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-摩克洛蛋白（alpha2m）的结构和功能，泛蛋白酶抑制剂和生长因子及其受体LRP的粘合剂。提出了四个具体目标，两个针对理解α2M和受体相关蛋白（RAP）与LRP的结合的特异性，而两个则针对理解α2M的结构特征，从而使其能够经历蛋白酶诱导的激活对受体识别的物种。当前在实验室中使用的一系列结构方法将被采用，包括高分辨率NMR和荧光光谱，等温滴定量热法和分析性超速离心（AU）（AU）应用于感兴趣的蛋白质及其复合物的良好定义域。目的1。确定LRP识别α2M的原子相互作用。 α2M的受体结合结构域（红色）的结构和来自LRP与高亲和力结合的一对补体样重复（CR3-CR4）将由NMR光谱确定。特定接触残基在确定亲和力和特异性中的作用将通过ITC的结合诱变和表征来分析。目标2。确定说唱与LRP结合的能力的基础。外源引入的受体荧光团和内源性色氨酸的荧光共振能量转移将用于绘制RAP中存在的三个域的组织以及RAP与LRP结合时发生的变化。目标3和4将检验以下假设：α2M由具有特定函数的离散域组成。 AIM 3将确定α2M的C末端的域组织。含有硫醇酯和生长因子结合区域的α2M的预测域将使用光谱和量热法在结构上表达并表征。这将包括检查硫醇酯形成残基之间的联系，使硫醇酯结构域的构象。还将检查α2M的C末端一半的其余部分的性质和功能，也将被检查为接头。 AIM 4。将确定结构域膜相互作用和构象变化在硫醇酯稳定和裂解以及受体结合结构域暴露中的作用。 AIM 3中确定的域的相互作用以及RBD将通过热力学和光谱方法检查，目的是了解暴露RBD并促进硫醇酯裂解的激活信号的性质。完整α2M的色氨酸变体将使荧光在激活α2M后用于绘制域的重组。