MeCP2 Structure and Function

描述(申请人提供)：MeCP2(甲基CpG结合蛋白2)是一个53 kDa的核蛋白，可以抑制转录，调节RNA剪接，并调节染色质结构。MeCP2的突变与衰弱的神经疾病Rett综合征(RTT)有关。目前的观点认为，MeCP2是一种多功能核调节蛋白，在健康和疾病中具有重要作用。我的实验室研究MeCP2如何影响染色质结构和功能。在上一次授予期间，我们证明了当MeCP2与甲基化和未甲基化的染色质纤维结合时，既能指导局部纤维凝聚，又能指导整体纤维与纤维之间的相互作用。此外，对纯化的重组蛋白进行了全面的生化鉴定。令人惊讶的是，MeCP2是溶液中的单体，60%的本质无序，约35%的2页/转角，并具有随机卷曲的三级结构。此外，MeCP2被组织成六个胰酶抗性结构域，每个结构域似乎包含一个或多个内在紊乱的长片段。这些进展提出了许多与我的长期目标有关的新问题，即了解MeCP2的结构以及它如何与RTT中MeCP2的多功能和功能障碍联系在一起。为了实现这一目标，我提出了以下具体目标：(1)通过研究单个结构域的结构、内在无序的作用和特定的MeCP2 RTT突变体来剖析MeCP2在溶液中的三级结构；(2)通过鉴定和分析MeCP2中的多个dsDNA和染色质结合位点，以及确定MeCP2在DNA结合时发生的结构变化，来表征MeCP2与dsDNA和单核小体的相互作用机制；(3)通过确定依赖于MeCP2的染色质纤维缩合过程中涉及的分子事件序列，建立MeCP2在体外组装浓缩超分子核蛋白复合体的途径。拟议的研究将在蛋白质、核小体和染色质纤维水平上产生重要进展。所提出的研究在结构的大小和规模上具有创新性：MeCP2单体为53 kDa，MeCP2-核小体复合体超过300 kDa，MeCP2-染色质纤维超结构可达数百兆吨。然而，所有这些结构都将作为纯重组成分在体外进行研究。拟议中的实验是由许多特定的假设驱动的，并将是首批剖析结构复杂、与疾病相关、本质上无序的蛋白质之一。总之，这些研究将提供前所未有的与MeCP2结构直接相关的信息，将首次对MeCP2如何影响基因组结构和功能产生严格的理解，并将开始为理解MeCP2在RTT分子发病机制中的作用奠定结构基础。公共卫生相关性：MeCP2蛋白的突变是一种衰弱的神经疾病Rett综合征的原因，Rett综合征是最常见的遗传性精神发育迟滞综合征。拟议的实验将极大地增加我们对MeCP2的结构以及它作为染色质结构蛋白的功能的理解。这些研究将为了解MeCP2在RTT分子发病机制中的作用奠定生化基础。

DESCRIPTION (provided by applicant): MeCP2 (methyl CpG Binding Protein 2) is a 53 kDa nuclear protein that can repress transcription, regulate RNA splicing, and modulate chromatin architecture. Mutations in MeCP2 have been causally linked to the debilitating neurological disorder, Rett Syndrome (RTT). The current view is that MeCP2 is a multifunctional nuclear regulatory protein with important roles in health and disease. My laboratory studies how MeCP2 affects chromatin structure and function. During the last grant period we demonstrated that MeCP2 functions to direct both local fiber condensation and global fiber-fiber interactions when bound to both methylated and unmethylated chromatin fibers. In addition, comprehensive biochemical characterization of the purified recombinant protein was completed. Surprisingly, MeCP2 is a monomer in solution, is 60% intrinsically disordered and ~35% 2-sheet/turn, and has a random coil-like tertiary structure. Further, MeCP2 is organized into six trypsin resistant domains, each of which appears to contain one or more long segments of intrinsic disorder. These advances raise many new questions related to my long-term objective of understanding MeCP2 structure and how it is linked to MeCP2 multifunctionality and dysfunction in RTT. To address this objective, I propose to the following Specific Aims: (1) to dissect the tertiary structure of MeCP2 in solution by studying the structure of the individual domains, the role of intrinsic disorder, and specific MeCP2 RTT mutants, (2) to characterize the mechanism of MeCP2 interaction with dsDNA and mononucleosomes by identifying and analyzing the multiple dsDNA and chromatin binding sites in MeCP2 and determining the structural changes that occur in MeCP2 upon DNA binding, and (3) to establish the pathway through which MeCP2 assembles condensed supramolecular nucleoprotein complexes in vitro by determining the sequence of molecular events involved in MeCP2-dependent chromatin fiber condensation. The proposed studies will yield important advances at both the protein, nucleosome, and chromatin fiber levels. The proposed research is innovative in the magnitude and scale of the structures being studied; the MeCP2 monomer is 53 kDa, the MeCP2-nucleosome complex exceeds 300 kDa, and the MeCP2-chromatin fiber suprastructures can be several hundred megadaltons. Nevertheless, all of these structures will be studied in vitro as pure recombinant components. The proposed experiments are driven by numerous specific hypotheses, and will be among the first to dissect a structurally perplexing, disease-related, intrinsically disordered protein. Collectively, these studies will provide an unprecedented body of information that is directly relevant to MeCP2 structure, will yield the first rigorous understanding of how MeCP2 influences genome structure and function, and will begin to lay a structural foundation for understanding the role of MeCP2 in the molecular pathogenesis of RTT. PUBLIC HEALTH RELEVANCE: Mutations in the MeCP2 protein are causative of a debilitating neurological disorder, Rett Syndrome, which is the most common inherited mental retardation syndrome. The proposed experiments will greatly increase our understanding of the structure of MeCP2, and how it functions as a chromatin architectural protein. In doing so, these studies will lay a biochemical foundation for understanding the role of MeCP2 in the molecular pathogenesis of RTT.