Xie Mingtang, Smith Jillian L, Ding Ziwei, Zhang Daqing, Cornell Rosemary B
Department of Molecular Biology and Biochemistry, Simon Fraser University, 8888 University Drive, Burnaby, British Columbia V5A 1S6, Canada.
J Biol Chem. 2004 Jul 2;279(27):28817-25. doi: 10.1074/jbc.M403311200. Epub 2004 Apr 6.
phosphocholine cytidylyltransferase (CCT), a key enzyme that controls phosphatidylcholine synthesis, is regulated by reversible interactions with membranes containing anionic lipids. Previous work demonstrated that CCT is a homodimer. In this work we show that the structure of the dimer interface is altered upon encountering membranes that activate CCT. Chemical cross-linking reactions were established which captured intradimeric interactions but not random CCT dimer collisions. The efficiency of capturing covalent cross-links with four different reagents was diminished markedly upon presentation of activating anionic lipid vesicles but not zwitterionic vesicles. Experiments were conducted to show that the anionic vesicles did not interfere with the chemistry of the cross-linking reactions and did not sequester available cysteine sites on CCT for reaction with the cysteine-directed cross-linking reagent. Thus, the loss of cross-linking efficiency suggested that contact sites at the dimer interface had increased distance or reduced flexibility upon binding of CCT to membranes. The regions of the enzyme involved in dimerization were mapped using three approaches: 1) limited proteolysis followed by cross-linking of fragments, 2) yeast two-hybrid analysis of interactions between select domains, and 3) disulfide bonding potential of CCTs with individual cysteine to serine substitutions for the seven native cysteines. We found that the N-terminal domain (amino acids 1-72) is an important participant in forming the dimer interface, in addition to the catalytic domain (amino acids 73-236). We mapped the intersubunit disulfide bond to the cystine 37 pair in domain N and showed that this disulfide is sensitive to anionic vesicles, implicating this specific region in the membrane-sensitive dimer interface.
磷酸胆碱胞苷转移酶（CCT）是一种控制磷脂酰胆碱合成的关键酶，它通过与含有阴离子脂质的膜发生可逆相互作用来调节。先前的研究表明CCT是一种同二聚体。在本研究中，我们发现二聚体界面的结构在遇到激活CCT的膜时会发生改变。建立了化学交联反应，该反应捕获二聚体内相互作用，但不捕获随机的CCT二聚体碰撞。当存在激活的阴离子脂质囊泡而非两性离子囊泡时，用四种不同试剂捕获共价交联的效率显著降低。进行的实验表明，阴离子囊泡不会干扰交联反应的化学过程，也不会隔离CCT上可用于与半胱氨酸定向交联试剂反应的半胱氨酸位点。因此，交联效率的降低表明，在CCT与膜结合后，二聚体界面处的接触位点距离增加或灵活性降低。使用三种方法绘制了参与二聚化的酶区域：1）有限蛋白酶解后片段交联；2）对选定结构域之间的相互作用进行酵母双杂交分析；3）对七个天然半胱氨酸进行单个半胱氨酸到丝氨酸替换的CCT的二硫键形成潜力分析。我们发现，除了催化结构域（氨基酸73 - 236）外，N端结构域（氨基酸1 - 72）是形成二聚体界面的重要参与者。我们将亚基间二硫键定位到结构域N中的37位半胱氨酸对，并表明该二硫键对阴离子囊泡敏感，这表明该特定区域参与了膜敏感的二聚体界面。
