IA sub(3), an Aspartic Proteinase Inhibitor from Saccharomyces cerevisiae, Is Intrinsically Unstructured in Solution

IA sub(3) is a highly specific and potent 68-amino acid endogenous inhibitor of yeast proteinase A (YprA), and X-ray crystallographic studies have shown that IA sub(3) binds to YprA as an alpha -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 IA sub(3) 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 IA sub(3) is unstructured in the absence of YprA. Specifically, IA sub(3) produced a CD spectrum characteristic of an unstructured peptide, and the super(15)N HSQC NMR spectra of IA sub(3) were characteristic of a polypeptide lacking intrinsic structure. We characterized the unstructured state of IA sub(3) by using singular-value decomposition (SVD) to analyze the CD data in the presence of TFE, by fully assigning the unbound IA sub(3) protein by NMR and comparing the chemical shifts to published random-coil values, and by measuring super(1)H- super(15)N heteronuclear NOEs, which are all consistent with an unfolded protein. The IA sub(3) samples used for NMR analyses were active and inhibited YprA with an inhibition constant (K sub(i)) of 1.7 nM, and the addition of YprA led to a large spectral transition in IA sub(3). Calorimetric (ITC) data also show that the overall enthalpy of the interaction between IA sub(3) and YprA is exothermic.