Expression, Crystallization, and Three-dimensional Structure of the Catalytic Domain of Human Plasma Kallikrein

Abstract

Plasma kallikrein is a serine protease that has many important functions, including modulation of blood pressure, complement activation, and mediation and maintenance of inflammatory responses. Although plasma kallikrein has been purified for 40 years, its structure has not been elucidated. In this report, we described two systems (Pichia pastoris and baculovirus/Sf9 cells) for expression of the protease domain of plasma kallikrein, along with the purification and high resolution crystal structures of the two recombinant forms. In the Pichia pastoris system, the protease domain was expressed as a heterogeneously glycosylated zymogen that was activated by limited trypsin digestion and treated with endoglycosidase H deglycosidase to reduce heterogeneity from the glycosylation. The resulting protein was chromatographically resolved into four components, one of which was crystallized. In the baculovirus/Sf9 system, homogeneous, crystallizable, and nonglycosylated protein was expressed after mutagenizing three asparagines (the glycosylation sites) to glutamates. When assayed against the peptide substrates, pefachrome-PK and oxidized insulin B chain, both forms of the protease domain were found to have catalytic activity similar to that of the full-length protein. Crystallization and x-ray crystal structure determination of both forms have yielded the first three-dimensional views of the catalytic domain of plasma kallikrein. The structures, determined at 1.85 Å for the endoglycosidase H-deglycosylated protease domain produced from P. pastoris and at 1.40 Å for the mutagenically deglycosylated form produced from Sf9 cells, show that the protease domain adopts a typical chymotrypsin-like serine protease conformation. The structural information provides insights into the biochemical and enzymatic properties of plasma kallikrein and paves the way for structure-based design of protease inhibitors that are selective either for or against plasma kallikrein.

Common variation in KLKB1 and essential hypertension risk: tagging-SNP haplotype analysis in a case-control study

“…Then plasma kallikrein acts on high molecular weight kininogen substrate to release bradykinin and converts prorenin to renin. By controlling the release of bradykinin (a potent vasodilator) and the activation of renin (the protease converting angiotensinogen to angiotensin I), plasma kallikrein is deeply involved in BP regulation (Dielis et al 2005;Marcondes and Antunes 2005;Schmaier 2003;Tang et al 2005). Based on the physiological eVects, human plasma kallikrein gene (KLKB1) encoding plasma kallikrein can be considered as a good candidate gene for essential hypertension.…”

The expanding diversity of serine hydrolases

“…Plasma kallikrein is a multidomain, glycosylated protein that is more homologous to factor XI than to tissue kallikrein with a single protease domain. The structural basis for the specificity of plasma kallikrein was revealed by its crystal structure [2]. Human tissue kallikreins (hK) belong to a closely related 15-member family.…”

Severe prekallikrein deficiencies due to homozygous C529Y mutations

“…It is likely that a small amount of the mutant is secreted but is inactive. The cysteine 529 residue is located in the vicinity of the catalytic domain involving the classical catalytic triad (H415, D464, S559; see Fig. 2 [13]) and may contribute to the organization of the substrate binding pocket. These data and the location and the conservation of the residue among species are in favor of a functional and/or structural role of this residue.…”

“…The residue C548 of the human sequence (capital case) is the mutated amino acid reported in this paper (C529 for the circulating polypeptide). Crystal structure of the human kallikrein precursor [13]. The crystal structure was represented using pyMol (PDB: 2ANY).…”