2anw: Difference between revisions
New page: left|200px<br /> <applet load="2anw" size="450" color="white" frame="true" align="right" spinBox="true" caption="2anw, resolution 1.85Å" /> '''Expression, crystal... |
No edit summary |
||
| Line 1: | Line 1: | ||
[[Image:2anw.gif|left|200px]]<br /> | [[Image:2anw.gif|left|200px]]<br /><applet load="2anw" size="350" color="white" frame="true" align="right" spinBox="true" | ||
<applet load="2anw" size=" | |||
caption="2anw, resolution 1.85Å" /> | caption="2anw, resolution 1.85Å" /> | ||
'''Expression, crystallization and three-dimensional structure of the catalytic domain of human plasma kallikrein: Implications for structure-based design of protease inhibitors'''<br /> | '''Expression, crystallization and three-dimensional structure of the catalytic domain of human plasma kallikrein: Implications for structure-based design of protease inhibitors'''<br /> | ||
==Overview== | ==Overview== | ||
Plasma kallikrein is a serine protease that has many important functions, including modulation of blood pressure, complement activation, and | 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. | ||
==Disease== | ==Disease== | ||
| Line 11: | Line 10: | ||
==About this Structure== | ==About this Structure== | ||
2ANW is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Homo_sapiens Homo sapiens] with BAM as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Plasma_kallikrein Plasma kallikrein], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.4.21.34 3.4.21.34] Full crystallographic information is available from [http:// | 2ANW is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Homo_sapiens Homo sapiens] with <scene name='pdbligand=BAM:'>BAM</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Plasma_kallikrein Plasma kallikrein], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.4.21.34 3.4.21.34] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2ANW OCA]. | ||
==Reference== | ==Reference== | ||
| Line 20: | Line 19: | ||
[[Category: Estevez, A.]] | [[Category: Estevez, A.]] | ||
[[Category: Jeffery, D.]] | [[Category: Jeffery, D.]] | ||
[[Category: Katz, B | [[Category: Katz, B A.]] | ||
[[Category: McGrath, M | [[Category: McGrath, M E.]] | ||
[[Category: Sampang, J.]] | [[Category: Sampang, J.]] | ||
[[Category: Shrader, W.]] | [[Category: Shrader, W.]] | ||
[[Category: Spencer, J | [[Category: Spencer, J R.]] | ||
[[Category: Sprengeler, P | [[Category: Sprengeler, P A.]] | ||
[[Category: Springman, E.]] | [[Category: Springman, E.]] | ||
[[Category: Tang, J.]] | [[Category: Tang, J.]] | ||
[[Category: Williams, S | [[Category: Williams, S R.]] | ||
[[Category: Young, W | [[Category: Young, W B.]] | ||
[[Category: Yu, C | [[Category: Yu, C L.]] | ||
[[Category: BAM]] | [[Category: BAM]] | ||
[[Category: trypsin-like serine protease; enzymatically deglycosylated]] | [[Category: trypsin-like serine protease; enzymatically deglycosylated]] | ||
''Page seeded by [http:// | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 16:29:12 2008'' | ||
Revision as of 17:29, 21 February 2008
|
Expression, crystallization and three-dimensional structure of the catalytic domain of human plasma kallikrein: Implications for structure-based design of protease inhibitors
OverviewOverview
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.
DiseaseDisease
Known diseases associated with this structure: Fletcher factor deficiency OMIM:[229000], Prekallikrein deficiency OMIM:[229000]
About this StructureAbout this Structure
2ANW is a Single protein structure of sequence from Homo sapiens with as ligand. Active as Plasma kallikrein, with EC number 3.4.21.34 Full crystallographic information is available from OCA.
ReferenceReference
Expression, crystallization, and three-dimensional structure of the catalytic domain of human plasma kallikrein., Tang J, Yu CL, Williams SR, Springman E, Jeffery D, Sprengeler PA, Estevez A, Sampang J, Shrader W, Spencer J, Young W, McGrath M, Katz BA, J Biol Chem. 2005 Dec 9;280(49):41077-89. Epub 2005 Sep 30. PMID:16199530
Page seeded by OCA on Thu Feb 21 16:29:12 2008