Inhibitory Specificity Change of the Ovomucoid Third Domain of the Silver Pheasant upon Introduction of an Engineered Cys14-Cys39 Bond
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- 作者：Hikaru Hemmi ; Takashi Kumazaki ; Toshimasa Yamazaki ; Shuichi Kojima ; Takuya Yoshida ; Yoshimasa Kyogoku ; Masataka Katsu ; Fumikazu Shinohara ; Hideyoshi Yokosawa ; Kin-ichiro Miura ; and Yuji Kobayashi
- 刊名：Biochemistry
- 出版年：2003
- 出版时间：March 11, 2003
- 年：2003
- 卷：42
- 期：9
- 页码：2524 - 2534
- 全文大小：186K
- 年卷期：v.42,no.9(March 11, 2003)
- ISSN：1520-4995
文摘
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 griseusprotease, subtilisin, and elastase. Structural studies have suggested that conformational flexibility in thereactive site loop of the free inhibitor may be related to broad specificity of the ovomucoid. On the basisof the structural homology between OMSVP3 and ascidian trypsin inhibitor (ATI), which has a cystine-stabilized 
-helical (CSH) motif in the sequence, we prepared the disulfide variant of OMSVP3, introducingan 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-chymotrypsin (CHT) and S. griseus proteases A and B (SGPA and SGPB), while this variant lost mostof its inhibitory activity toward porcine pancreatic elastase (PPE). We determined the solution structureof 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 inhibitoryspecificity change. For the molecular core consisting of a central -helix and a three-stranded antiparallel
-sheet, essentially no structural difference was detected between the two (pairwise rmsd value = 0.41Å). In contrast to this, a significant difference was detected in the loop from Cys8 to Thr17, where inP14C/N39C it has drawn approximately 4 Å nearer the central helix to form the engineered Cys14-Cys39bond. Concomitantly, the Tyr11-Pro12 cis-peptide linkage, which is highly conserved in ovomucoid thirddomains, was isomerized to the trans configuration. Such structural change in the loop near the reactivesite may possibly affect the inhibitory specificity of P14C/N39C for the corresponding proteases.
-helical (CSH) motif in the sequence, we prepared the disulfide variant of OMSVP3, introducingan 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-chymotrypsin (CHT) and S. griseus proteases A and B (SGPA and SGPB), while this variant lost mostof its inhibitory activity toward porcine pancreatic elastase (PPE). We determined the solution structureof 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 inhibitoryspecificity change. For the molecular core consisting of a central -helix and a three-stranded antiparallel-sheet, essentially no structural difference was detected between the two (pairwise rmsd value = 0.41Å). In contrast to this, a significant difference was detected in the loop from Cys8 to Thr17, where inP14C/N39C it has drawn approximately 4 Å nearer the central helix to form the engineered Cys14-Cys39bond. Concomitantly, the Tyr11-Pro12 cis-peptide linkage, which is highly conserved in ovomucoid thirddomains, was isomerized to the trans configuration. Such structural change in the loop near the reactivesite may possibly affect the inhibitory specificity of P14C/N39C for the corresponding proteases.