Decoding the structures and lipid binding specificity of small GTPase membrane anchors

项目摘要 我们最近发现K-Ras膜锚的多碱基结构域（PDB）采用定义的 PM上的动态结构允许与膜脂质的高度选择性相互作用。我们还发现 具有不同一级序列的PBD具有编码不同脂质结合特异性的能力。 因此，PBDs与质膜（PM）的相互作用比简单的要复杂得多 静电学进一步的分析表明，K-Ras锚的脂质结合特异性是K-Ras在细胞中表达的关键。 信号输出的决定因素。与此同时，我们发现了一个有趣的发现，即PM电位选择性地 调节特定带负电荷脂质的扩散动力学和空间组织， 调节K-Ras纳米簇和信号传导的程度。这种新颖的机制使得电信号 可以通过蛋白质-脂质相互作用调节经典信号通路是电调节的基础 基于脂质的信号平台。在这项新的资助中，我们将在这些令人兴奋的初步观察的基础上， 进一步明确K-Ras膜锚结构和功能的分子细节，研究K-Ras膜的结构 PM上其他小GTP酶的动态。我们的核心假设是， K-Ras可推广至具有含PBD的膜锚的其它小GTP酶。在Aim 1中 我们将检验这样一个假设，即K-Ras PM锚比被动电荷复杂得多 探测器使用广泛的实验技术和分子模拟。我们将展示PBD 序列和构象动力学产生对功能至关重要的脂质结合特异性。我们将测试 通过分析Ras超家族的其他成员，证明了目标2中这一假设的普遍性。我们还将 定义同一组蛋白质的PM空间组织。目标3将研究分子 多个小GTP酶的膜相互作用机制和膜依赖性 潜力结果将产生新的见解如何脂质结合特异性是在膜编码 锚的小GTP酶，使他们受到PM脂质组成和组织。因为小 GTP酶调节细胞生物学的许多方面，它们的功能障碍与多种病理学有关，包括 肿瘤发生，结果将有广泛的影响。

PROJECT SUMMARY We recently discovered that the polybasic domain (PDB) of the K-Ras membrane anchor adopts defined dynamic structures on the PM that allow for highly selective interactions with membrane lipids. We also found that PBDs with different primary sequences have the capacity to encode different lipid binding specificities. Thus interactions of PBDs with the plasma membrane (PM) are considerably more complex than simple electrostatics. Further analysis showed that the lipid binding specificity of the K-Ras anchor is a key determinant of signal output. In parallel we made the intriguing discovery that PM potential selectively modulates the diffusional dynamics and spatial organization of specific negatively charged lipids and thereby regulates the extent of K-Ras nanoclustering and signaling. This novel mechanism whereby electrical signals can modulate classical signaling pathways through protein-lipid interactions is the basis of electrical regulation of lipid based signaling platforms. In this new grant we will build on these exciting preliminary observations to further define the molecular details of K-Ras membrane anchor structure and function and study the structural dynamics of other small GTPases on the PM. Our core hypothesis is that the observations we have made with K-Ras are generalizable to other small GTPases that have a PBD-containing membrane anchor. Thus in Aim 1 we will test the hypothesis that the K-Ras PM anchor is substantially more complex than a passive charge detector using a wide-range of experimental techniques and molecular simulations. We will show that PBD sequence and conformational dynamics generate lipid-binding specificity that is critical for function. We will test the generalizability of this hypothesis in Aim 2 by analyzing other members of the Ras superfamily. We will also define the PM spatial organization of the same set of proteins. Aim 3 will investigate the molecular mechanisms and the dependence of membrane interactions of multiple small GTPases on membrane potential. The results will yield new insights into how lipid-binding specificity is encoded in the membrane anchors of small GTPases to render them subject to PM lipid composition and organization. Since small GTPases regulate many aspects of cell biology and their dysfunction is linked to multiple pathologies, including oncogenesis, the results will have wide-ranging implications.