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

Christine Luong Yu,Wendy Young,Paul A. Sprengeler,Jun Sampang,Steven R. Williams,Jie Tang,Alberto Estevez,Bradley A. Katz,Jeff Spencer,Douglas Jeffery,William Shrader,Eric Springman,Mary McGrath

doi:10.1074/jbc.m506766200

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 A for the endoglycosidase H-deglycosylated protease domain produced from P. pastoris and at 1.40 A 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.

Highlights

The name “kallikrein” was originally introduced in the 1930s to describe the proteolytic activity from the pancreas responsible for cleavage of kininogen [1, 2]
The N-terminal heavy chain of 371 amino acids contains four Apple domains whose homologues are found in factor XI [9], whereas the C-terminal light chain forms the protease domain
We describe the expression of the protease domain of plasma kallikrein in the methylotrophic yeast Pichia pastoris and the production of crystallizable protein by removal of Endo H-sensitive glycosylation

Summary

Introduction

The name “kallikrein” was originally introduced in the 1930s to describe the proteolytic activity from the pancreas responsible for cleavage of kininogen [1, 2]. The purified light chain has amidolytic activity similar to that of the native enzyme, but it is less active in kaolin-dependent coagulation assays. Both the light and heavy chains are glycosylated; PK from human blood contains 15% carbohydrate and has five potential sites for N-glycosylation. Plasma kallikrein plays a central role in the kinin-generating pathways (PK system) and in the surface-mediated “contact system” [8, 12]. It is synthesized predominantly in the liver as a proenzyme, prekallikrein, known as prokallikrein [13]. The PK1⁄7HK complex binds to a multiprotein com-

Methods

Results

Conclusion