Solution structure and dynamics of polyubiquitin chains

描述(申请人提供)：这项建议是为了描述多泛素(PolyUb)链的溶液结构、动力学和相互作用，多泛素(PolyUb)链在调节一系列细胞过程中发挥信号分子的作用，从细胞周期的进展到转录激活、抗原处理和蛋白质的囊泡运输。底物与不同连接的PolyUb链的结合使目标蛋白在细胞中有不同的命运。特别是，Lys48连接的PolyUb在泛素-蛋白酶体蛋白分解途径中起到普遍信号的作用，泛素-蛋白酶体蛋白分解途径是短暂蛋白质周转的主要调节机制，影响各种重要的细胞事件。了解26S蛋白酶体和其他下游效应分子是如何识别PolyUb链的，对于我们理解调控机制是至关重要的。尽管关于多泛素化调控的细胞过程的信息越来越丰富，以及大量将多聚Ub信号与下游事件联系在一起的Ub结合蛋白的鉴定，但Ub介导的信号多样性的分子基础仍然不清楚。在了解PolyUb信号的特异性来源之前，不同的PolyUb链对不同结果的信号传递能力的潜在机制仍有待阐明。获得这样的信息是绝对必要的，以便发展对不同链如何能够作为特定信号的分子理解。这项拟议工作的目的是表征各种类型的PolyUb链的结构和识别特性，以便在分子水平上了解Ub作为多功能细胞信号的能力的结构基础。我们将集中于所谓的非正则链：通过Lys48或Lys63以外的赖氨酸连接(例如，Lys11)，具有异质连接的链(例如，Lys11，Lys48，Lys63，头尾相连)，以及由Ub和Ub样蛋白组成的异源链(Rub1/Nedd8)。我们还将分析与一个模型底物蛋白连接的规范的Lys48连接的PolyUb链，以检查连接对PolyUb标签和底物的影响。此外，我们将表征(聚)Ub与ubistatins的复合物，ubistatins是一种小分子抑制剂，可以阻断蛋白酶体对PolyUb信号的识别，以帮助设计下一代ubistatin衍生物。我们将使用现代核磁共振方法来确定这些链在溶液中的三维结构，并表征它们的结合性质和与受体的络合物。这将使用由自旋松弛和剩余偶极耦合测量得出的长程、取向约束的组合来执行，并辅之以来自NOE的距离信息和顺磁松弛增强以及顺磁标记引入的假接触位移。核磁共振数据将与小角X射线和中子散射(SAXS，SANS)数据相结合，以更好地表征聚Ub链的结构和构象系综。

DESCRIPTION (provided by applicant): This proposal is to characterize the solution structure, dynamics, and interactions of polyubiquitin (polyUb) chains, which function as signaling molecules in the regulation of a host of cellular processes, ranging from progression through the cell cycle, to transcriptional activation, antigen processing and vesicular trafficking of proteins. Conjugation of substrates to polyUb chains of different linkages commits the target protein to distinct fates in the cell. In particular, Lys48-linked polyUb acts as a universal signal in the ubiquitin-proteasome proteolytic pathway, the principal regulatory mechanism for the turnover of short-lived proteins that influences a variety of vital cellular events. Understanding how polyUb chains are recognized by the 26S proteasome and other downstream effector molecules is central to our understanding of the mechanisms of regulation. Despite an increasing wealth of information on the cellular processes regulated by polyubiquitination and the identification of numerous Ub-binding proteins that tie the polyUb signal to downstream events, the molecular basis of diversity in Ub-mediated signaling remains unclear. The mechanisms underlying the ability of different polyUb chains to signal for distinct outcomes remain to be elucidated before the origin of specificity in polyUb signaling is understood. Obtaining such information is absolutely necessary in order to develop a molecular understanding of how different chains are able to act as specific signals. The objective of the proposed work is to characterize the structure and recognition properties of various types of polyUb chains, in order to understand at a molecular level the structural basis for Ub's ability to serve as a versatile cellular signal. We will focus on the so-called non-canonical chains: linked via lysines other than Lys48 or Lys63 (e.g., Lys11), chains with heterogeneous linkages (e.g., Lys11, Lys48, Lys63, head-to-tail), both linear and branched, as well as heterologous chains composed of Ub and Ub-like proteins (Rub1/Nedd8). We will also analyze the canonical, Lys48-linked polyUb chain conjugated to a model substrate protein, in order to examine the effect of the conjugation on both the polyUb tag and the substrate. In addition, we will characterize (poly)Ub complexes with ubistatins, small molecule inhibitors that block polyUb signal recognition by the proteasome, in order to assist in designing the next generation of ubistatin derivatives. We will use modern NMR approaches to determine the three-dimensional structure of these chains in solution and characterize their binding properties and complexes with receptors. This will be performed using a combination of long-range, orientational constraints derived from spin-relaxation and residual dipolar coupling measurements, complemented with distance information from NOEs and paramagnetic relaxation enhancements and pseudocontact shifts introduced by paramagnetic labels. NMR data will be combined with small-angle X-ray and neutron scattering (SAXS, SANS) data, to better characterize the structure and the conformational ensemble of polyUb chains.