PDBsum entry 2anw

Blood clotting, hydrolase

PDB id: 2anw

Contents

Protein chain

238 a.a. *

Ligands

BEN

Waters ×642

* Residue conservation analysis

PDB id:

2anw

Name:

Blood clotting, hydrolase

Title:

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

Structure:

Plasma kallikrein, light chain. Chain: a. Fragment: protease domain, enzymatically deglycosylated. Synonym: kininogenin. Fletcher factor. Engineered: yes. Mutation: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: klkb1, klk3. Expressed in: spodoptera frugiperda. Expression_system_taxid: 7108.

Resolution:

1.85Å R-factor: 0.221 R-free: 0.283

Authors:

J.Tang,C.L.Yu,S.R.Williams,E.Springman,D.Jeffery,P.A.Sprengeler, A.Estevez,J.Sampang,W.Shrader,J.R.Spencer,W.B.Young,M.E.Mcgrath, B.A.Katz

Key ref:

J.Tang et al. (2005). Expression, crystallization, and three-dimensional structure of the catalytic domain of human plasma kallikrein. J Biol Chem, 280, 41077-41089. PubMed id: 16199530 DOI: 10.1074/jbc.M506766200

Date:

11-Aug-05 Release date: 11-Oct-05

Protein chain

P03952  (KLKB1_HUMAN) -  Plasma kallikrein from Homo sapiens

Seq: 638 a.a.

Struc: 238 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.3.4.21.34 - plasma kallikrein.
• Reaction: Cleaves selectively Arg-|-Xaa and Lys-|-Xaa bonds, including Lys-|-Arg and Arg-|-Ser bonds in (human) kininogen to release bradykinin.

DOI no: 10.1074/jbc.M506766200

J Biol Chem 280:41077-41089 (2005)

PubMed id: 16199530

Expression, crystallization, and three-dimensional structure of the catalytic domain of human plasma kallikrein.

J.Tang, C.L.Yu, S.R.Williams, E.Springman, D.Jeffery, P.A.Sprengeler, A.Estevez, J.Sampang, W.Shrader, J.Spencer, W.Young, M.McGrath, B.A.Katz.

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.

Selected figure(s)

Figure 6.
FIGURE 6. Model of CG-05 bound to plasma kallikrein, showing expected interaction of the diacid group of the inhibitor with Lys192 of the enzyme, along with the multicentered short hydrogen bond array observed at the active site of the trypsin complex. Short hydrogen bonds are shown in cyan.

Figure 8.
FIGURE 8. Model of CG-01 bound to plasma kallikrein, showing the expected location of the terminal phenyl group of the inhibitor in the S1' site.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2005, 280, 41077-41089) copyright 2005.

Figures were selected by an automated process.