Capturing structure and dynamics of transmembrane signaling proteins

获资助奖项项目摘要(R01GM141298) 为了感知环境，细胞依赖于嵌入膜的受体。受体酪氨酸 蛋白激酶(RTK)家族是一个庞大、多样的信号蛋白家族，对人类和人类都具有重要意义 发展中的疾病和癌症。到目前为止，证据支持一种模型，即信号通道 通过RTKs由胞外区的结构变化启动，然后进行 通过跨膜结构域(TMD)和膜旁结构域(JMD)连接到细胞质激酶 域。受体通常以二聚体的形式被激活。大量RTK突变带来 疾病，例如成纤维细胞生长过程中~30%的TMD残留物的单点突变 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 会导致癌症。了解FGFR3和TrkA信号TMD的结构相互作用 因此，JMD对基础生物学和未来治疗方法的发展至关重要 可能以这些路径为目标。原子分辨的TMD+JMD二聚体结构是主要的 这个项目的目标。传统计算和结晶学方法的应用是 被膜环境的流动性质所阻碍。我们的目标是开发新的高效 指导和最大限度地利用核磁共振、FRET和细胞内实验的计算方法 数据并应用这些方法捕获FGFR3和TrkA TMD和TMD+JMD二聚体 野生型和突变致病形式的结构。在目标1中，我们将结合我们的小说 高流动性膜模拟模型，能够自发捕获候选TMD 二聚体结构，具有一种新的最小偏差的方式来应用减少的数量 基于实验距离测量的计算限制。由此产生的TMD 二聚体结构将通过比较计算和实验测量来验证 参数。这些结构将揭示突变在RTK动力学中所起的作用。在目标2中，我们 将使用我们的计算-实验方法来确定靠近膜的角色 域在RTK信令中发挥作用。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。我们的新型计算 方法结合了核磁共振领域合作研究人员和合作者的广泛专业知识， FRET、RTK信号和膜相关现象，为我们的发展提供了独特的定位 并应用这一方法。我们还将开发一个开源的、用户友好的工作流插件 对于广泛使用的软件套件，该软件套件将允许 科学界。具体目标的完成将提高我们有效获得 关于RTK的结构信息，并将为研究机制开辟新的研究途径 跨膜信号在健康和疾病中的作用，导致新治疗方法的发展。

Project Summary of the Funded Award (R01GM141298) 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.