Structural and Functional Study of an Anemonia Elastase Inhibitor, a "Nonclassical" Kazal-Type Inhibitor from Anemonia sulcata
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- 作者：Hikaru Hemmi ; Takashi Kumazaki ; Kumiko Yoshizawa-Kumagaye ; Yuji Nishiuchi ; Takuya Yoshida ; Tadayasu Ohkubo ; and Yuji Kobayashi
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：July 19, 2005
- 年：2005
- 卷：44
- 期：28
- 页码：9626 - 9636
- 全文大小：303K
- 年卷期：v.44,no.28(July 19, 2005)
- ISSN：1520-4995
文摘
Anemonia elastase inhibitor (AEI) is a "nonclassical" Kazal-type elastase inhibitor fromAnemonia sulcata. Unlike many nonclassical inhibitors, AEI does not have a cystine-stabilized 
-helical(CSH) motif in the sequence. We chemically synthesized AEI and determined its three-dimensional solutionstructure by two-dimensional NMR spectroscopy. The resulting structure of AEI was characterized by acentral -helix and a three-stranded antiparallel 
-sheet of a typical Kazal-type inhibitor such as silverpheasant ovomucoid third domain (OMSVP3), even though the first and fifth half-cystine residues forminga disulfide bond in AEI are shifted both toward the C-terminus in comparison with those of OMSVP3.Synthesized AEI exhibited unexpected strong inhibition toward Streptomyces griseus protease B (SGPB).Our previous study [Hemmi, H., et al. (2003) Biochemistry 42, 2524-2534] demonstrated that the site-specific introduction of the engineered disulfide bond into the OMSVP3 molecule to form the CSH motifcould produce an inhibitor with a narrower specificity. Thus, the CSH motif-containing derivative of AEI(AEI analogue) was chemically synthesized when a Cys4-Cys34 bond was changed to a Cys6-Cys31bond. The AEI analogue scarcely inhibited porcine pancreatic elastase (PPE), even though it exhibitedalmost the same potent inhibitory activity toward SGPB. For the molecular scaffold, essentially no structuraldifference was detected between the two, but the N-terminal loop from Pro5 to Ile7 near the putativereactive site (Met10-Gln11) in the analogue moved by 3.7 Å toward the central helix to form the introducedCys6-Cys31 bond. Such a conformational change in the restricted region correlates with the specificitychange of the inhibitor.
-helical(CSH) motif in the sequence. We chemically synthesized AEI and determined its three-dimensional solutionstructure by two-dimensional NMR spectroscopy. The resulting structure of AEI was characterized by acentral -helix and a three-stranded antiparallel -sheet of a typical Kazal-type inhibitor such as silverpheasant ovomucoid third domain (OMSVP3), even though the first and fifth half-cystine residues forminga disulfide bond in AEI are shifted both toward the C-terminus in comparison with those of OMSVP3.Synthesized AEI exhibited unexpected strong inhibition toward Streptomyces griseus protease B (SGPB).Our previous study [Hemmi, H., et al. (2003) Biochemistry 42, 2524-2534] demonstrated that the site-specific introduction of the engineered disulfide bond into the OMSVP3 molecule to form the CSH motifcould produce an inhibitor with a narrower specificity. Thus, the CSH motif-containing derivative of AEI(AEI analogue) was chemically synthesized when a Cys4-Cys34 bond was changed to a Cys6-Cys31bond. The AEI analogue scarcely inhibited porcine pancreatic elastase (PPE), even though it exhibitedalmost the same potent inhibitory activity toward SGPB. For the molecular scaffold, essentially no structuraldifference was detected between the two, but the N-terminal loop from Pro5 to Ile7 near the putativereactive site (Met10-Gln11) in the analogue moved by 3.7 Å toward the central helix to form the introducedCys6-Cys31 bond. Such a conformational change in the restricted region correlates with the specificitychange of the inhibitor.