Nitric oxide modification of rat brain neurogranin - Identification of the cysteine residues involved in intramolecular disulfide bridge formation using site-directed mutagenesis
Nitric oxide modification of rat brain neurogranin - Identification of the cysteine residues involved in intramolecular disulfide bridge formation using site-directed mutagenesis
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DOI：
10.1074/jbc.271.46.28798
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发表时间：
1996-11-15
影响因子：
4.8
通讯作者：
Huang, KP
中科院分区：
文献类型：
--
作者：
Mahoney, CW;Pak, JH;Huang, KP
神经颗粒蛋白（Ng）是一种神经元特异性蛋白激酶C选择性底物，在低水平的Ca 2+下以去磷酸化形式结合钙调蛋白（CaM）。该蛋白质含有氧化还原活性Cys残基，其容易被几种一氧化氮（NO）供体和其他氧化剂氧化以形成分子内二硫化物。通过定点突变检测了参与NO介导的分子内二硫键形成的大鼠脑Ng的Cys残基，Cys（3），Cys（4），Cys（9）和Cys（51）的鉴定。所有四个Cys残基的突变或Cys（51）的单个突变阻断了氧化剂介导的分子内二硫键形成，如通过非还原SDS-聚丙烯酰胺凝胶电泳下的向下迁移率移位所监测的。Cys（3）、Cys（4）或Cys（9）的单突变或这三个Cys残基中的任何一对的双突变不阻断这种分子内二硫键形成，尽管这些突变蛋白的氧化速率不同。因此，Cys（51）是Ng中NO介导的分子内二硫键形成的重要配对伴侣。Cys（3）、Cys（4）和Cys（9）可单独与Cys（51）形成二硫键，其反应活性顺序为Cys（9）> Cys（4）> Cys（3），表明Cys（9）和Cys（51）形成优先二硫键。在所有测试的情况下，分子内二硫键桥接的Ng蛋白显示出显著减弱的CaM结合亲和力和类似于2-3倍弱的蛋白激酶C底物磷酸化活性。数据表明，在溶液中N-末端Cys（3）、Cys（4）和Cys（9）与C-末端Cys（51）非常接近。Ng的N-和C-末端结构域之间的二硫桥使得处于固定构象的中央CaM结合和磷酸化位点结构域不利于与CaM结合并且作为蛋白激酶C的底物。
Neurogranin (Ng) is a neuron-specific protein kinase C-selective substrate, which binds calmodulin (CaM) in the dephosphorylated form at low levels of Ca2+. This protein contains redox active Cys residues that are readily oxidized by several nitric oxide (NO) donors and other oxidants to form intramolecular disulfide. Identification of the Cys residues of rat brain Ng, Cys(3), Cys(4), Cys(9), and Cys(51), involved in NO-mediated intramolecular disulfide bridge formation was examined by site-directed mutagenesis. Mutation of all four Cys residues or single mutation of Cys(51) blocked the oxidant-mediated intramolecular disulfide formation as monitored by the downward mobility shift under nonreducing SDS-polyacrylamide gel electrophoresis. Single mutation of Cys(3), Cys(4), or Cys(9) or double mutation of any pair of these three Cys residues did not block such intramolecular disulfide formation, although the rates of oxidation of these mutant proteins were different. Thus, Cys(51) is an essential pairing partner in NO-mediated intramolecular disulfide formation in Ng. Cys(3), Cys(4), and Cys(9) individually could pair with Cys(51), and the order of reactivity was Cys(9) > Cys(4) > Cys(3), suggesting that Cys(9) and Cys(51) form the preferential disulfide bridge. In all cases tested, the intramolecularly disulfide bridged Ng proteins displayed dramatically attenuated CaM-binding affinity and similar to 2-3-fold weaker protein kinase C substrate phosphorylation activity. The data indicate that the N-terminal Cys(3), Cys(4), and Cys(9) are in close proximity to the C-terminal Cys(51) in solution. The disulfide bridge between the N- and C-terminal domains of Ng renders the central CaM-binding and phosphorylation site domain in a fixed conformation unfavorable for binding to CaM and as a substrate of protein kinase C.