IA3, an aspartic proteinase inhibitor from Saccharomyces cerevisiae, is intrinsically unstructured in solution.
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.
Green
Terry B
TB
Department of Biochemistry and Molecular Biology, Box 100245, University of Florida, Gainesville, Florida 32610-0245, USA.
Ganesh
Omjoy
O
Perry
Kyle
K
Smith
Leif
L
Phylip
Lowri H
LH
Logan
Timothy M
TM
Hagen
Stephen J
SJ
Dunn
Ben M
BM
Edison
Arthur S
AS
D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D002942:Circular Dichroism; D008024:Ligands; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D011480:Protease Inhibitors / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism; D012996:Solutions; D013379:Substrate Specificity; D013816:Thermodynamics
D008024:Ligands; C018134:PAI3 protein, S cerevisiae; D011480:Protease Inhibitors; D029701:Saccharomyces cerevisiae Proteins; D012996:Solutions; C022325:aspartic proteinase A; D016282:Aspartic Acid Endopeptidases