title: The potency and specificity of the interaction between the IA3 inhibitor and its target aspartic proteinase from Saccharomyces cerevisiae.
abstract: The yeast IA3 polypeptide consists of only 68 residues, and the free inhibitor has little intrinsic secondary structure. IA3 showed subnanomolar potency toward its target, proteinase A from Saccharomyces cerevisiae, and did not inhibit any of a large number of aspartic proteinases with similar sequences/structures from a wide variety of other species. Systematic truncation and mutagenesis of the IA3 polypeptide revealed that the inhibitory activity is located in the N-terminal half of the sequence. Crystal structures of different forms of IA3 complexed with proteinase A showed that residues in the N-terminal half of the IA3 sequence became ordered and formed an almost perfect alpha-helix in the active site of the enzyme. This potent, specific interaction was directed primarily by hydrophobic interactions made by three key features in the inhibitory sequence. Whereas IA3 was cut as a substrate by the nontarget aspartic proteinases, it was not cleaved by proteinase A. The random coil IA3 polypeptide escapes cleavage by being stabilized in a helical conformation upon interaction with the active site of proteinase A. This results, paradoxically, in potent selective inhibition of the target enzyme.
authors.affiliation: School of Biosciences, Cardiff University, P. O. Box 911, Cardiff CF10 3US, Wales, United Kingdom.
authors.forename: L H
authors.initials: LH
authors.lastname: Phylip
authors.forename: W E
authors.initials: WE
authors.lastname: Lees
authors.forename: B G
authors.initials: BG
authors.lastname: Brownsey
authors.forename: D
authors.initials: D
authors.lastname: Bur
authors.forename: B M
authors.initials: BM
authors.lastname: Dunn
authors.forename: J R
authors.initials: JR
authors.lastname: Winther
authors.forename: A
authors.initials: A
authors.lastname: Gustchina
authors.forename: M
authors.initials: M
authors.lastname: Li
authors.forename: T
authors.initials: T
authors.lastname: Copeland
authors.forename: A
authors.initials: A
authors.lastname: Wlodawer
authors.forename: J
authors.initials: J
authors.lastname: Kay
mesh_terms: D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D011480:Protease Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins
chemical_list: D005656:Fungal Proteins; C018134:PAI3 protein, S cerevisiae; D010455:Peptides; D011480:Protease Inhibitors; D029701:Saccharomyces cerevisiae Proteins; D016282:Aspartic Acid Endopeptidases