2ANW

Expression, crystallization and three-dimensional structure of the catalytic domain of human plasma kallikrein: Implications for structure-based design of protease inhibitors

2ANW の概要

• エントリーDOI: 10.2210/pdb2anw/pdb
• 関連するPDBエントリー: 2ANY
• 分子名称: plasma kallikrein, light chain, BENZAMIDINE (3 entities in total)
• 機能のキーワード: trypsin-like serine protease; enzymatically deglycosylated, blood clotting, hydrolase
• 由来する生物種: Homo sapiens (human)
• 細胞内の位置: Secreted: P03952
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 27219.96

構造登録者

Tang, J.,Yu, C.L.,Williams, S.R.,Springman, E.,Jeffery, D.,Sprengeler, P.A.,Estevez, A.,Sampang, J.,Shrader, W.,Spencer, J.R.,Young, W.B.,McGrath, M.E.,Katz, B.A. (登録日: 2005-08-11, 公開日: 2005-10-11, 最終更新日: 2024-10-30)

主引用文献

Tang, J.,Yu, C.L.,Williams, S.R.,Springman, E.,Jeffery, D.,Sprengeler, P.A.,Estevez, A.,Sampang, J.,Shrader, W.,Spencer, J.,Young, W.,McGrath, M.,Katz, B.A.
Expression, crystallization, and three-dimensional structure of the catalytic domain of human plasma kallikrein.
J.Biol.Chem., 280:41077-41089, 2005

PubMed 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.

PubMed: 16199530
DOI: 10.1074/jbc.M506766200

実験手法

X-RAY DIFFRACTION (1.85 Å)