Structural Studies of Large Dynamic Complexes

大型动态复合体的结构研究

• 批准号: 10402366
• 负责人: HELEN JANE DYSON
• 金额: $ 51.47万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10402366
• 关键词: Acquired Immunodeficiency Syndrome; Apoptotic; Arthritis; Asthma; Cell Extracts; Cell Nucleus; Cell physiology; Cell-Free System; Cells; Cellular Structures; Cessation of life; Characteristics; Client; Collaborations; Complex; Cryoelectron Microscopy; Crystallography; Cytoplasm; DNA; Diabetes Mellitus; Dissociation; Estrogen Receptors; Eukaryotic Cell; Family; Feedback; Fluorescence; Genes; Genetic Transcription; I Kappa B-Alpha; Immune response; Inflammatory; Kinetics; Label; Ligand Binding Domain; Literature; Macromolecular Complexes; Malignant Neoplasms; Mammalian Cell; Membrane; Methods; Molecular; Molecular Chaperones; Movement; Nuclear; Phosphorylation; Phosphotransferases; Preparation; Process; Proteins; Protocols documentation; RNA; Reporting; Research; Rest; Roentgen Rays; Signal Transduction; Structure; System; Techniques; Ubiquitination; Untranslated RNA; Viral; Virus Diseases; biological adaptation to stress; biophysical techniques; cell growth; inhibitor; innovation; macromolecule; member; overexpression; physical state; reconstitution; response; solid state; transcription factor

The major overall aim of the research described in this MIRA proposal is to elucidate the structural and dynamic characteristics of large macromolecules and their complexes, which generally contain both well- structured and less-structured dynamic or unfolded regions, using a combination of solid-state methods such as crystallography or cryo-EM and solution methods such as NMR, SAXS and fluorescence. Two major systems have been under study in our lab for several years. Members of the NFκB family of transcription factors are held in the cytoplasm in an inactive state by their bound inhibitors (IκBs) until the cell receives an external signal. NFκB is activated by phosphorylation and ubiquitination of the IκBα, which is then targeted for proteasomal degradation, releasing the NFκB to translocate into the nucleus. In a classical negative feedback mechanism, NFκB upregulates transcription of IκBα in addition to signal-specific stress-response genes: newly-synthesized IκBα kinetically enhances NFκB dissociation from the DNA in a process we have termed molecular stripping. A transient ternary complex intermediate is formed during the stripping process and in an exciting new observation, it was recently shown that the stability of the resting NFκB-IκBα complex in the cytoplasm is enhanced by interaction with a specific long non-coding RNA (lncRNA), which appears to form a stable ternary complex analogous to the transient NFκB-IκBα-DNA complex formed in the nucleus during molecular stripping. We propose the structural characterization of this NFκB-IκBα-RNA complex using a variety of biophysical techniques, including NMR, cryo-electron microscopy, in collaboration with Dr. Gabriel Lander, and small-angle X-ray scattering, in collaboration with Dr. John Tainer, and will probe the structural and dynamic differences between the binary and ternary complexes of NFκB, IκBα and DNA, and the ternary NFκB-IκBα-RNA complex using specifically methyl-labeled proteins. Although a great deal is known about chaperone and co-chaperone structure, the structural basis for the interaction between Hsp90 and its clients remains unknown. The fundamental problem is that we still do not understand the physical state of the client protein when it is bound to the Hsp90 chaperone, and we have only a rough idea of where on the Hsp90 molecule the client protein makes contact. We propose an innovative method of preparation of a client protein-Hsp90 complex, by reconstituting the chaperone cascade of the eukaryotic cell, but in the context of a cell-free expression system employing bacterial cell extracts. We will prepare the complex of the estrogen receptor ligand-binding domain and Hsp90, adapting methods that have been used in the literature to demonstrate the presence of this interaction in mammalian cell extracts. Our cell-free protocol will include the use of a range of separate bacterial cell extracts containing the over-expressed, folded co-chaperones required according to literature reports for the formation of a stable complex with Hsp90. The advantage of this cell-free system is that it can be tuned to the optimization of complex formation by varying the relative amounts of the component cell extracts.

该MIRA建议中描述的研究的主要总体目的是阐明结构和 大型大分子及其复合物的动态特征，通常包含既有良好的 结构化和结构较低的动态或展开的区域，使用固态方法的组合 晶体学或冷冻EM和溶液方法，例如NMR，SAXS和荧光。两个主要系统 在我们的实验室已经研究了几年。持有NFκB家族的转录因子的成员 在细胞质中，其结合抑制剂（IκB）在不活动状态下，直到细胞接收外部信号。 NFκB被IκBα的磷酸化和泛素化激活，然后将其靶向蛋白酶体 退化，释放NFκB以转移到核us中。在经典的负面反馈机制中， NFκB除了信号特异性应力响应基因外，还上调了IκBα的转录：新合成的 在我们称为分子剥离的过程中，IκBα运动可以增强与DNA的NFκB分离。一个 瞬态三元复合体中间是在剥离过程中形成的，在令人兴奋的新观察中， 最近显示，静息NFκB-IκBα复合物在细胞质中的稳定性得到了增强 与特定长的非编码RNA（LNCRNA）的相互作用，该RNA似乎形成稳定的三元络合物 类似于分子剥离过程中核中形成的瞬时NFκB-IκBα-DNA复合物。我们 提出使用多种生物物理技术的NFκB-IκBα-RNA复合物的结构表征， 包括NMR，冷冻电子显微镜，与Gabriel Lander博士和小角X射线合作 散射，与约翰·泰纳（John Tainer）博士合作，并将探讨结构和动态差异 NFκB，IκBα和DNA的二元和三元复合物，以及三元NFκB-IκBα-RNA复合物使用 虽然对伴侣和副酮有很多了解 结构，HSP90及其客户之间相互作用的结构基础仍然未知。这 基本问题是，当客户蛋白限制时，我们仍然不了解其物理状态 HSP90伴侣，我们只对HSP90分子的位置有一个粗略的了解。 接触。我们提出了一种创新的方法来制备客户蛋白-HSP90复合物，通过重新建立 真核细胞的伴侣级联反应，但在无细胞表达系统的背景下 细菌细胞提取物。我们将准备雌激素受体配体结合结构域和HSP90的复合物， 文献中已用于证明这种相互作用的适应方法 哺乳动物细胞提取物。我们的无细胞方案将包括使用一系列单独的细菌细胞提取物 根据文献报告，包含所需的过表达，折叠的副伴侣 HSP90的稳定复合物。该无单元系统的优点是可以调整为优化 通过改变组分细胞提取物的相对量来形成复合物。