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

背景：许多临床相关的蛋白复合物在细胞中以低丰度短暂形成，以响应外部刺激。此外，生长信号蛋白通过上游信号调控异质磷酸化，严格调节其构象、亚基组成和细胞内定位。对于传统的结构测定技术，如x射线晶体学和核磁共振（NMR）光谱学，要达到毫克蛋白质的数量，这些调节类的蛋白质中的许多都不适合过度表达。因此，靶向内源性水平表达的天然复合物至关重要。然而，由于内源性蛋白质的化学和构象异质性，另一层次的复杂性出现了。为了克服这一挑战，我们试图利用翻译后修饰状态和支架亚基来定义功能、亚基组成和细胞内定位。哺乳动物雷帕霉素靶蛋白（mTOR）被发现与调控支架蛋白RPTOR或RICTOR相关，它们分别将激酶靶向mTORC1或mTORC2，具有明确的化学成分。事实上，这些支架蛋白已经被用来选择性地拉低内源性mTOR复合物。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受到上游激酶磷酸化的严格控制。在饥饿细胞中，TSC位于溶酶体表面，在那里它下调mTORC1的活性。胰岛素刺激后，TSC被Akt迅速磷酸化，并从溶酶体转运到细胞质，在那里它在胰岛素刺激后保持催化活性状态长达1小时。目的：提出了TOR通路蛋白组分的结构研究计划，重点研究酵母和人tsc中的TORC2。1)聚合物包被多孔EM网格的开发与优化。2)酵母基因标记TORC2的冷冻处理。3) TSC的信号集成亲和低温电镜。意义：mTORCs和TSC全全息复合物的原子分辨率结构知识严重缺乏，这有助于设计选择性激动剂和拮抗剂。详细的TORC2结构知识对于生成选择性抑制剂用于mTORC2依赖性信号通路的基础生物学研究是必要的。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亚基之间的界面及其与上游激酶的相互作用。我们的低温样品制备方案将适用于其他稀缺配合物。

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.