Structural and mechanistic basis of AAGAB-controlled AP2 adaptor assembly

项目总结 在网状蛋白介导的内吞作用(CME)中，外壳蛋白网状蛋白依靠接头来募集货物蛋白 内吞的部位。CME中主要的笼状蛋白适配器是异四聚体AP2适配器复合体，它 由两个大亚基(和β)、一个中亚基(µ)和一个小亚基()组成。它一般都是 认为AP2适配器等多聚体运输适配器是自发组装的。然而，我们最近 发现AP2接头组装是一个受α和伽马接头结合控制的有序过程 蛋白质(AAGAB)。如果没有AAGAB的帮助，AP2接头就无法形成，从而导致CME缺陷。这些 研究结果揭示了笼状蛋白介导的人口贩运中一条以前未被认识的途径。然而，它仍然 不清楚AAGAB如何识别AP2亚基，以及AAGAB-AP2相互作用如何驱动AP2适配器组装。 在我们的初步研究中，我们表达和纯化了游离AAGAB和AAGAB：AP2组装中间体， 对它们的结构和功能有了初步的了解。在这项研究中，我们将利用这些优势 初步数据，以确定AAGAB控制的AP2适配器组件的结构和机械基础。 我们将首先表征游离AAGAB和AAGAB：AP2组装中间体的生化性质 使用重组的系统。然后我们将确定蛋白质和蛋白质复合体的原子结构 使用X射线结晶学和单粒子低温电子显微镜。接下来，我们将验证生理上的 使用基于细胞的遗传分析的生化和结构结果的相关性。最后，我们将确定 AAGAB的功能如何受到致病突变的影响。圆满完成这项建议 研究将填补我们对膜蛋白运输知识的一个重大空白。这项工作也将作为一项 总体上理解贩运适配子组装的范例。最终，这些发现将 促进开发由AAGAB突变引起的人类疾病的新治疗策略。

PROJECT SUMMARY In clathrin-mediated endocytosis (CME), the coat protein clathrin relies on adaptors to recruit cargo proteins to endocytic sites. The predominant clathrin adaptor in CME is the heterotetrameric AP2 adaptor complex, which is comprised of two large subunits ( and β), one medium subunit (µ), and one small subunit (). It is generally thought that multimeric trafficking adaptors such as AP2 adaptor assemble spontaneously. However, we recently discovered that AP2 adaptor assembly is an ordered process controlled by alpha and gamma adaptin binding protein (AAGAB). Without the assistance of AAGAB, AP2 adaptor fails to form, leading to CME defects. These findings revealed a previously unrecognized pathway in clathrin-mediated trafficking. However, it remains unclear how AAGAB recognizes AP2 subunits and how AAGAB-AP2 interactions drive AP2 adaptor assembly. In our preliminary studies, we expressed and purified free AAGAB and AAGAB:AP2 assembly intermediates, and gained initial insights into their structures and functions. In this research, we will take advantage of these preliminary data to determine the structural and mechanistic basis of AAGAB-controlled AP2 adaptor assembly. We will first characterize the biochemical properties of free AAGAB and AAGAB:AP2 assembly intermediates using reconstituted systems. We will then determine the atomic structures of the proteins and protein complexes using X-ray crystallography and single-particle cryo-electron microscopy. Next, we will validate the physiological relevance of the biochemical and structural findings using cell-based genetic assays. Finally, we will determine how AAGAB functions are impacted by disease-causing mutations. Successful completion of this proposed research will fill a major gap in our knowledge of membrane protein trafficking. This work will also serve as a paradigm for understanding the assembly of trafficking adaptors in general. Ultimately, these findings will facilitate the development of novel therapeutic strategies for human diseases caused by AAGAB mutations.