Dynamics of lipid-anchored proteins

通常认为，脂类锚定蛋白通过疏水的脂类非特异性地附着在膜上。 修改过的部分。我们认为，要有效地组织脂质锚定的组织，需要的不仅仅是脂质修饰 靶膜上的蛋白质。我们将使用脂质锚定的小分子RAS超家族来验证这一假设 GTP酶(LaSGs)作为模型系统。RAS超家族由RAS、RHO、ARF和RAB家族组成 调节多种细胞过程的分子开关，控制细胞的生长、运动和 贩卖人口。这些蛋白质的结构由一个保守的催化结构域、一个柔性连接子和一个脂质组成。 抛锚。人们对内在无序连接区域的确切作用知之甚少。相比之下，它是很好的 确定了在活跃的GTP结合的和非活跃的GDP结合的构象状态之间的循环 催化结构域调节功能。突变或基因缺陷破坏这一循环会导致许多疾病。 包括癌症和发育障碍。拟议中的工作将为开发奠定基础 直接针对这些蛋白质的有效疗法。在强劲的初步数据支持下，我们假设 单体LaSGs通过下列机制之一与膜接触：(I)具有 脂质锚和G结构域之间的中长(~20aa)柔性连接子采用多个不同的 相对于膜平面的取向，催化结构域在表面“摆动”和“滚动”。 (Ii)具有长的柔性接头的那些保持G-结构域远离膜，而长接头坍塌 就像意大利面挂在墙上一样。(Iii)具有短的(和刚性的)接头的那些，例如与GTP结合的Arf1，接合 膜呈单一取向，G-结构域能够在膜表面滚动，但不能摆动。(四) 双脂锚定的LaSGs的G-结构域不重定向。我们将使用状态-来检验这些假设 最先进的分子模拟和模拟指导实验。在目标1中，我们将定义顺序和 用原子分子动力学研究LaSGs膜结合和重定向的结构决定因素 模拟绘制Rheb，RhoA，Rab11A和Arf1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-的功能角色 使用突变和细胞信号分析、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将评估以下方面的影响 G-结构域膜相互作用和重定向对我们模型系统的可药性的影响。结果将会是 阐明LaSGs和LaSGs的动态膜相互作用的原理和结构区域 确定它们的膜重新定向的决定因素，并潜在地开辟新的治疗机会 治疗许多难治的疾病。

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.