MeCP2 Structure and Function

描述（由申请人提供）：MeCP 2（甲基CpG结合蛋白2）是一种53 kDa的核蛋白，可抑制转录、调节RNA剪接和调节染色质结构。MeCP 2的突变与使人衰弱的神经系统疾病Rett综合征（RTT）有因果关系。目前的观点是，MeCP 2是一种多功能的核调节蛋白，在健康和疾病中具有重要作用。我的实验室研究MeCP 2如何影响染色质结构和功能。在最后一个补助期间，我们证明了MeCP 2的功能，以指导当地的纤维凝聚和全球的纤维-纤维的相互作用时，结合到甲基化和未甲基化的染色质纤维。此外，对纯化的重组蛋白进行了全面的生化表征。令人惊讶的是，MeCP 2是溶液中的单体，是60%的固有无序和~35%的2-折叠/转角，并且具有无规螺旋状三级结构。此外，MeCP 2被组织成六个胰蛋白酶抗性结构域，每个结构域似乎含有一个或多个长的内在紊乱片段。这些进展提出了许多新的问题，这些问题与我的长期目标有关，即理解MeCP 2结构以及它如何与RTT中的MeCP 2多功能性和功能障碍联系在一起。为了实现这一目标，我提出以下具体目标：（1）通过研究各个结构域的结构、内在紊乱的作用和特定的MeCP 2 RTT突变体来剖析MeCP 2在溶液中的三级结构，（二）通过鉴定和分析MeCP 2中的多个dsDNA和染色质结合位点，并确定DNA结合后MeCP 2中发生的结构变化，以及（3）通过确定MeCP 2依赖性染色质纤维凝聚中涉及的分子事件序列，建立MeCP 2在体外组装凝聚的超分子核蛋白复合物的途径。这些研究将在蛋白质、核小体和染色质纤维水平上取得重要进展。拟议的研究是创新的幅度和规模的结构正在研究; MeCP 2单体是53 kDa，MeCP 2-核小体复合物超过300 kDa，和MeCP 2-染色质纤维超结构可以是几百兆道尔顿。尽管如此，所有这些结构将在体外作为纯重组组分进行研究。拟议的实验是由许多特定的假设驱动的，并且将是第一个解剖结构复杂，疾病相关，内在无序蛋白质的实验。总的来说，这些研究将提供与MeCP 2结构直接相关的前所未有的信息体，将产生对MeCP 2如何影响基因组结构和功能的第一个严格理解，并将开始为理解MeCP 2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：MeCP 2蛋白的突变是导致衰弱性神经系统疾病Rett综合征的原因，Rett综合征是最常见的遗传性智力低下综合征。拟议的实验将大大增加我们对MeCP 2结构的理解，以及它如何作为染色质结构蛋白发挥作用。这些研究将为了解MeCP 2在RTT分子发病机制中的作用奠定生物化学基础。

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.