Capturing structure and dynamics of transmembrane signaling proteins

项目摘要 为了感知环境，细胞依赖于嵌入膜的受体。受体酪氨酸激酶(RTK) 信号蛋白家族是大的、多样的，并且对人类发育疾病和 癌症。到目前为止，证据支持一种模型，即通过RTK的信号通路是由结构变化启动的 在胞外区，然后通过跨膜(TMD)和近膜(JMD)进行 结构域到细胞质激活域。受体通常以二聚体的形式被激活。数不胜数 RTK突变与疾病有关，例如成纤维细胞TMD~30%的残留物中的单点突变 生长因子受体3(FGFR3)是致病的，而原肌球蛋白受体激酶A的突变可以导致 敬癌症。从而理解FGFR3和TrkA信号的结构相互作用 对于基础生物学和可能针对这些途径的疗法的未来发展至关重要。 原子分辨TMD+JMD二聚体结构是该项目的主要目标。应用程序 传统的计算和结晶学方法受到膜的流体性质的阻碍。 环境。我们的目标是开发新的高效的计算方法，以指导和最大限度地利用 核磁共振、FRET和细胞内实验数据，并应用这些方法捕获FGFR3和TrkA TMD和 TMD+JMD二聚体结构为野生型和突变致病形式。在目标1中，我们将结合我们的 一种新型的可自发捕获候选TMD二聚体的高流动性膜模拟模型 结构，以一种新的最小偏差的方式应用减少数量的计算约束基于 在实验距离测量上。得到的TMD二聚体结构将通过比较来验证 计算和实验测量的参数。这些结构将揭示突变在 RTK动态。在目标2中，我们将使用计算-实验方法来确定 膜旁结构域在RTK信号转导中发挥作用。突变的二聚体的分解结构将有助于 了解受体激活的病理和机制。我们的新计算方法 结合合作研究人员和合作者在核磁共振、FRET、RTK信令和 膜相关现象，为我们开发和应用这一方法学提供了独特的条件。我们还将 为广泛使用的软件套件开发一个开源、用户友好的工作流插件，以实现高效 科学界对拟议方案的使用。具体目标的完成将增加我们的 能够有效地获取RTK的结构信息，并将为调查开辟新的研究途径 跨膜信号在健康和疾病中的机制导致新治疗方法的发展。

Project Summary To sense the environment, cells rely on membrane-embedded receptors. The receptor tyrosine kinase (RTK) family of signaling proteins is large, diverse, and centrally important both to human development diseases and cancers. Evidence so far supports a model that signal passage through RTKs is initiated by a structural change in the extracellular domain and then conducted through the transmembrane (TMD) and juxtamembrane (JMD) domains to the cytoplasmic kinase domain. The receptors usually are activated in the dimer form. Numerous RTK mutations confer diseases, e.g. single point mutations in ~30% of residues of the TMD of the fibroblast growth factor receptor 3 (FGFR3) are pathogenic, while mutations of tropomyosin receptor kinase A can lead to cancers. Understanding the structural interactions of the FGFR3 and TrkA signaling TMD and JMD therefore is crucial for fundamental biology and for future development of therapies that may target these pathways. Atomistically resolved TMD+JMD dimer structures are the major objective of this project. Application of traditional computational and crystallographic methods is hindered by the fluid nature of the membrane environment. Our goal is to develop novel efficient computational methods that guide and maximally leverage NMR, FRET, and in-cell experimental data and apply these methods to capture the FGFR3 and TrkA TMD and TMD+JMD dimer structures for the wild type and mutated pathogenic forms. In Aim 1, we will combine our novel highly mobile membrane mimetic model, capable of spontaneously capturing candidate TMD dimer structures, with a novel minimally biased way of applying a reduced number of computational restraints based on experimental distance measurements. The resulting TMD dimer structures will be validated by comparing computed and experimentally measured parameters. These structures will reveal the role mutations play in RTK dynamics. In Aim 2, we will use our computational-experimental approach to determine the role that juxtamembrane domains play in RTK signaling. The resolved structures of the mutated dimers will facilitate understanding of the pathology and mechanisms of receptor activation. Our novel computational approaches combined with extended expertise of co-investigators and collaborators in NMR, FRET, RTK signaling, and membrane-associated phenomena, uniquely position us to develop and apply this methodology. We will also develop an open-source, user friendly workflow plugin for a widely-used software suite that will allow efficient use of the proposed protocols by the scientific community. Completion of the specific aims will increase our ability to efficiently gain structural information on RTKs and will open new research avenues for investigating mechanisms of transmembrane signaling in health and disease leading to development of new treatments.