Crystallography of Biological Macromolecules

Carboxypeptidase Y (CPY) inhibitor I from the yeast, consisting of 204 amino acid residues, belongs to the phosphatidylethanolaminebinding protein (PEBP) family. The 2.7 Å crystal structure of the ICPY complex has been solved by molecular replacement [1, 2]. The structure of I consists of a major -type domain and an Nterminal helical segment. I has two CPY-binding sites: the N-terminal inhibitory reactive site and the secondary CPY-binding site which interact with the S1 substrate-binding site of CPY and the hydrophobic surface flanked by the active site of the enzyme, respectively. I also has the ligand-binding site, the putative binding site of the polar head group of phospholipid, which is conserved among PEBPs and accommodates a sulfate ion in the crystal structure. Along with the complex structure of I, its mutational analyses for inhibitory activity and binding to CPY demonstrate that the Nterminal inhibitory reactive site is essential for the complex formation with CPY as well as enzyme inhibition and that the I binding to CPY forms a novel mode of the proteinase-protein inhibitor interaction. The unique binding mode of Ic toward CPY gives insights into not only the inhibitory mechanism of PEBPs toward serine proteinases but also the biological functions of I belonging to the PEBP family.