神经营养因子受体p75NTR跨膜区自组装及其相互作用的分子机制研究

Transmembrane proteins are involved in a variety of signal pathways key to survival and function of both individual cells and multicellular organisms as receptors for signal transduction. Transmembrane protein p75 neurotrophin receptor (p75NTR) plays a range of critical roles in the nervous system, and inducing cell death is the most remarkable biological function of p75NTR. However, the activation mechanism of p75NTR is still elusive. Oligomerization of single span transmembrane receptors is well established as a general structural mechanism for receptor activation, therefore, this project mainly focused on the self-assembly and interaction of transmembrane domain of p75NTR in different membranes. Our previous studies proposed a scissorlike opening formed by the disulfide-linked transmembrane (TM) p75NTR homodimer, which may contain two structural conformations. To confirm the structural basis accounted for p75NTR dimerization by multiple biochemical and biophysical methods is our next goal. Molecular dynamics simulation will be applied as well to collect and analyze the structural information, and energetically favorable conformation will be screened out by PMF calculation. Then we will further investigate whether the conformation switch of p75NTR homodimer is ligand-dependent. Our project aims to better understand the molecular mechanism of p75NTR signal transduction and provide insight into its activation mechanism, based on the study of association and interaction of its transmembrane domain.

跨膜蛋白作为信号分子受体对大量通路进行精密调控，细胞存活及功能实现均与其密切相关。跨膜蛋白神经营养因子受体 p75(p75NTR)最显著的生物学功能是诱导细胞凋亡。p75NTR的激活在神经系统中发挥关键作用，然而其激活机制至今仍不清楚。单跨膜受体的低聚自组装是受体激活的一种重要结构基础，因此本项目将在多种膜环境中对p75NTR跨膜区多肽自组装及相互作用进行探究。我们前期工作表明p75NTR跨膜区能形成同源二聚体且可能存在剪刀状开合两种构象，在此基础上我们将采用多种生物化学、生物物理手段研究其二聚作用结构基础，同时开展分子动力学模拟采集分子结构信息，计算平均力势得出能量优势构象，进一步揭示其自组装机制。并以此为契机在细胞内测定p75NTR两种二聚构象转变与其配体结合的关系。基于上述研究，本项目将为p75NTR激活机制相关研究奠定重要基础，并为跨膜受体自组装相互作用对其激活的影响提供理论依据。

大量单跨膜受体的跨膜区低聚自组装及不同构象间的转换是其行使功能及调控功能的重要结构基础。跨膜蛋白神经营养因子受体p75NTR 能够和大量配体结合从而介导众多生理功能，在细胞存活、轴突生长、细胞融合等进程中均具有重要意义，而其信号转导可能受到跨膜区二聚作用的调控，因此本课题围绕 p75NTR 跨膜区的自组装及相互作用分子机制开展研究。首先利用生物学蛋白纯化表达以及化学固相合成多肽的方法，建立了高效表达和高效合成跨膜区多肽的平台，并建立了在大肠杆菌细胞膜环境中研究跨膜区多肽相互作用的研究方法，通过CAT活性遴选出二聚表现最优的p75NTR跨膜区野生型序列。其次完成了多种膜环境中p75NTR跨膜区多肽的自组装以及相互作用研究。在我们构建的多种膜环境如去污剂体系、大肠杆菌细胞膜、分子动力学模拟DPPC双分子层、脂筏结构混合膜体系中p75NTR跨膜区多肽均能形成同源二聚体，且不依赖跨膜区保守残基C257位二硫键的形成。通过粗粒化模拟实验我们发现了几种潜在的p75NTR跨膜区二聚体构象，其二聚作用驱动力分别来自保守残基C257形成的二硫键、GxxxG基元、以及疏水作用形成的亮氨酸拉链。在缺失二硫键的情况下，基于A262xxxG266二聚界面的右手装配构象（交叉角为24°）成为优势二聚构象。最后通过p75NTR二聚体不同构象自由能的计算确认在缺失二硫键情况下基于A262xxxG266二聚界面的形成的右手装配二聚体最为稳定，此外我们发现脂筏结构能够通过其中的信号分子通过蛋白质-脂质相互作用招募p75NTR跨膜区多肽并促进其构象转换。综上，本项目为我们进一步揭示p75NTR跨膜区二聚作用对其信号转导调控机制的关键作用提供了理论依据和分子结构信息，同时也为未来以p75NTR跨膜区为靶点诱导p75NTR构象变化从而调控其功能的小分子药物开发提供了研究基础。