Fan J S, Zhang Q, Li M, Tochio H, Yamazaki T, Shimizu M, Zhang M
Department of Biochemistry, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, People's Republic of China.
J Biol Chem. 1998 Dec 11;273(50):33472-81. doi: 10.1074/jbc.273.50.33472.
Neuronal nitric-oxide synthase (nNOS) is the primary nitric oxide (NO) regulator in neurons. The activity of the enzyme is inhibited by a protein inhibitor called PIN. We were able to purify large quantities of PIN overexpressed in bacterial cells. Analytical ultracentrifugation and chemical cross-linking studies showed that PIN exists as a monomer at low concentrations. The protein forms a high order aggregate at elevated concentrations. We have shown, using NMR spectroscopy, that the previously identified PIN-binding domain (PINB) of nNOS (residues 161-245) adopts a random coil structure in solution. By titrating 15N-labeled PINB with unlabeled PIN, the PIN-binding region of nNOS was precisely mapped to a 17-residue peptide fragment from Met-228 to His-244 of nNOS. NMR titration experiments also showed that PIN binds to nNOS with a 1:2 stoichiometry. A synthetic peptide corresponding to the identified PIN-binding region of nNOS was used to study the interaction between PIN and nNOS in detail. The functional implications of the results obtained from this study are discussed.
神经元型一氧化氮合酶（nNOS）是神经元中一氧化氮（NO）的主要调节因子。该酶的活性受到一种名为PIN的蛋白质抑制剂的抑制。我们能够纯化在细菌细胞中过表达的大量PIN。分析超速离心和化学交联研究表明，PIN在低浓度下以单体形式存在。该蛋白质在浓度升高时形成高阶聚集体。我们使用核磁共振光谱表明，先前鉴定的nNOS的PIN结合结构域（PINB，第161 - 245位氨基酸残基）在溶液中呈无规卷曲结构。通过用未标记的PIN滴定15N标记的PINB，nNOS的PIN结合区域被精确地定位到nNOS从Met - 228到His - 244的一个17个氨基酸残基的肽段。核磁共振滴定实验还表明，PIN与nNOS以1:2的化学计量比结合。使用与鉴定出的nNOS的PIN结合区域相对应的合成肽详细研究了PIN与nNOS之间的相互作用。讨论了本研究所得结果的功能意义。
