Department of Chemistry , Massachusetts Institute of Technology , 77 Massachusetts Avenue , Cambridge , Massachusetts 02139 , United States.
Lawrence Livermore National Laboratory , Livermore , California , United States.
Biochemistry. 2019 Jan 29;58(4):286-294. doi: 10.1021/acs.biochem.8b00089. Epub 2018 Apr 6.
The epidermal growth factor receptor (EGFR) is critical to normal cellular signaling pathways. Moreover, it has been implicated in a range of pathologies, including cancer. As a result, it is the primary target of many anticancer drugs. One limitation to the design and development of these drugs has been the lack of molecular-level information about the interactions and conformational dynamics of EGFR. To overcome this limitation, this work reports the construction and characterization of functional, fluorescently labeled, and full-length EGFR in model membrane nanolipoprotein particles (NLPs) for in vitro fluorescence studies. To demonstrate the utility of the system, we investigate ATP-EGFR interactions. We observe that ATP binds at the catalytic site providing a means to measure a range of distances between the catalytic site and the C-terminus via Förster resonance energy transfer (FRET). These ATP-based experiments suggest a range of conformations of the C-terminus that may be a function of the phosphorylation state for EGFR. This work is a proof-of-principle demonstration of single-molecule studies as a noncrystallographic assay for EGFR interactions in real-time and under near-physiological conditions. The diverse nature of EGFR interactions means that new tools at the molecular level have the potential to significantly enhance our understanding of receptor pathology and are of utmost importance for cancer-related drug discovery.
表皮生长因子受体 (EGFR) 对正常细胞信号通路至关重要。此外，它还与多种病理学有关，包括癌症。因此，它是许多抗癌药物的主要靶点。这些药物的设计和开发受到限制的一个原因是缺乏关于 EGFR 相互作用和构象动力学的分子水平信息。为了克服这一限制，本工作报告了在模型膜纳米脂蛋白颗粒 (NLP) 中构建和表征功能性、荧光标记和全长 EGFR 用于体外荧光研究。为了证明该系统的实用性，我们研究了 ATP-EGFR 相互作用。我们观察到 ATP 结合在催化位点上，提供了一种通过Förster 共振能量转移 (FRET) 测量催化位点和 C 末端之间一系列距离的方法。这些基于 ATP 的实验表明 C 末端可能存在一系列构象，这可能是 EGFR 磷酸化状态的函数。这项工作是一个原理证明，证明了单分子研究作为一种实时和近生理条件下的非晶体学测定方法，用于 EGFR 相互作用。EGFR 相互作用的多样性意味着分子水平的新工具有可能极大地增强我们对受体病理学的理解，对于癌症相关的药物发现至关重要。
