Inhibitory Specificity Change of the Ovomucoid Third Domain of the Silver Pheasant upon Introduction of an Engineered Cys¹⁴−Cys³⁹ Bond

Abstract

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.

Functional analysis of a pancreatic secretory trypsin inhibitor-like protein in insects: Silencing effects resemble the human pancreatic autodigestion phenotype

“…For example the number of amino acids between the first two cysteine residues is lower in invertebrates, resulting in a less flexible conformation causing the inhibitors to be more protease specific (Hemmi et al, 2003). These differences (and similarities) are excellently reviewed by Rimphanitchayakit (Rimphanitchayakit and Tassanakajon, 2010).…”

Canonical protein inhibitors of serine proteases

“…Vol. 60,2003 Review Article 2431 also, there is an inhibitor with a single disulfide bond instead of the two typically observed among members of this family [65]. Worthy of note is that engineering of an additional disulfide bond near the reactive site of the silver pheasant third domain (Kazal inhibitor) left intact its potent inhibitory activity toward chymotrypsin, Streptomyces griseus proteases A and B, but almost abolished it toward pancreatic elastase [66].…”

Increasing the hydrolysis constant of the reactive site upon introduction of an engineered Cys14Cys39bond into the ovomucoid third domain from silver pheasant

“…Introduction of the non‐natural disulfide into the reactive site loop has been applied to serpins19, but not to a small‐sized and compact inhibitor such as the ovomucoid third domains other than P14C/N39C. In the study elucidating the structural basis of the broad specificity of ovomucoid, we have unexpectedly found that P14C/N39C loses almost all inhibitory activity toward PPE, while it retains the potent inhibitory activities toward CHT, SGPA and SGPB17. Schematic representation of main chain folding of wild type OMSVP3 overlaid with that of P14C/N39C are shown in Figure 1B.…”

“…Double‐directed arrow points out two residues forming an engineered disulfide bond in P14C/N39C. Schematic representation of main chain folding of wild type OMSVP3 (red) overlaid with that of P14C/N39C (green) (B)17. Note that the six N‐terminal residues of wild type and P14C/N39C have been omitted from the models.…”

“…P14C/N39C is the disulfide variant of OMSVP3 introducing an engineered Cys 14 Cys 39 bond near the reactive site based on the sequence homology between OMSVP3 and ascidian trypsin inhibitor with the CSH‐motif17, 18. Introduction of the non‐natural disulfide into the reactive site loop has been applied to serpins19, but not to a small‐sized and compact inhibitor such as the ovomucoid third domains other than P14C/N39C.…”