Solution structure and dynamics of polyubiquitin chains

描述（由申请人提供）：该提案是表征多泛素（polybiquitin, polyb）链的溶液结构、动力学和相互作用，多泛素（polybiquitin, polyb）链作为信号分子在一系列细胞过程的调控中起作用，从细胞周期的进展到转录激活、抗原加工和蛋白质的囊泡运输。底物与不同键的聚脲链的结合使靶蛋白在细胞中有不同的命运。特别是，lys48连接的polyb在泛素-蛋白酶体蛋白水解途径中作为一个通用信号，泛素-蛋白酶体蛋白水解途径是影响各种重要细胞事件的短寿命蛋白周转的主要调节机制。了解polyb链如何被26S蛋白酶体和其他下游效应分子识别是我们理解调控机制的核心。尽管关于多泛素化调控的细胞过程的信息越来越丰富，并且发现了许多将多泛素化信号与下游事件联系在一起的ub结合蛋白，但ub介导的信号传导多样性的分子基础仍不清楚。在了解聚脲b信号的特异性起源之前，不同聚脲b链对不同结果发出信号的能力的机制仍有待阐明。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。这项工作的目的是表征各种类型的聚脲b链的结构和识别特性，以便在分子水平上了解聚脲b作为多功能细胞信号的能力的结构基础。我们将重点关注所谓的非规范链：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，从头到尾），线性和分支链，以及由Ub和Ub样蛋白组成的异源链（Rub1/Nedd8）。我们还将分析与模型底物蛋白结合的典型的lys48连接的polyb链，以检验这种结合对polyb标签和底物的影响。此外，我们将表征（多）Ub配合物与ubi抑素，小分子抑制剂，阻断polyUb信号识别的蛋白酶体，以协助设计下一代的ubi抑素衍生物。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，并表征它们的结合特性和与受体的复合物。这将使用来自自旋弛豫和剩余偶极耦合测量的远程定向约束，以及noe的距离信息和顺磁标签引入的顺磁弛豫增强和伪接触位移的组合来完成。核磁共振数据将与小角度x射线和中子散射（SAXS， SANS）数据相结合，以更好地表征聚脲链的结构和构象系。

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.