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PDBsum entry 2anw
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Blood clotting, hydrolase
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PDB id
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2anw
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References listed in PDB file
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Key reference
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Title
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Expression, Crystallization, And three-Dimensional structure of the catalytic domain of human plasma kallikrein.
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Authors
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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.
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Ref.
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J Biol Chem, 2005,
280,
41077-41089.
[DOI no: ]
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PubMed id
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Abstract
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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.
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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.
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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.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2005,
280,
41077-41089)
copyright 2005.
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