling of Kallistatin-Tissue Kallikrein Complex-Human tissue kallikrein was recently crystallized, but the structural coordinates of the "kallikrein loop" spanning from residue Asp 95[102] to Glu 98 [115] could not be traced due to a disorder of this region (26). Human tissue kallikrein has a large sequence insertion in this loop as compared with porcine pancreatic kallikrein and other serine proteinases (33).…”

“…These studies showed that the P2 residue with a bulky and hydrophobic side chain, particularly Phe, is a critical determinant for tissue kallikrein specificity. Crystallographic analysis of porcine tissue kallikrein suggested that the hydrophobic phenyl side chain of Phe at P2 could be optimally accommodated into a hydrophobic crevice between Tyr 99 and Trp 215 of tissue kallikrein (25,26). 1 However, the knowledge on P3 specificity of tissue kallikrein is limited.…”

Sweetened kallikrein-related peptidases (KLKs): glycan trees as potential regulators of activation and activity

“…In contrast to KLK1 expressed in Escherichia coli, natural urinary KLK1 was confirmed to be glycosylated, as well as the variant expressed in the yeast Pichia pastoris (Angermann et al, 1989;Chan et al, 1998). This expression system with heterogeneous glycosylation caused problems in crystallization of human KLK1, even after enzymatic deglycosylation, requiring the mutation of the sequons for a successful X-ray structure determination (Katz et al, 1998). In the crystal structure of human apo-KLK1 only a single GlcNAc at Asn95 was visible in the electron density, despite the presence of up to three GlcNAc 2 Man 3 (Fuc) trees (Figure 3) (Laxmikanthan et al, 2005).…”