Mapping the Dynamic Architecture of the Human Mediator Complex

描述（由申请人提供）：人类细胞利用大蛋白复合物来调节正常功能所必需的基本过程。这些复合物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以介导其生物功能。也许这方面最引人注目的例子是人类介体复合物，其结构状态在结合不同的DNA结合转录因子（包括p53肿瘤抑制因子）后发生改变。值得注意的是，结构变化发生在整个1.2 MDa，26亚基复合物，表明大规模重组的亚基-亚基架构。更重要的是，p53诱导的结构转变改变了介体复合物的功能：p53结合的介体激活转录，而无激活子的介体（采用不同的结构状态）则不激活。p53诱导的结构转变和p53靶基因表达的激活之间的相关性表明，通过靶向传播结构转变所需的关键蛋白质界面来控制p53活性（及其在p53功能获得性癌细胞中的致癌潜力）的可能性。目前，由于缺乏适当的技术，不可能研究大型复合体（如Mediator）的动态架构。为了解决这一关键的未满足的需求，我们开发了一种集成的化学控制交联质谱（CXMS）和计算方法，可以映射大型蛋白质复合物的结构。我们的战略围绕着一种创新的、双功能的、MS不稳定的交联试剂（称为BDRG），以及高质量准确度的MS分析。本提案的目的是评估使用BDRG CXMS映射人类中介复合体的动态架构的可行性。在目的中，我们将应用BDRG CXMS方法来映射人类介体复合物在两种不同结构和功能状态下的亚基结构。具体来说，我们将研究无激活剂（无活性）的介体复合物和与p53激活结构域结合的介体复合物（有活性）。这项研究是重要的，因为如果成功的话，交联数据将揭示关键的“控制点”，代表p53定向结构转变所需的介体亚基-亚基界面。使用现有的生物化学或生物物理方法不能容易地获得这些信息，并且将为控制癌细胞中的p53功能提供新的策略和靶点（未来的研究）。此外，该结果将首次阐明人类介体（一种全基因组转录调节因子）的亚基组织。最后，将BDRG CXMS技术成功应用于人类介体复合物将牢固地确立这种方法作为询问大型动态组装内的结构-功能关系的有效手段。这将为该技术更广泛地应用于其他大型癌症相关分子机器（如DNA修复复合物或剪接体）打开大门。

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.