Capturing structure and dynamics of transmembrane signaling proteins

项目摘要 为了感知环境，细胞依赖于膜嵌入受体。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 癌的到目前为止的证据支持一个模型，即通过RTK的信号通道是由结构变化启动的 在细胞外结构域，然后通过跨膜（TMD）和跨膜（JMD）进行， 结构域到胞质激酶结构域。受体通常以二聚体形式被激活。许多 RTK突变导致疾病，例如成纤维细胞TMD的~30%残基中的单点突变 生长因子受体3（FGFR 3）是致病性的，而原肌球蛋白受体激酶A的突变可导致 到癌症因此，理解FGFR 3和TrkA信号转导TMD和JMD的结构相互作用 对于基础生物学和未来可能针对这些途径的疗法的发展至关重要。 原子分辨的TMD+JMD二聚体结构是本项目的主要目标。的应用 传统的计算和晶体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有效的计算方法，指导和最大限度地利用 NMR、FRET和细胞内实验数据，并应用这些方法捕获FGFR 3和TrkA TMD， 野生型和突变的致病形式的TMD+JMD二聚体结构。在目标1中，我们将联合收割机 新型高移动的膜模拟模型，能够自发捕获候选TMD二聚体 结构，与一种新的最小偏置的方式，应用减少数量的计算约束的基础上， 实验性的距离测量所得TMD二聚体结构将通过比较 计算和实验测量的参数。这些结构将揭示突变在 RTK动力学在目标2中，我们将使用我们的计算-实验方法来确定 质膜结构域在RTK信号传导中起作用。突变的二聚体的解析结构将促进 了解受体激活的病理学和机制。我们新颖的计算方法 结合合作研究者和合作者在NMR、FRET、RTK信号传导和 膜相关的现象，独特的定位我们开发和应用这种方法。我们还将 为广泛使用的软件套件开发一个开源的、用户友好的工作流程插件， 科学界使用拟议的议定书。具体目标的完成将提高我们的 能够有效地获得结构信息的RTK，并将开辟新的研究途径，调查 健康和疾病中的跨膜信号传导机制导致新治疗方法的开发。

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.