Determinants of Apolipoprotein E/LDL Receptor Binding

了解apoE与低密度脂蛋白受体（LDLR）相互作用的结构基础，将阐明该受体家族中多种蛋白质如何识别配体，并将有助于设计具有治疗潜力的受体模拟物。本建议的目的是：i)通过测试静电相互作用是LDLR和apoE相互作用的主要手段的假设，确定特定LDLR残基对配体结合的贡献；ii)为LDLR创建一个无脂多价配体，以检验高亲和力相互作用主要是由于apoE受体结合域的多个拷贝的出现而产生的假设，独立于重大的结构改变。iii)辨别表皮生长因子前体（EGFP）结构域触发酸依赖性配体释放的机制。结合将首先使用固相结合试验进行分析，然后将开发一种方法，如荧光能量转移或表面等离子体共振，以进行更定量的结合分析。点突变将被引入到最小长度LDLR中，以研究带电残基、钙配位和暴露的疏水表面对配体结合的重要性。对配体多价性的要求将通过表达重组融合蛋白来研究，该融合蛋白由载脂蛋白e的n端结构域连接到定义的低聚物状态的卷曲结构域。通过监测pH对含有EGFP结构域关键残基突变的LDLR构建体的配体结合亲和力的影响，将探索酸依赖性配体释放。

Understanding the structural basis for the interaction of apoE with the LDL receptor (LDLR) will clarify hove the diverse array of proteins in this receptor family recognize ligands and will aid in the design of receptor mimics with therapeutic potential. The aims of this proposal are to: i) determine the contribution of specific LDLR residues to ligand binding by testing the hypothesis that electrostatic interactions are the primary means by which LDLR and apoE interact, ii) create a lipid-free multivalent ligand for the LDLR to examine the hypothesis that high affinity interaction arises primarily due to the presentation of multiple copies of apoE receptor binding domain, independent of significant structural alterations, and iii) discern the mechanism by which the epidermal growth factor precursor (EGFP) domain triggers acid- dependent ligand release. Binding will be initially analyzed using a solid phase binding assay, then a method such as fluorescence energy transfer or surface plasmon resonance will be developed for a more quantitative analysis of binding. Point mutations will be introduced into a minimal length LDLR to investigate the importance of charged residues, calcium coordination, and an exposed hydrophobic surface for ligand binding. The requirement for ligand multivalency will be investigated by expressing recombinant fusion proteins that consist of the N-terminal domain of apoE connected to coiled coil domains of defined oligomeric state. Acid-dependent ligand release will be explored by monitoring the effects of pH on the ligand binding affinity of LDLR constructs containing mutations of key residues in the EGFP domain.