1iy6

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Solution structure of OMSVP3 variant, P14C/N39C

Overview

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.

About this Structure

1IY6 is a Single protein structure of sequence from Lophura nycthemera. Full crystallographic information is available from OCA.

Reference

Inhibitory specificity change of the ovomucoid third domain of the silver pheasant upon introduction of an engineered Cys14-Cys39 bond., Hemmi H, Kumazaki T, Yamazaki T, Kojima S, Yoshida T, Kyogoku Y, Katsu M, Shinohara F, Yokosawa H, Miura K, Kobayashi Y, Biochemistry. 2003 Mar 11;42(9):2524-34. PMID:12614146

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