Quantitative structure activity relationship of IA₃‐like peptides as aspartic proteinase inhibitors

Abstract

The IA(3) polypeptide inhibitor from Saccharomyces cerevisiae interacts potently and selectively with its target, the S. cerevisiae vacuolar aspartic proteinase (ScPr). Upon encountering the enzyme, residues 2-32 of the intrinsically unstructured IA(3) polypeptide become ordered into an almost-perfect alpha-helix. In previous IA(3) mutagenesis studies, we identified important characteristics of the enzyme inhibitor interactions and generated a large dataset of variants with K(i) values determined experimentally at pH 3.1 and 4.7. Using this information, the three-dimensional structure of each variant was modelled in silico with the correct protonation for each experimental pH value. A set of descriptors of the inhibitor/ScPr interactions was then calculated and used to establish mathematical models relating the variant sequences to their inhibitory activities at each pH. Cross-validation, external-set validation and five separate selections of the training and test samples confirmed the robustness of the equations. A major contributor to the structure-activity relationship was the free energy of binding calculated by the FoldX program. The mathematical models were challenged further (i) by in silico alanine-scanning mutagenesis of residues 2-32 in IA(3) and relating binding energy to experimentally derived inhibition constants for selected representatives of these variants; and (ii) by predicting inhibitory-potencies for two novel IA(3)-variants. The predictions of the equations for these new IA(3)-variants with ScPr matched almost precisely the kinetic data determined experimentally. The models described represent valuable tools for the future design of novel inhibitor variants active against ScPr and other aspartic proteinases.

Hydrogen Bonding Compensation on the Convex Solvent-Exposed Helical Face of IA3, an Intrinsically Disordered Protein

“…This structure–sequence relationship appears optimized for helical propensity and binding to YPRA in a manner that interacts with one aspartic acid residue in the active site while evading interaction with the other, likely due to smaller amino acid volumes. Our results are also consistent with earlier inhibitory studies of modified N-terminal and chimera IA 3 sequences, ,, in that substitutions that we find to lower TFE-induced helical propensity also have reduced binding affinities for YPRA.…”

“…As expected, I11M retains near WT helicity (Figure 2B,D). We recognize that our structure− function comparisons are being made between a TFE-induced helix, which may not be stabilized by the exact same forces as IA 3 showed ∼4× decrease in K i for YPRA, with the double mutant having ∼3000× lessened K i . 6 Both S9 and Q13 reside in the convex hydrophilic surfaceexposed face of IA 3 and incorporation of nonpolar alanine at S9A (almost equivalent volume and equivalent hydrophobicity) 22,26 and at Q13A (smaller volume and less polar) reduce the hydrophobic moment of the formed helix and remove potential hydrogen-bonding acceptor and donor interactions (Table S2 (amino acid volume), S3 (hydrophobicity), and S4 (hydrogen bonding)).…”

“…YPRA is a member of the aspartic protease family, which are enzymes present in many species. Aspartic proteases play important roles in numerous pathologies, including fungal infections, HIV infection, and hypertension, and as such, there remains a desire for the development of novel inhibitors for aspartic proteases. − The mechanism of inhibition of IA 3 is unique in that it is a peptide inhibitor of its parent organism proteinase but digested by other proteinases. ,, This unique selectivity and function occurs, because in solution, IA 3 is unstructured (i.e., random coil secondary structure) and thus susceptible to cleavage by the other proteases. Yet, when IA 3 binds to its parent organism proteinase, it adopts a helical structure that bulges from the active site.…”

“…cerevisiae YPRA/IA 3 complex reveals a curvature to the helical N-terminal domain (NTD) that evades peptide cleavage by the active site aspartic acid residues (Figure A). With an inhibition constant, K i of ∼1 nM, ,,, residues 8–26 of the NTD of IA 3 are resolved in the complex and contained within the binding cleft of YPRA in a helical conformation, with a calculated hydrophobic moment of 0.337 μH . The resolved YPRA-bound NTD helix of IA 3 contains a concave hydrophobic surface that faces in toward the active site and a convex hydrophilic solvent-exposed face that contains several hydrogen-bonding interactions among neighboring amino acid residues as well as to YPRA (Figure A).…”

Multi-Scaled Explorations of Binding-Induced Folding of Intrinsically Disordered Protein Inhibitor IA3 to its Target Enzyme

“…Even fewer structures of inhibitor-enzyme complexes have been determined. One complex that has been studied is that of the yeast peptidase A with its naturally occurring peptide inhibitor, IA3 [18]. Free IA3 is a 68-residue peptide that lacks a stable structure in solution.…”