IA3, an aspartic proteinase inhibitor from Saccharomyces cerevisiae, is intrinsically unstructured in solution

Biochemistry. 2004 Apr 13;43(14):4071-81. doi: 10.1021/bi034823n.


IA(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(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(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(3) is unstructured in the absence of YprA. Specifically, IA(3) produced a CD spectrum characteristic of an unstructured peptide, and the (15)N HSQC NMR spectra of IA(3) were characteristic of a polypeptide lacking intrinsic structure. We characterized the unstructured state of IA(3) by using singular-value decomposition (SVD) to analyze the CD data in the presence of TFE, by fully assigning the unbound IA(3) protein by NMR and comparing the chemical shifts to published random-coil values, and by measuring (1)H-(15)N heteronuclear NOEs, which are all consistent with an unfolded protein. The IA(3) 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 IA(3). Calorimetric (ITC) data also show that the overall enthalpy of the interaction between IA(3) and YprA is exothermic.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Aspartic Acid Endopeptidases / antagonists & inhibitors*
  • Aspartic Acid Endopeptidases / metabolism
  • Circular Dichroism
  • Ligands
  • Molecular Sequence Data
  • Nuclear Magnetic Resonance, Biomolecular
  • Protease Inhibitors / chemistry*
  • Protease Inhibitors / metabolism
  • Protein Binding
  • Protein Conformation
  • Protein Structure, Secondary
  • Saccharomyces cerevisiae Proteins / chemistry*
  • Saccharomyces cerevisiae Proteins / metabolism
  • Solutions
  • Substrate Specificity
  • Thermodynamics


  • Ligands
  • PAI3 protein, S cerevisiae
  • Protease Inhibitors
  • Saccharomyces cerevisiae Proteins
  • Solutions
  • aspartic proteinase A
  • Aspartic Acid Endopeptidases