Dynamics of lipid-anchored proteins

通常认为，脂质锚定蛋白通过疏水性脂质非特异性地附着于膜， 修饰的部分。我们认为，要有效地组织脂质锚定的 靶膜上的蛋白质。我们将使用Ras超家族的脂质锚定小分子来验证这一假设。 GTP酶（laSG）作为模型系统。Ras超家族由Ras、Rho、Arf和Rab家族组成。 分子开关介导多种细胞过程，控制细胞生长、运动和 贩卖人口这些蛋白质的结构由保守的催化结构域、柔性接头和脂质组成 锚。关于内在无序连接区的确切作用知之甚少。相比之下， 建立了活性GTP结合和非活性GDP结合构象状态之间的循环， 催化结构域调节功能。突变或遗传缺陷破坏了这个循环，就会导致许多疾病 包括癌症和发育障碍。拟议的工作将为发展奠定基础 直接针对这些蛋白质的有效疗法。在强有力的初步数据支持下，我们假设 单体laSG通过以下机制之一与膜接合：（i）具有 在脂质锚和G-结构域之间的中等长度（~ 20 aa）柔性接头采用多个不同的 相对于膜平面的取向，催化域在表面上“摆动”和“滚动”。 (ii)那些具有长的柔性连接子的分子使G结构域远离膜，长的连接子塌陷 就像意大利面挂在墙上一样(iii)那些具有短（和刚性）接头的，如GTP结合的Arf 1， 在一个单一的方向与G-域能够滚动，但不摆动的膜表面上的膜。（四） 双重脂质锚定的IaSG的G结构域不重定向。我们将使用状态测试这些假设- 最先进的分子模拟和模拟引导实验。在目标1中，我们将定义序列， 用原子分子动力学研究laSGs的膜结合和重取向的结构决定因素 模拟绘制Rheb，RhoA，Rab 11 A和Arf 1的构象和能量景观，代表 Ras、Rho、Rab和Arf家族蛋白。在目标2中，我们将确定G的功能作用。 结构域膜接合和重定向，共聚焦成像， 电子显微镜（EM）和天然脂质纳米盘中的单分子FRET。目标3将评估 G-结构域膜相互作用和重定向对我们的模型系统的可药用性。结果将 阐明负责laSGs的动态膜相互作用的原理和结构区域， 确定其膜重定向的决定因素，并可能开辟新的治疗机会 治疗许多疑难杂症。

It is often assumed that lipid-anchored proteins non-specifically attach to membranes by the hydrophobic lipid- modified moiety. We propose that it takes more than lipid-modification to productively organize lipid-anchored proteins on target membranes. We will test this hypothesis using the Ras superfamily of lipid-anchored small GTPases (laSGs) as model systems. The Ras superfamily consists of the Ras, Rho, Arf and Rab family of molecular switches that mediate a wide variety of cellular processes controlling cell growth, motility and trafficking. The structure of these proteins consists of a conserved catalytic domain, a flexible linker, and a lipid anchor. Little is known about the precise roles of the intrinsically disordered linker region. By contrast, it is well established that cycling between active GTP-bound and inactive GDP-bound conformational states of the catalytic domain regulate function. Disruption of this cycle by mutation or genetic defects causes many diseases including cancer and developmental disorders. The proposed work will lay the foundation for the development of effective therapies that directly target these proteins. Supported by strong preliminary data, we hypothesize that monomeric laSGs engage membranes through one of the following mechanisms: (i) Those with a moderately long (~20aa) flexible linker between the lipid-anchor and the G-domain adopt multiple distinct orientations with respect to the membrane plane, with the catalytic domain ‘swinging’ and ‘rolling’ on the surface. (ii) Those with a long flexible linker keep the G-domain distal from the membrane, with the long linker collapsing on it as spaghetti would on a wall. (iii) Those with a short (and rigid) linker, such as GTP-bound Arf1, engage membranes in a single orientation with the G-domain able to roll but not swing on the membrane surface. (iv) The G-domain of dually lipid-anchored laSGs does not reorient. We will test these hypotheses using state-of- the-art molecular simulations and simulation-guided experiments. In Aim 1 we will define the sequence and structural determinants of membrane binding and reorientation of laSGs using atomistic molecular dynamics simulations to map the conformational and energy landscapes of Rheb, RhoA, Rab11A and Arf1, representing the Ras, Rho, Rab and Arf family proteins, respectively. In Aim 2, we will determine the functional roles of G- domain membrane engagement and reorientation using mutations and cell signaling assays, confocal imaging, electron microscopy (EM), and single molecule FRET in native lipid nanodiscs. Aim 3 will assess the impact of G-domain membrane interaction and reorientation on the druggability of our model systems. The results will elucidate the principles and structural regions responsible for the dynamic membrane interaction of laSGs and define the determinants of their membrane reorientation, and potentially open up novel therapeutic opportunities to treat many intractable diseases.