K-Ras G-domain binding with signaling lipid phosphatidylinositol (4,5)-phosphate (PIP2): membrane association, protein orientation, and function
K-Ras G-domain binding with signaling lipid phosphatidylinositol (4,5)-phosphate (PIP2): membrane association, protein orientation, and function
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DOI：
10.1074/jbc.ra118.004021
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发表时间：
2019-04-26
影响因子：
4.8
通讯作者：
Buck, Matthias
中科院分区：
文献类型：
--
作者：
Cao, Shufen;Chung, Stacey;Buck, Matthias
Ras基因是人类癌症的潜在驱动力，其中突变的原癌基因GT3 KRAS 4 B（K-Ras 4 B）是最丰富的同种型。致癌基因产物的靶向抑制被认为是当今癌症治疗的圣杯，最近发现的小分子KRas 4 B抑制剂是由于对这种GT3的结构和动力学有了更深入的了解。由于与生物膜的相互作用是Ras功能的关键，Ras-脂质相互作用已成为一个主要的焦点，特别是因为这种相互作用显然涉及脂质锚定的Ras C末端及其G蛋白结构域。在这里，使用NMR光谱和分子动力学模拟，辅以生物物理和细胞生物学测定，我们研究了K-Ras 4 B与信号脂质磷脂酰肌醇（4，5）-磷酸（PIP 2）之间的相互作用。我们发现，2和3股以及螺旋4和5的GTGFG结构域结合到PIP 2，并确定了在这些相互作用中使用的这些结构元件中的特定残基，可能发生在两个K-Ras 4 B相对于膜的取向状态。重要的是，我们发现这些已知在突变时致癌的残基中的一些（D47 K、D92 N、K104 M和D126 N）对于K-Ras介导的成纤维细胞转化是关键的，但基本上不影响基础和辅助核苷酸水解和交换。此外，K104 M取代消除了K-Ras在质膜上的定位。这些发现表明，特定的G结构域残基可以通过介导与膜相关的PIP 2脂质的相互作用来关键地调节Ras功能;这些见解可能会为未来设计靶向Ras活性的治疗试剂提供信息。
Ras genes potently drive human cancers, with mutated proto-oncogene GTPase KRAS4B (K-Ras4B) being the most abundant isoform. Targeted inhibition of oncogenic gene products is considered the holy grail of present-day cancer therapy, and recent discoveries of small-molecule KRas4B inhibitors were made thanks to a deeper understanding of the structure and dynamics of this GTPase. Because interactions with biological membranes are key for Ras function, Ras-lipid interactions have become a major focus, especially because such interactions evidently involve both the Ras C terminus for lipid anchoring and its G-protein domain. Here, using NMR spectroscopy and molecular dynamics simulations complemented by biophysical- and cell-biology assays, we investigated the interaction between K-Ras4B with the signaling lipid phosphatidylinositol (4,5)-phosphate (PIP2). We discovered that the 2 and 3 strands as well as helices 4 and 5 of the GTPase G-domain bind to PIP2 and identified the specific residues in these structural elements employed in these interactions, likely occurring in two K-Ras4B orientation states relative to the membrane. Importantly, we found that some of these residues known to be oncogenic when mutated (D47K, D92N, K104M, and D126N) are critical for K-Ras-mediated transformation of fibroblast cells, but do not substantially affect basal and assisted nucleotide hydrolysis and exchange. Moreover, the K104M substitution abolished localization of K-Ras to the plasma membrane. The findings suggest that specific G-domain residues can critically regulate Ras function by mediating interactions with membrane-associated PIP2 lipids; these insights that may inform the future design of therapeutic reagents targeting Ras activity.