Structural Biology of Membrane Scaffolds

膜支架的结构生物学

• 批准号: 8331498
• 负责人: VINZENZ UNGER
• 金额: $ 39.19万
• 依托单位: NORTHWESTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8331498
• 关键词: Actins; Amaze; Architecture; Binding; Binding Proteins; Biochemical; Biogenesis; Biological; Biological Process; Cell division; Cell physiology; Cells; Complex; Cryoelectron Microscopy; Cytoskeleton; DNA Sequence Rearrangement; Data; Diabetes Mellitus; Disease; Dynamin; Educational process of instructing; Electron Spin Resonance Spectroscopy; Epilepsy; Foundations; Generations; Goals; Guanosine Triphosphate Phosphohydrolases; Imagery; In Vitro; Kinetics; Learning; Length; Life; Lipid Bilayers; Macromolecular Complexes; Malignant Neoplasms; Membrane; Modeling; Molecular Machines; Movement; N-WASP protein; N-terminal; Organelles; Outcome; Play; Positioning Attribute; Procedures; Process; Protein Binding Domain; Protein Family; Proteins; Recruitment Activity; Regulation; Resolution; Role; SH3 Domains; Set protein; Shapes; Solutions; Specificity; Structure; Tertiary Protein Structure; Testing; Thinking; Time; Vesicle; Wiskott-Aldrich Syndrome; Work; amphiphysin; base; cdc42 GTP-Binding Protein; cell motility; design; dimer; human disease; image reconstruction; insight; member; molecular assembly/self assembly; molecular scale; protein complex; public health relevance; reconstitution; research study; scaffold; structural biology; success; trafficking

DESCRIPTION (provided by applicant): A vast number of cellular processes depend on the cell's ability to change the shape of their membranes with astounding spatial and temporal accuracy. At a biochemical level, many of the players that participate in these processes are known. What remains unknown, however, is how ensembles of several proteins with often overlapping functions and interaction specificities reproducibly accomplish defined biological outcomes. The largest hurdle towards resolving the mysteries of membrane remodeling is to obtain structural information about the membrane-associated scaffolds that orchestrate every aspect of these processes from changing membrane curvature to membrane fission, and recruitment of the actin cytoskeleton. Overcoming this limitation, we have demonstrated that electron cryomicroscopy provides access to the architecture of membrane-associated scaffolds at resolutions sufficient for the generation of detailed mechanistic models. Exploiting this advance, the long term goals of this project are to understand how members of the BAR superfamily (bin-amphihpysin-rvs family) of proteins generate/stabilize/sense membrane curvature, and how these molecules can selectively recruit interaction partners from a pool of promiscuous multidomain proteins such as the fission GTPase dynamin and the cytoskeletal activator N-WASP. We will use a combination of electron cryomicroscopy, low angle scattering, electron paramagnetic resonance spectroscopy and in vitro biophysical structure-function experiments to pursue three specific aims: (1) we will expand the number of experimentally determined scaffold structures, which will teach us much about their design principles and how these designs contribute to membrane curvature generation and selection of interaction partners, (2) we will exploit what we already learned to test mechanistic models of early steps in scaffold assembly, which may provide vital clues how scaffold assembly is regulated and (3) we will lay the foundation for structural work on higher order macromolecular complexes that BAR-domain proteins form with two of their most important effectors: dynamin and N-WASP. Taken together, these studies will allow us to greatly advance understanding of one of the most fundamental aspects of life: the ability of cells to change the shape of their membranes with amazing spatial and temporal resolution. Understanding these processes will be essential to appreciate how imbalances and errors in membrane remodeling contribute to a broad spectrum of human diseases ranging from epilepsy to diabetes and cancer. PUBLIC HEALTH RELEVANCE: In order to live, cells must continuously change the shape of their membranes with high precision. How cells accomplish this complex task is poorly understood because we have almost no information about how the molecular machines that are responsible for these processes interact with the membranes they reshape. Overcoming this limitation, we established a procedure to visualize membrane-remodeling molecules as they are engaged to their targets. This - for the first time - allows us to closely examine how these molecules function, and how they interact with additional proteins whose recruitment results in a specific biological effect. Visualizing these interactions is key to understanding how errors in these processes can contribute to diseases as varied as epilepsy, diabetes and cancer.

描述（由申请人提供）：大量的细胞过程取决于细胞以惊人的空间和时间精度改变细胞膜形状的能力。在生化水平上，参与这些过程的许多参与者都是已知的。然而，目前尚不清楚的是，具有经常重叠的功能和相互作用特异性的几种蛋白质的集合如何可重复地实现确定的生物学结果。解决膜重塑之谜的最大障碍是获得有关膜相关支架的结构信息，这些支架协调这些过程的各个方面，从改变膜曲率到膜裂变，以及肌动蛋白细胞骨架的招募。克服了这一限制，我们证明电子冷冻显微镜可以以足以生成详细机械模型的分辨率来了解膜相关支架的结构。利用这一进展，该项目的长期目标是了解蛋白质 BAR 超家族（bin-amphihpysin-rvs 家族）的成员如何生成/稳定/感知膜曲率，以及这些分子如何从池中选择性地招募相互作用伙伴混杂的多结构域蛋白，例如裂变 GTP 酶动力和细胞骨架激活剂 N-WASP。我们将结合使用电子冷冻显微镜、低角散射、电子顺磁共振波谱和体外生物物理结构功能实验来实现三个具体目标：（1）我们将扩大实验确定的支架结构的数量，这将给我们带来很多启发关于他们的设计原理以及这些设计如何有助于膜曲率的生成和相互作用伙伴的选择，（2）我们将利用我们已经学到的知识来测试支架组装早期步骤的机械模型，这可能提供如何调节支架组装的重要线索(3) 我们将为 BAR 结构域蛋白与其两个最重要的效应子（动力蛋白和 N-WASP）形成的高阶大分子复合物的结构研究奠定了基础。总而言之，这些研究将使我们能够极大地增进对生命最基本方面之一的理解：细胞以惊人的空间和时间分辨率改变其细胞膜形状的能力。了解这些过程对于理解膜重塑的不平衡和错误如何导致从癫痫到糖尿病和癌症等广泛的人类疾病至关重要。 公共健康相关性：为了生存，细胞必须持续高精度地改变其细胞膜的形状。人们对细胞如何完成这一复杂任务知之甚少，因为我们几乎没有关于负责这些过程的分子机器如何与它们重塑的膜相互作用的信息。克服了这一限制，我们建立了一种程序来可视化膜重塑分子，因为它们与目标结合。这第一次使我们能够仔细研究这些分子如何发挥作用，以及它们如何与其他蛋白质相互作用，这些蛋白质的招募会导致特定的生物效应。可视化这些相互作用是理解这些过程中的错误如何导致癫痫、糖尿病和癌症等多种疾病的关键。