Capturing structure and dynamics of transmembrane signaling proteins

资助奖项目摘要（R 01 GM 141298） 为了感知环境，细胞依赖于膜嵌入受体。受体酪氨酸 信号蛋白激酶（RTK）家族是一个庞大的、多样的、对人类神经系统和神经系统都非常重要的信号蛋白家族。 发育疾病和癌症。迄今为止的证据支持一种模型， 通过RTK是由胞外结构域的结构变化引发的，然后进行 通过跨膜（TMD）和跨膜（JMD）结构域到胞质激酶 域受体通常以二聚体形式被激活。许多RTK突变赋予 疾病，例如成纤维细胞生长的TMD的~30%残基中的单点突变 因子受体3（FGFR 3）是致病性的，而原肌球蛋白受体激酶A的突变可以 导致癌症。了解FGFR 3和TrkA信号转导TMD的结构相互作用 因此，JMD对于基础生物学和未来治疗的发展至关重要， 可以针对这些途径。原子分辨的TMD+JMD二聚体结构是主要的 这个项目的目标。传统的计算和晶体学方法的应用， 受到膜环境的流体性质的阻碍。我们的目标是开发新型高效的 指导和最大限度地利用NMR，FRET和细胞内实验的计算方法 数据并应用这些方法捕获FGFR 3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，我们将联合收割机 高度移动的膜模拟模型，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 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值和实验测量值来验证 参数这些结构将揭示突变在RTK动力学中的作用。在目标2中， 将使用我们的计算-实验方法来确定细胞膜的作用， 域在RTK信令中起作用。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。我们的新型计算 方法与合作研究者和合作者在NMR方面的扩展专业知识相结合， FRET，RTK信号和膜相关现象，使我们能够开发 并应用这种方法。我们还将开发一个开源的，用户友好的工作流程插件 一个广泛使用的软件套件，将允许有效地使用拟议的协议， 科学界。具体目标的完成将提高我们的能力， RTKs的结构信息，并将为调查机制开辟新的研究途径 跨膜信号在健康和疾病中的作用，导致新的治疗方法的发展。

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.