SAXS REVEALS THE MULTI-DOMAIN ASSEMBLY STATES OF HCK IN SOLUTION

SAXS 在解决方案中揭示了 HCK 的多域组装状态

• 批准号: 8168639
• 负责人: Sichun Yang
• 金额: $ 0.54万
• 依托单位: ILLINOIS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-01 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8168639
• 关键词: Binding; Catalytic Domain; Cereals; Complex; Computer Retrieval of Information on Scientific Projects Database; Data; Enzymes; Equilibrium; Funding; Grant; Institution; Link; Methods; Pathway interactions; Pattern; Peptide Signal Sequences; Population; Procedures; Relative (related person); Research; Research Personnel; Resources; Roentgen Rays; SH3 Domains; Signal Transduction; Solutions; Source; United States National Institutes of Health; Yang; base; cellular transduction; novel; response; simulation; src-Family Kinases

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The Src tyrosine kinases are large multi-domain enzymes [SH3-SH2-catalytic domain] involved in cellular signaling. Their ability to alternate between catalytically active (high-regulated) and inactive (down-regulated) states in response to specific signals provides a central switching mechanism in cellular transduction pathways. The activity of Src kinases is controlled by the assembly of this multi-domain enzyme. We propose an approach combining small-angle X-ray solution scattering (SAXS) with coarse-grained simulations to characterize quantitatively the multi-domain assembly states of Hck in solution. First, a basis set comprising a small number (~10) of assembly state "classes" is generated by clustering the configurations obtained from extensive coarse-grained simulations of Hck. Second, the average theoretical SAXS profile for each class of assembly state in the basis set is calculated by using the coarse-grained Fast-SAXS method [Yang et al, Biophys. J. 96:4449 (2009)]. Finally, the relative population of the different classes of assembly states is determined by using a Bayesian-based Monte Carlo procedure seeking to minimize the difference between the theoretical scattering pattern and SAXS data. This novel integrated approach linking experimental SAXS data and simulations is able to resolve the states of assembly of multi-domain Hck in solution under various conditions. The analysis reveals a shift in the equilibrium population of the assembly states upon the binding of various signaling peptides binding to the SH2 or SH3 domains. This integrated approach provides a new way to investigate complex multi-domain assemblies in solution.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 Src 酪氨酸激酶是参与细胞信号传导的大型多结构域酶 [SH3-SH2 催化结构域]。它们响应特定信号而在催化活性（高调节）和非活性（下调）状态之间交替的能力提供了细胞转导途径中的中心切换机制。 Src 激酶的活性由这种多结构域酶的组装控制。我们提出了一种将小角度 X 射线溶液散射 (SAXS) 与粗粒度模拟相结合的方法，以定量表征溶液中 Hck 的多域组装状态。首先，通过对从 Hck 的广泛粗粒度模拟获得的配置进行聚类，生成包含少量（~10）组装状态“类”的基础集。其次，使用粗粒度 Fast-SAXS 方法计算基础集中每类组装状态的平均理论 SAXS 轮廓 [Yang 等人，Biophys. J.96:4449(2009)]。最后，通过使用基于贝叶斯的蒙特卡罗程序来确定不同类别组装状态的相对总体，力求最小化理论散射模式与 SAXS 数据之间的差异。这种连接实验 SAXS 数据和模拟的新颖集成方法能够解析解决方案中各种条件下多域 Hck 的组装状态。分析揭示了在各种信号肽与 SH2 或 SH3 结构域结合后，组装状态的平衡群体发生了变化。这种集成方法提供了一种研究解决方案中复杂的多域程序集的新方法。