Structural Studies of Large Dynamic Complexes

大型动态复合体的结构研究

基本信息

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
项目类别：
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

项目摘要

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.

MIRA 提案中描述的研究的主要总体目标是阐明结构和大分子及其复合物的动态特性，通常包含良好的结构化和非结构化动态或展开区域，使用固态方法的组合，例如晶体学或冷冻电镜和溶液方法，例如 NMR、SAXS 和荧光两个主要系统。我们的实验室对 NFκB 转录因子家族的成员进行了多年的研究。其结合抑制剂 (IκB) 在细胞质中处于非活性状态，直到细胞接收到外部信号。 NFκB 通过 IκBα 的磷酸化和泛素化而被激活，然后被靶向蛋白酶体降解，释放 NFκB 转位到细胞核中，在经典的负反馈机制中，除了信号特异性应激反应基因外，NFκB 还上调 IκBα 的转录：新合成 IκBα 在动力学上增强 NFκB 从 DNA 的解离，这一过程我们称为分子剥离 A。在剥离过程中和令人兴奋的新观察中形成瞬态三元复合中间体，最近表明，细胞质中静止的 NFκB-IκBα 复合物的稳定性通过与特定的长非编码 RNA (lncRNA) 相互作用，似乎形成稳定的三元复合物类似于分子剥离过程中细胞核中形成的瞬时 NFκB-IκBα-DNA 复合物。提出使用多种生物物理技术对 NFκB-IκBα-RNA 复合物进行结构表征，包括与 Gabriel Lander 博士合作的 NMR、冷冻电子显微镜和小角度 X 射线与 John Tainer 博士合作，将探讨散射之间的结构和动态差异 NFκB、IκBα 和 DNA 的二元和三元复合物，以及 NFκB-IκBα-RNA 三元复合物尽管我们对分子伴侣和共分子伴侣有很多了解。结构，Hsp90 与其客户之间相互作用的结构基础仍然未知。根本问题是我们仍然不了解客户蛋白结合时的物理状态 Hsp90 分子伴侣，我们对客户蛋白在 Hsp90 分子上的位置只有一个粗略的了解我们提出了一种通过重构来制备客户蛋白-Hsp90 复合物的创新方法。真核细胞的伴侣级联，但在使用无细胞表达系统的情况下我们将制备雌激素受体配体结合结构域和 Hsp90 的复合物，采用文献中使用的方法来证明这种相互作用的存在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。含有根据文献报告形成所需的过度表达、折叠的共伴侣与 Hsp90 的稳定复合物，这种无细胞系统的优点是它可以调整以优化通过改变成分细胞提取物的相对量来形成复合物。