Mapping the Dynamic Architecture of the Human Mediator Complex

描述(申请人提供)：人类细胞利用大的蛋白质复合体来调节对正常功能至关重要的基本过程。这些复合体的一个共同特征是它们的动态性质：它们必须经历结构转变才能调节其生物学功能。也许最明显的例子是人类介体复合体，它的结构状态在与不同的DNA结合的转录因子结合时发生改变，包括p53肿瘤抑制因子。值得注意的是，结构变化发生在1.2MDA26亚基复合体中，暗示着亚基-亚单位结构的大规模重组。更重要的是，P53诱导的结构变化改变了介体复合体的功能：P53结合的介体激活转录，而无激活剂的介体(采用不同的结构状态)不能。P53诱导的结构改变和P53靶基因表达激活之间的相关性表明，有可能通过靶向传播结构改变所需的关键蛋白质接口来控制P53的活性(及其在P53功能获得癌细胞中的致癌潜力)。目前，由于缺乏适当的技术，无法研究像Mediator这样的大型复合体的动态体系结构。为了解决这一关键的未得到满足的需求，我们开发了一种集成的化学控制交联质谱(CXMS)和计算方法，能够绘制大型蛋白质复合体的结构图。我们的战略以一种名为BDRG的创新、双功能、MS不稳定的交联剂为核心，结合高质量精度的MS分析。这项建议的目标是评估使用BDRG CXMS绘制人类调解人复合体的动态架构的可行性。在这个目的中，我们将应用BDRG CXMS方法来映射两种不同结构和功能状态下的人类介体复合体的亚单位结构。具体地说，我们将研究无激活剂(非活性)的介体复合体和与P53(活性)激活域结合的介体复合体。这项研究意义重大，因为如果成功，交联数据将揭示关键的“控制点”，这些控制点代表了P53导向的结构转变所需的介体亚单位-亚单位接口。这些信息不能用现有的生化或生物物理方法轻易获得，并将为控制癌细胞中的P53功能提供新的策略和目标(未来的研究)。此外，这些结果将首次阐明人类介体的亚单位组织，这是一种全基因组的转录调节因子。最后，将BDRG CXMS技术成功地应用到人工中介复合体中，将坚定地将该方法确立为询问大型动态装配中结构-功能关系的有效手段。这将为更广泛地将该技术应用于其他与癌症相关的大型分子机器打开大门，例如DNA修复复合体或剪接体。

DESCRIPTION (provided by applicant): Human cells utilize large protein complexes to regulate basic processes that are essential for normal function. A shared feature of these complexes is their dynamic nature: they must undergo structural transitions to mediate their biological functions. Perhaps the most striking example of this is the human Mediator complex, whose structural state is altered upon binding distinct DNA-binding transcription factors, including the p53 tumor suppressor. Remarkably, the structural shifts occur throughout the 1.2 MDa, 26-subunit complex, suggesting a massive re-organization of subunit-subunit architecture. More important, the p53-induced structural shift alters the function of the Mediator complex: p53-bound Mediator activates transcription, whereas activator-free Mediator (which adopts a distinct structural state) does not. The correlation between the p53-induced structural shift and activation of p53 target gene expression suggests the possibility of controlling p53 activity (and its oncogenic potential in p53 gain-of-function cancer cells) by targeting key protein interfaces that are required to propagate the structural shift. Currently, it is not possible to study the dynamic architecture of large complexes such as Mediator due to a lack of appropriate technology. To address this critical unmet need, we developed an integrated chemical control crosslinking-mass spectrometric (CXMS) and computational approach that enables mapping the architecture of large protein complexes. Our strategy centers around an innovative, bi-functional, MS-labile crosslinking reagent called BDRG, coupled with high mass accuracy MS analysis. The objective of this proposal is to assess the feasibility of using BDRG CXMS to map the dynamic architecture of the human Mediator complex. In the Aim we will apply the BDRG CXMS approach to map the subunit architecture of the human Mediator complex in two distinct structural and functional states. Specifically, we will examine activator-free (inactive) Mediator complexes and Mediator complexes bound to the activation domain of the p53 (active). The research is significant because, if successful, the crosslinking data will reveal key "control points" that represent Mediator subunit-subunit interfaces required for p53-directed structural shifts. Such information could not be readily attained using existing biochemical or biophysical approaches, and would provide new strategies and targets for controlling p53 function in cancer cells (future studies). Furthermore, the results would for the first time elucidate the subunit organization of human Mediator, a genome-wide regulator of transcription. Finally, successful application of the BDRG CXMS technology to the human Mediator complex would firmly establish this approach as an effective means to interrogate structure- function relationships within large, dynamic assemblies. This would open the door for more general application of the technology to other large, cancer-relevant molecular machines, such as DNA-repair complexes or the spliceosome.