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

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 Src酪氨酸激酶是参与细胞信号转导的大分子多域酶[SH3-SH2-催化域]。它们能够在催化活性(高调节)和非活性(低调节)状态之间切换，以响应特定的信号，为细胞转导途径提供了一个中心切换机制。这种多结构域酶的组装控制着Src激酶的活性。我们提出了一种结合小角X射线溶液散射(SAXS)和粗粒度模拟的方法来定量表征HCK在溶液中的多域组装状态。首先，通过对从大量的HCK粗粒度模拟获得的构型进行聚类来生成包含少量(~10)个组装状态类的基集。其次，使用粗粒度的Fast-SAXS方法计算了基组中每类组装状态的平均理论SAXS分布[Yang等人，生物物理]。J.96：4449(2009)]。最后，使用基于贝叶斯的蒙特卡罗方法来确定不同类别组装态的相对总体，以求最小化理论散射模式和SAXS数据之间的差异。这种将实验SAXS数据和模拟相结合的新方法能够解析不同条件下多结构域HCK在溶液中的组装状态。分析表明，当结合到SH2或SH3结构域的各种信号肽结合时，组装状态的平衡布居发生了变化。这种集成的方法为研究解决方案中复杂的多域组装提供了一种新的方法。

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.