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的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH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 中心，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[SH 3-SH 2-催化结构域]。它们响应特定信号在催化活性（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。我们提出了一种结合小角X射线溶液散射（SAXS）与粗粒度模拟定量表征Hck在溶液中的多域组装状态的方法。首先，通过对从Hck的广泛粗粒度模拟获得的配置进行聚类，生成包括少量（~10）组装状态“类”的基集。第二，通过使用粗粒度Fast-SAXS方法计算基组中每类组装状态的平均理论SAXS曲线[Yang等人，Biophys. J. 96：4449（2009）]。最后，不同类别的组装状态的相对人口是通过使用贝叶斯为基础的蒙特卡罗程序寻求最小化理论散射模式和SAXS数据之间的差异。这种新的集成方法连接实验SAXS数据和模拟是能够解决在各种条件下的解决方案中的多域Hck的组装状态。该分析揭示了在组装状态的平衡群体的各种信号肽结合到SH 2或SH 3结构域的结合后的转变。这种集成的方法提供了一种新的方法来研究复杂的多域组件的解决方案。

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.