DNA based platform for visualization of scarce protein assemblies with electron cryo-microscopy

背景：*许多临床上相关的蛋白质复合体在细胞内对外界刺激的反应是短暂的低丰度。此外，生长信号蛋白通过上游信号严格调节其构象、亚基组成和细胞内定位而经历异质性磷酸化。对于X射线结晶学和核磁共振光谱学等传统结构测定技术而言，获得毫克蛋白质量所必需的过度表达不是这些“调节类”蛋白质中的许多蛋白质所必需的。因此，以内源水平表达的天然复合体为靶点是至关重要的。然而，由于内源性蛋白质的化学和构象异质性，出现了另一种程度的并发症。为了克服这一挑战，我们试图利用翻译后修饰状态和脚手架亚基来定义功能、亚基组成和细胞内定位。哺乳动物靶标雷帕霉素(MTOR)蛋白被发现与调控支架蛋白RPTOR或RICTOR有关，它们分别将激酶靶向mTORC1或mTORC2，具有明确的化学组成。事实上，这些支架蛋白已经被用来选择性地下拉内源性mTOR复合体。另一方面，结节性硬化症复合体(TSC)的功能、复合体的形成和亚细胞定位是通过上游激酶的磷酸化严格控制的。在饥饿的细胞中，TSC存在于溶酶体表面，在那里它下调mTORC1的活性。在胰岛素刺激下，TSC被Akt迅速磷酸化，并从溶酶体转移到细胞质，在那里它在胰岛素刺激后保持催化活性状态长达1小时。*目标：*我们提出了一项研究TOR途径蛋白质组分的计划，重点是酵母和人TSC中的TORC2。*1)聚合物涂层孔状EM网格的开发和优化。*2)酿酒酵母TORC2基因标签的冷冻电子显微镜。*3)TSC的信号整合亲和蛋白。*意义：*mTORCs和TSC全息复合体的原子分辨结构知识严重缺乏，有助于设计选择性激动剂和拮抗剂。TORC2的详细结构知识对于产生选择性抑制剂是必要的，这对于基础生物学研究mTORC2依赖的信号通路是必要的。类似的TSC结构，将揭示疾病相关突变的机制基础，并确定不同TSC亚单位之间的界面及其与上游激酶的相互作用。我们的低温EM样品制备方案将适用于其他稀缺的络合物。

Background:***Many clinically relevant protein complexes form transiently in cells at low abundance in response to external stimuli. Additionally, growth signaling proteins undergo heterogeneous phosphorylation regulated via upstream signaling that strictly regulate their conformation, subunit composition, and intra-cellular localization within cells. Many of these ‘regulatory class' of proteins are not amenable to over-expression that is necessary to achieve milligram protein quantities for traditional structure determination techniques such as X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. Therefore, it is critical to target native complexes expressed at endogenous levels. However, another level of complication arises due to chemical and conformational heterogeneity of endogenous proteins. To overcome this challenge, we sought to take advantage of post-translationally modified states and scaffolding subunits that define function, subunit composition and intra-cellular localization. Mammalian target of rapamycin (mTOR) protein is found in association with regulatory scaffold proteins RPTOR or RICTOR that target the kinase to mTORC1 or mTORC2, respectively, with define chemical composition. Indeed, these scaffolding proteins have been used to selectively pull-down endogenous mTOR complexes. On the other hand, tuberous sclerosis complex (TSC) function, complex formation, and sub-cellular localization is strictly controlled via phosphorylation by upstream kinases. In starving cells, TSC is found on the lysosomal surface where it down-regulates mTORC1 activity. Upon insulin stimulation, TSC is rapidly phosphorylated by Akt and translocates from lysosomes to the cytoplasm where it remains in a catalytically active state for up to 1h post insulin stimulation. ***Objectives:***We proposed a research program for strtuctural investigation of protein components of TOR pathway, with emphasis on TORC2 from yeast and human TSC.***1) Development and optimization of polymer-coated holey EM grids. ***2) cryoEM of genetically-tagged TORC2 from yeast Saccharomyces cerevisiae. ***3) Signal-integrated affinity cryoEM of TSC. ***Significance:***Atomic-resolution structural knowledge is acutely lacking for mTORCs and TSC holo-complexes and is instrumental in design of selective agonist and antagonist. A detailed structural knowledge of TORC2 is necessary for generation of selective inhibitors for basic biological research into mTORC2 dependent signaling pathways. Similarly TSC structure, will reveal the mechanistic basis of disease associated mutations and identify interfaces between different TSC subunits and their interaction with upstream kinases. Our cryoEM sample preparation scheme will be adoptable to other scarce complexes.