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Title: STRUCTURE FUNCTION STUDIES OF IA3, AN INHIBITOR OF YEAST PROTEINASE A

Author
item Green, Terry
item GANESH, OMJOY - UNIV OF FLORIDA
item PERRY, KYLE - UNIV OF FLORIDA
item SMITH, LEIF - UNIV OF FLORIDA
item PHYLIP, LOWRI - CARDIFF UNIV
item LOGAN, TIMOTHY - FLORIDA STATE UNIV
item HAGEN, STEPHEN - UNIV OF FLORIDA
item DUNN, BEN - UNIV OF FLORIDA
item EDISON, ARTHUR - UNIV OF FLORIDA

Submitted to: Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/9/2004
Publication Date: 3/18/2004
Citation: Green, T.B., Ganesh, O., Perry, K., Smith, L., Phylip, L.H., Logan, T.M., Hagen, S.J., Dunn, B.M., Edison, A.S. 2004. Structure function studies of ia3, an inhibitor of yeast proteinase a. Biochemistry. 43:4071-4081.

Interpretive Summary: IA3 is a 68 amino acid inhibitor that is specific for only Yeast Proteinase A (YprA). Proteinases are attractive targets for drug design against human pathogens because they are essential to the life cycle of the pathogen. Yeast Proteinase A has significant structural and mechanistic similarities to Candida, HIV, and Plasmodium proteinases, all of which lead to serious life threatening infections that cause candidiasis, AIDS, and malaria, respectively. The IA3 inhibitor is unstructured in solution but undergoes a structural transition to an '-helix in the presence of YprA. This mechanism of binding is unprecedented due to the fact that all other inhibitors bind in an extended '-strand conformation. The characterization of IA3 binding to YprA using biophysical methods such as circular dichroism, crystallography, kinetic analysis, nuclear magnetic resonance, and isothermal titration calorimetry, has provided critical molecular details on this novel interaction that will be used to design inhibitors to various pathogenic aspartic proteinases.

Technical Abstract: IA3 is a highly specific and potent endogenous inhibitor of yeast proteinase A (YprA). X-ray crystallographic studies have shown that IA3 binds to YprA as an '-helix in the active site. Surprisingly, only residues 2-32 of the 68 amino acid inhibitor are observed in the x-ray structure that forms an amphipathic helix. Circular dichrism (CD), size-exclusion chromatography, and nuclear magnetic resonance (NMR) spectroscopy 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 protein lacking intrinsic structure. In addition, all the amide protons exchanged with deuterium oxide (D20) in less than 3 minutes, signifying that IA3 is unstructured due to solvent exposure. The unstructured state of IA3 was fully analyzed and characterized by fully assigning the unbound IA3 protein 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. In the presence of YprA, NMR data indicate that IA3 not only undergoes a conformational change but also makes C-terminal contacts to the enzyme, which were not elucidated in the x-ray structure. In addition, CD and NMR data show that IA3 undergoes a coil to helix transition in the presence of trifluoroethanol (TFE). The coil to helix transition suggests that the thermodynamics of IA3 binding and inhibiting YprA are very interesting. A van't Hoff enthalpy of 76 kJ/mol was obtained using inhibition data of IA3 binding to YprA while an enthalpy of (') 86 kJ/mol was revealed using isothermal titration calorimetry (ITC). These data suggest that the thermodynamics of IA3 folding and binding to YprA are complex and that a simple two-state binding model oversimplifies the interaction.