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

项目总结 我们最近发现，K-RAS膜锚定的多碱结构域(PDB)采用定义的 PM上的动态结构，允许与膜脂高度选择性地相互作用。我们还发现 具有不同一级序列的PBD具有编码不同的脂结合特异性的能力。 因此，PBDS与质膜(PM)的相互作用要比简单的复杂得多 静电学。进一步的分析表明，K-RAS锚点的脂结合特异性是一个关键 信号输出的决定因素。与此同时，我们发现了一个耐人寻味的发现，即PM有选择地 调节特定负电荷脂质的扩散动力学和空间组织，从而 调节K-RAS纳米聚集和信号传递的程度。这种新的机制使电信号 能通过蛋白质-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。在这笔新的赠款中，我们将以这些令人兴奋的初步观察为基础 进一步明确K-RAS膜锚的分子细节结构和功能，并对其结构进行研究 质膜上其他小分子GTP酶的动态变化我们的核心假设是，我们所做的观察 K-RAS可推广到其他具有含PBD膜锚的小GTP酶。因此，在目标1中 我们将测试K-RAS PM锚比被动电荷复杂得多的假设 探测器使用了广泛的实验技术和分子模拟。我们将向您展示PBD 序列和构象动力学产生对功能至关重要的脂结合特异性。我们将测试 通过分析RAS超家族的其他成员，这一假设在目标2中的普适性。我们还将 定义同一组蛋白质的PM空间组织。目标3号将研究分子 多个小分子GTP酶的膜相互作用机制及其对膜的依赖性 潜力。这些结果将为脂质结合特异性如何在膜上编码提供新的见解 小的GTP酶的锚定，使它们受制于PM的脂质组成和组织。从小到小 GTP酶调节细胞生物学的许多方面，其功能障碍与多种病理有关，包括 对于肿瘤的发生，这一结果将产生广泛的影响。

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.