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 experimentall… Show more
“…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.…”
Section: Introductionsupporting
confidence: 93%
“…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)).…”
Section: ■ Materials and Methodsmentioning
confidence: 99%
“…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.…”
Section: Introductionmentioning
confidence: 99%
“…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).…”
Saccharomyces cerevisiae IA 3 is a 68 amino acid peptide inhibitor of yeast proteinase A (YPRA) characterized as a random coil when in solution, folding into an N-terminal amphipathic alpha helix for residues 2−32 when bound to YPRA, with residues 33−68 unresolved in the crystal complex. Circular dichroism (CD) spectroscopy results show that amino acid substitutions that remove hydrogen-bonding interactions observed within the hydrophilic face of the N-terminal domain (NTD) of IA 3 -YPRA crystal complex reduce the 2,2,2-trifluoroethanol (TFE)-induced helical transition in solution. Although nearly all substitutions decreased TFE-induced helicity compared to wild-type (WT), each construct did retain helical character in the presence of 30% (v/v) TFE and retained disorder in the absence of TFE. The NTDs of 8 different Saccharomyces species have nearly identical amino acid sequences, indicating that the NTD of IA 3 may be highly evolved to adopt a helical fold when bound to YPRA and in the presence of TFE but remain unstructured in solution. Only one natural amino acid substitution explored within the solvent-exposed face of the NTD of IA 3 induced TFE-helicity greater than the WT sequence. However, chemical modification of a cysteine by a nitroxide spin label that contains an acetamide side chain did enhance TFE-induced helicity. This finding suggests that non-natural amino acids that can increase hydrogen bonding or alter hydration through side-chain interactions may be important to consider when rationally designing intrinsically disordered proteins (IDPs) with varied biotechnological applications.
“…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.…”
Section: Introductionsupporting
confidence: 93%
“…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)).…”
Section: ■ Materials and Methodsmentioning
confidence: 99%
“…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.…”
Section: Introductionmentioning
confidence: 99%
“…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).…”
Saccharomyces cerevisiae IA 3 is a 68 amino acid peptide inhibitor of yeast proteinase A (YPRA) characterized as a random coil when in solution, folding into an N-terminal amphipathic alpha helix for residues 2−32 when bound to YPRA, with residues 33−68 unresolved in the crystal complex. Circular dichroism (CD) spectroscopy results show that amino acid substitutions that remove hydrogen-bonding interactions observed within the hydrophilic face of the N-terminal domain (NTD) of IA 3 -YPRA crystal complex reduce the 2,2,2-trifluoroethanol (TFE)-induced helical transition in solution. Although nearly all substitutions decreased TFE-induced helicity compared to wild-type (WT), each construct did retain helical character in the presence of 30% (v/v) TFE and retained disorder in the absence of TFE. The NTDs of 8 different Saccharomyces species have nearly identical amino acid sequences, indicating that the NTD of IA 3 may be highly evolved to adopt a helical fold when bound to YPRA and in the presence of TFE but remain unstructured in solution. Only one natural amino acid substitution explored within the solvent-exposed face of the NTD of IA 3 induced TFE-helicity greater than the WT sequence. However, chemical modification of a cysteine by a nitroxide spin label that contains an acetamide side chain did enhance TFE-induced helicity. This finding suggests that non-natural amino acids that can increase hydrogen bonding or alter hydration through side-chain interactions may be important to consider when rationally designing intrinsically disordered proteins (IDPs) with varied biotechnological applications.
“…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.…”
Biomolecular function is realized by recognition, and increasing evidence shows that recognition is determined not only by structure but also by flexibility and dynamics. We explored a biomolecular recognition process that involves a major conformational change – protein folding. In particular, we explore the binding-induced folding of IA3, an intrinsically disordered protein that blocks the active site cleft of the yeast aspartic proteinase saccharopepsin (YPrA) by folding its own N-terminal residues into an amphipathic alpha helix. We developed a multi-scaled approach that explores the underlying mechanism by combining structure-based molecular dynamics simulations at the residue level with a stochastic path method at the atomic level. Both the free energy profile and the associated kinetic paths reveal a common scheme whereby IA3 binds to its target enzyme prior to folding itself into a helix. This theoretical result is consistent with recent time-resolved experiments. Furthermore, exploration of the detailed trajectories reveals the important roles of non-native interactions in the initial binding that occurs prior to IA3 folding. In contrast to the common view that non-native interactions contribute only to the roughness of landscapes and impede binding, the non-native interactions here facilitate binding by reducing significantly the entropic search space in the landscape. The information gained from multi-scaled simulations of the folding of this intrinsically disordered protein in the presence of its binding target may prove useful in the design of novel inhibitors of aspartic proteinases.
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