Global response of diacylglycerol kinase towards substrate binding observed by 2D and 3D MAS NMR
Global response of diacylglycerol kinase towards substrate binding observed by 2D and 3D MAS NMR
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DOI：
10.1038/s41598-019-40264-8
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发表时间：
2019-03-08
影响因子：
4.6
通讯作者：
Glaubitz, Clemens
中科院分区：
文献类型：
--
作者：
Moebius, Kristin;Kazemi, Sina;Glaubitz, Clemens
大肠杆菌二酰基甘油激酶（DGK）是一种完整的膜蛋白，它催化atp依赖性的二酰基甘油（DAG）磷酸化为磷酸（PA）。它是一种独特的三聚体酶，与典型的激酶不具有序列同源性。尽管它的体积很小，但在结构和功能上都表现出明显的复杂性。在这里，基于3D MAS NMR，使用手动和自动分析协议，对脂质双层内的野生型DGK进行了化学位移分配。在核苷酸结合时，可以观察到广泛的化学位移扰动。这些数据为对称DGK三聚体的三个活性位点同时被占用提供了证据。此外，我们可以检测到核苷酸底物诱导了实质性的构象变化，很可能引导DGK进入其催化活性形式。此外，通过dnp增强的MAS NMR结合定点诱变和功能分析，鉴定了功能相关的蛋白质间相互作用。
Escherichia coli diacylglycerol kinase (DGK) is an integral membrane protein, which catalyses the ATP-dependent phosphorylation of diacylglycerol (DAG) to phosphatic acid (PA). It is a unique trimeric enzyme, which does not share sequence homology with typical kinases. It exhibits a notable complexity in structure and function despite of its small size. Here, chemical shift assignment of wild-type DGK within lipid bilayers was carried out based on 3D MAS NMR, utilizing manual and automatic analysis protocols. Upon nucleotide binding, extensive chemical shift perturbations could be observed. These data provide evidence for a symmetric DGK trimer with all of its three active sites concurrently occupied. Additionally, we could detect that the nucleotide substrate induces a substantial conformational change, most likely directing DGK into its catalytic active form. Furthermore, functionally relevant interprotomer interactions are identified by DNP-enhanced MAS NMR in combination with site-directed mutagenesis and functional assays.