Determinants of Apolipoprotein E/LDL Receptor Binding

了解apoE与LDL受体（LDLR）相互作用的结构基础将阐明该受体家族中各种蛋白质识别配体的方式，并将有助于设计具有治疗潜力的受体模拟物。这项建议的目的是：i）通过检验静电相互作用是LDLR和apoE相互作用的主要方式的假设来确定特异性LDLR残基对配体结合的贡献，ii）为LDLR产生无脂质多价配体以检验主要由于apoE受体结合结构域的多个拷贝的呈递而产生高亲和力相互作用的假设，独立于显著的结构改变，和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。结合最初将使用固相结合测定法进行分析，然后将开发荧光能量转移或表面等离子体共振等方法以进行更定量的结合分析。将在最小长度LDLR中引入点突变，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。将通过表达重组融合蛋白来研究配体多价性的要求，所述重组融合蛋白由连接至限定寡聚体状态的卷曲螺旋结构域的apoE的N-末端结构域组成。将通过监测pH对含有EGFP结构域中关键残基突变的LDLR构建体的配体结合亲和力的影响来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.