Structure/function of Alpha2M and its receptor LRP

Alpha2M及其受体LRP的结构/功能

• 批准号: 7153526
• 负责人: PETER G.W. GETTINS
• 金额: $ 30.1万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-05-01 至 2009-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7153526
• 关键词: Affinity; Binding; Binding Proteins; C-terminal; Calorimetry; Cells; Complement; Complex; Endopeptidases; Esters; Fluorescence; Fluorescence Resonance Energy Transfer; Fluorescence Spectroscopy; Goals; Growth Factor; Human; LDL-Receptor Related Protein 1; Link; Lipoprotein Receptor; Macroglobulins; Macrophage-1 Antigen; Maps; Molecular; Molecular Chaperones; Molecular Conformation; Mutagenesis; NMR Spectroscopy; Nature; Neurites; Pan Genus; Peptide Hydrolases; Plasma; Process; Property; Protease Inhibitor; Protein Binding; Protein Tyrosine Kinase; Proteins; Range; Receptor Signaling; Regulation; Resolution; Role; Signal Transduction; Specificity; Structure; Sulfhydryl Compounds; Tertiary Protein Structure; Testing; Thermodynamics; Thinking; Titrations; Transforming Growth Factors; Tryptophan; Variant; Venus; alpha 2-Glucoproteins; analytical ultracentrifugation; aspergillopepsin II; base; cytokine; domain mapping; fluorophore; in vivo; interest; platelet-derived growth factor BB; protein folding; receptor; receptor binding; release of sequestered calcium ion into cytoplasm

DESCRIPTION (provided by applicant): The long term goals of this proposal are to understand the structure and function of human alpha-2-macroglobulin (alpha2M), a pan-proteinase inhibitor and binder of growth factors and its receptor LRP. Four specific aims are proposed, two directed at understanding the specificity of binding of alpha2M and the receptor associated protein (RAP) to LRP, and two directed at understanding structural features of alpha2M that enable it to undergo proteinase-induced activation to a receptor-recognized species. A range of structural approaches that are currently used in the lab will be employed, including high resolution NMR and fluorescence spectroscopies, isothermal titration calorimetry and analytical ultracentrifugation (AU) applied to well-defined domains of the proteins of interest and to their complexes. Aim 1. To determine the atomic interactions involved in recognition of alpha2M by LRP. The structure of the receptor binding domain (RED) of alpha2M and a pair of complement-like repeats (CR3-CR4) from LRP to which it binds with high affinity will be determined by NMR spectroscopy. The role of specific contact residues in determining affinity and specificity will be analyzed by mutagenesis and characterization of the binding by ITC. Aim 2. To determine the basis for RAP's ability to bind to LRP. Fluorescence resonance energy transfer between exogenously-introduced acceptor fluorophores and endogenous tryptophans will be used to map the organization of the three domains present in RAP and the changes that occur when RAP binds to LRP. Aims 3 and 4 will test the hypothesis that alpha2M is composed of discrete domains that have specific functions. Aim 3 will determine the domain organization of the C-terminal half of alpha2M. Predicted domains of alpha2M that contain the thiol ester and the growth factor binding region will be expressed and characterized structurally using spectroscopic and calorimetric approaches. This will include examination of the link between the thiol ester forming residues abd the conformation of the thiol ester domain. The nature and function of the remaining parts of the C-terminal half of alpha2M that may serve as linkers will also be examined. Aim 4. The role of domain-domain interactions and conformational changes in thiol ester stabilization and cleavage and in receptor binding domain exposure will be determined. The interactions of domains identified in aim 3, as well as the RBD will be examined by thermodynamic and spectroscopic approaches with the goal of understanding the nature of the activating signal that exposes RBD and promotes thiol ester cleavage. Tryptophan variants of intact alpha2M will enable fluorescence to be used to map domain reorganizations upon activation of the alpha2M.

描述（由申请人提供）：该提案的长期目标是了解人α-2-巨球蛋白（α2M）的结构和功能，α2M是一种泛蛋白酶抑制剂和生长因子及其受体LRP的结合剂。提出了四个具体目标，两个旨在了解 alpha2M 和受体相关蛋白 (RAP) 与 LRP 结合的特异性，两个旨在了解 alpha2M 的结构特征，使其能够经历蛋白酶诱导的受体识别激活物种。将采用实验室目前使用的一系列结构方法，包括高分辨率核磁共振和荧光光谱、等温滴定量热法和分析超速离心 (AU)，应用于目标蛋白质及其复合物的明确结构域。目标 1. 确定 LRP 识别 alpha2M 所涉及的原子相互作用。 α2M 的受体结合结构域 (RED) 和来自 LRP 的一对补体样重复序列 (CR3-CR4) 的结构将通过 NMR 光谱确定，LRP 与它以高亲和力结合。将通过 ITC 的诱变和结合表征来分析特定接触残基在确定亲和力和特异性中的作用。目标 2. 确定 RAP 与 LRP 结合能力的基础。外源引入的受体荧光团和内源色氨酸之间的荧光共振能量转移将用于绘制 RAP 中存在的三个结构域的组织以及 RAP 与 LRP 结合时发生的变化。目标 3 和 4 将检验 alpha2M 由具有特定功能的离散域组成的假设。目标 3 将确定 alpha2M C 端一半的结构域组织。将使用光谱和量热方法表达和表征包含硫羟酸酯和生长因子结合区域的 alpha2M 的预测结构域。这将包括检查硫羟酸酯形成残基与硫羟酸酯结构域的构象之间的联系。 α2M C 端剩余部分（可充当接头）的性质和功能也将被检查。目标 4. 将确定结构域-结构域相互作用以及构象变化在硫羟酸酯稳定和裂解以及受体结合结构域暴露中的作用。目标 3 中确定的结构域以及 RBD 的相互作用将通过热力学和光谱方法进行检查，目的是了解暴露 RBD 并促进硫羟酸酯裂解的激活信号的性质。完整 alpha2M 的色氨酸变体将使荧光能够用于在 alpha2M 激活时绘制结构域重组图。