Structural and Functional Analysis of Calcium Ion Mediated Binding of 5-Lipoxygenase to Nanodiscs.
Structural and Functional Analysis of Calcium Ion Mediated Binding of 5-Lipoxygenase to Nanodiscs.
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DOI：
10.1371/journal.pone.0152116
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Jegerschöld C
中科院分区：
文献类型：
--
作者：
Kumar RB;Zhu L;Idborg H;Rådmark O;Jakobsson PJ;Rinaldo-Matthis A;Hebert H;Jegerschöld C
白三烯家族炎症介质产生的一个重要步骤是 Ca2+ 介导的 5 脂氧合酶 (5LO) 向核膜的募集。为了在体外研究这种反应，使用了纳米盘的天然膜模拟环境。用脂质 POPC 和膜支架蛋白 MSP1E3D1 制成内径 10.5 nm 的纳米圆盘。研究了单体和二聚体 5LO。单体 5LO 与 Ca2+ 和纳米圆盘混合可形成稳定的复合物，1) 从花生四烯酸中产生预期的白三烯产物，2) 首次可以通过非变性凝胶电泳和负染色透射电子显微镜观察到，3) 每个纳米圆盘显示两个 5LO 的最高比例。我们将其解释为光盘每一侧都有一个 5LO。 5LO 的二聚体通过负染色透射电子显微镜观察到，并且显示不与纳米圆盘结合。这项研究展示了纳米圆盘在获得单位膜蛋白与膜之间复合物的基本结构信息以及功能信息方面的优势。
An important step in the production of inflammatory mediators of the leukotriene family is the Ca2+ mediated recruitment of 5 Lipoxygenase (5LO) to nuclear membranes. To study this reaction in vitro, the natural membrane mimicking environment of nanodiscs was used. Nanodiscs with 10.5 nm inner diameter were made with the lipid POPC and membrane scaffolding protein MSP1E3D1. Monomeric and dimeric 5LO were investigated. Monomeric 5LO mixed with Ca2+ and nanodiscs are shown to form stable complexes that 1) produce the expected leukotriene products from arachidonic acid and 2) can be, for the first time, visualised by native gel electrophoresis and negative stain transmission electron microscopy and 3) show a highest ratio of two 5LO per nanodisc. We interpret this as one 5LO on each side of the disc. The dimer of 5LO is visualised by negative stain transmission electron microscopy and is shown to not bind to nanodiscs. This study shows the advantages of nanodiscs to obtain basic structural information as well as functional information of a complex between a monotopic membrane protein and the membrane.