IA3, an Aspartic Proteinase Inhibitor from Saccharomyces cerevisiae, Is Intrinsically Unstructured in Solution

IA3 is a highly specific and potent 68-amino acid endogenous inhibitor of yeast proteinase A (YprA), and X-ray crystallographic studies have shown that IA3 binds to YprA as an α-helix [Li, M., Phylip, L. H., Lees, W. E., Winther, J. R., Dunn, B. M., Wlodawer, A., Kay, J., and Gustchina, A. (2000) Nat. Struct. Biol. 7, 113−117]. Surprisingly, only residues 2−32 of IA3 are seen in the X-ray structure, and the remaining residues are believed to be disordered in the complex. We have used circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy to show that IA3 is unstructured in the absence of YprA. Specifically, IA3 produced a CD spectrum characteristic of an unstructured peptide, and the 15N HSQC NMR spectra of IA3 were characteristic of a polypeptide lacking intrinsic structure. We characterized the unstructured state of IA3 by using singular-value decomposition (SVD) to analyze the CD data in the presence of TFE, by fully assigning the unbound IA3 protein by NMR and comparing the chemical shifts to published random-coil values, and by measuring 1H−15N heteronuclear NOEs, which are all consistent with an unfolded protein. The IA3 samples used for NMR analyses were active and inhibited YprA with an inhibition constant (K i) of 1.7 nM, and the addition of YprA led to a large spectral transition in IA3. Calorimetric (ITC) data also show that the overall enthalpy of the interaction between IA3 and YprA is exothermic.