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PDBsum entry 1iy6
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Contents |
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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Inhibitory specificity change of the ovomucoid third domain of the silver pheasant upon introduction of an engineered cys14-Cys39 bond.
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Authors
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H.Hemmi,
T.Kumazaki,
T.Yamazaki,
S.Kojima,
T.Yoshida,
Y.Kyogoku,
M.Katsu,
F.Shinohara,
H.Yokosawa,
K.Miura,
Y.Kobayashi.
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Ref.
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Biochemistry, 2003,
42,
2524-2534.
[DOI no: ]
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PubMed id
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Abstract
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The ovomucoid third domain from silver pheasant (OMSVP3), a typical Kazal-type
inhibitor, strongly inhibits different serine proteases of various
specificities, i.e., chymotrypsin, Streptomyces griseus protease, subtilisin,
and elastase. Structural studies have suggested that conformational flexibility
in the reactive site loop of the free inhibitor may be related to broad
specificity of the ovomucoid. On the basis of the structural homology between
OMSVP3 and ascidian trypsin inhibitor (ATI), which has a cystine-stabilized
alpha-helical (CSH) motif in the sequence, we prepared the disulfide variant of
OMSVP3, introducing an engineered disulfide bond between positions 14 and 39
near the reactive site (Met18-Glu19) by site-directed mutagenesis. The disulfide
variant P14C/N39C retained potent inhibitory activities toward
alpha-chymotrypsin (CHT) and S. griseus proteases A and B (SGPA and SGPB), while
this variant lost most of its inhibitory activity toward porcine pancreatic
elastase (PPE). We determined the solution structure of P14C/N39C, as well as
that of wild-type OMSVP3, by two-dimensional nuclear magnetic resonance (2D NMR)
methods and compared their structures to elucidate the structural basis of the
inhibitory specificity change. For the molecular core consisting of a central
alpha-helix and a three-stranded antiparallel beta-sheet, essentially no
structural difference was detected between the two (pairwise rmsd value = 0.41
A). In contrast to this, a significant difference was detected in the loop from
Cys8 to Thr17, where in P14C/N39C it has drawn approximately 4 A nearer the
central helix to form the engineered Cys14-Cys39 bond. Concomitantly, the
Tyr11-Pro12 cis-peptide linkage, which is highly conserved in ovomucoid third
domains, was isomerized to the trans configuration. Such structural change in
the loop near the reactive site may possibly affect the inhibitory specificity
of P14C/N39C for the corresponding proteases.
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