Key Features Determining the Specificity of Aspartic Proteinase Inhibition by the Helix-forming IA3 Polypeptide

Abstract

The 68-residue IA 3 polypeptide from Saccharomyces cerevisiae is essentially unstructured. It inhibits its target aspartic proteinase through an unprecedented mechanism whereby residues 2-32 of the polypeptide adopt an amphipathic ␣-helical conformation upon contact with the active site of the enzyme. This potent inhibitor (K i < 0.1 nM) appears to be specific for a single target proteinase, saccharopepsin. Mutagenesis of IA 3 from S. cerevisiae and its ortholog from Saccharomyces castellii was coupled with quantitation of the interaction for each mutant polypeptide with saccharopepsin and closely related aspartic proteinases from Pichia pastoris and Aspergillus fumigatus. This identified the charged K18/D22 residues on the otherwise hydrophobic face of the amphipathic helix as key selectivity-determining residues within the inhibitor and implicated certain residues within saccharopepsin as being potentially crucial. Mutation of these amino acids established Ala-213 as the dominant specificity-governing feature in the proteinase. The side chain of Ala-213 in conjunction with valine 26 of the inhibitor marshals Tyr-189 of the enzyme precisely into a position in which its side-chain hydroxyl is interconnected via a series of water-mediated contacts to the key K18/D22 residues of the inhibitor. This extensive hydrogen bond network also connects K18/D22 directly to the catalytic Asp-32 and Tyr-75 residues of the enzyme, thus deadlocking the inhibitor in position. In most other aspartic proteinases, the amino acid at position 213 is a larger hydrophobic residue that prohibits this precise juxtaposition of residues and eliminates these enzymes as targets of IA 3 . The exquisite specificity exhibited by this inhibitor in its interaction with its cognate folding partner proteinase can thus be readily explained.

N‐terminal extension of the yeast IA3 aspartic proteinase inhibitor relaxes the strict intrinsic selectivity

“…Gene‐encoded inhibitors of aspartic proteinases are rather rare in nature. Thus, there is a need to understand the mechanisms of action of the few that are known, in order to exploit their therapeutic potential [1]. We have described previously one such inhibitor: the IA 3 protein from Saccharomyces cerevisiae [1–4].…”

“…Thus, there is a need to understand the mechanisms of action of the few that are known, in order to exploit their therapeutic potential [1]. We have described previously one such inhibitor: the IA 3 protein from Saccharomyces cerevisiae [1–4]. This remarkable polypeptide not only is a highly potent inhibitor of its target enzyme, saccharopepsin, but also appears to be completely specific for this sole target proteinase [1,2].…”

“…We have described previously one such inhibitor: the IA 3 protein from Saccharomyces cerevisiae [1–4]. This remarkable polypeptide not only is a highly potent inhibitor of its target enzyme, saccharopepsin, but also appears to be completely specific for this sole target proteinase [1,2]. Crystal structures solved for complexes of IA 3 with saccharopepsin revealed an unprecedented mechanism of action [2,3].…”

“…The free inhibitor is essentially unstructured [5,6] but, upon contacting its target enzyme, residues 2–32 become ordered and adopt an alpha helical conformation occupying the active site cleft of the proteinase [2,3]. This absolute selectivity for saccharopepsin was shown to be conferred by a combination of the K18 and D22 residues in the S. cerevisiae IA 3 sequence coupled with the requirement for an alanine residue to be present at position 213 in saccharopepsin [1].…”

“…Included among these enzymes are a number with sequences that are very closely related to that of saccharopepsin (e.g. the vacuolar aspartic proteinase from P. pastoris ; PpPr) [1]. This shares a sequence identity of 77% with saccharopepsin and essentially all of the active site residues of saccharopepsin that contact the helical IA 3 inhibitor are identical in PpPr.…”