The role of dynamics in E3 ligase function

动力学在 E3 连接酶功能中的作用

• 批准号: 1817774
• 负责人: Elizabeth Komives
• 金额: $ 89.99万
• 依托单位: University of California-San Diego
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1817774&HistoricalAwards=false
• 关键词: role; dynamics; E3; ligase; function

The project will explore the motions of a large cellular machine that labels proteins for destruction. The label is a small protein called ubiquitin (Ub), which is attached to the condemned protein to target it to the proteasome where it is degraded. The cellular machines (E3 ligases) that are responsible for tagging proteins with Ub are very large, nearly 300 kilodaltons in size, and they contain eight to ten protein subunits that all work together to put the Ub tag on the condemned protein. These machines function in all higher life forms, so it is essential to understand how they work. Structural and dynamic experiments will explore the mechanism of how these machines work in vitro using an assembled, purified, active machine. Two graduate students will work full-time on the project, and they will learn both protein biochemistry and biophysical experimental methods. High school students will work in the lab during the summer months. The high school students, some of whom are from disadvantaged backgrounds, will gain knowledge of cutting edge mass spectrometry methods used to understand protein dynamics. The project focuses on ASB-CUL5-RING E3s (ASB-CRL), a large class of E3 ligases. They consist of one of 18 Ankyrin and SOCS box proteins (ASBs) as substrate receptors, elongins B and C (ELOB/C), cullin 5 (CUL5), and the E2-binding protein (RBX2). Each ASB binds 10-15 substrates. No structures are available for ASB-CRLs. The project aims to understand the structure, dynamics, and function of the ASB9-CRL. Thus far, full-length ASB9-CRL has been purified following strategic co-expression in E. coli. The purified complex is active in vitro and ubiquitylation of two substrates, creatine kinase (CK) and histone octamers has been demonstrated. Structural models of CRLs predict a distance of over 60 angstroms between Ub and the substrate, and it remains unknown how Ub is transferred across such a long distance. Additionally, neddylation has been shown to regulate the ubiquitylation activity of CRLs. In the first part of the project, ubiquitylation assays will be used to quantitatively compare the rate of Ub-transfer to CK and to histone octamers by the neddylated and un-neddylated ligase. The Ub attachment sites and extent of poly-ubiquitylation will be measured. In the second part of the project, hydrogen/deuterium exchange mass spectrometry (HDXMS) will be used to probe the internal and global conformational dynamics that drive the long distance conformational changes required for Ub transfer by the ASB9-CRL. In the third part of the project, cryo-electron microscopy (cryoEM) will be used to obtain high-resolution structures of the full ASB-CRL complex and important sub-complexes. The populations and rates of interconversion of the conformational states will be investigated by single molecule FRET (smFRET). Together, the results will reveal the internal dynamics and global dynamics of the ASB9-CRL that are required to achieve processive ubiquitylation of protein substrates.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

该项目将探索一个大型细胞机器的运动，该机器标记蛋白质以进行破坏。标签是一种称为泛素（Ub）的小蛋白质，它附着在被谴责的蛋白质上，使其靶向蛋白酶体，在那里被降解。负责用Ub标记蛋白质的细胞机器（E3连接酶）非常大，大小接近300千道尔顿，它们包含8到10个蛋白质亚基，这些亚基一起工作，将Ub标签放在被谴责的蛋白质上。这些机器在所有更高的生命形式中发挥作用，因此了解它们是如何工作的至关重要。结构和动态实验将探索这些机器如何在体外使用组装，纯化，活性机器工作的机制。两名研究生将全职从事该项目，他们将学习蛋白质生物化学和生物物理实验方法。高中生将在夏季的几个月里在实验室工作。高中生，其中一些人来自弱势背景，将获得用于了解蛋白质动力学的尖端质谱方法的知识。 该项目的重点是ASB-CUL 5-RING E3（ASB-CRL），这是一大类E3连接酶。它们由作为底物受体的18种锚蛋白和SOCS盒蛋白（ASB）之一、延伸蛋白B和C（EIB B/C）、cullin 5（CUL 5）和E2结合蛋白（RBX 2）组成。每个ASB结合10-15个底物。没有可用于ASB-CRL的结构。该项目旨在了解ASB 9-CRL的结构，动态和功能。到目前为止，全长ASB 9-CRL已经在E.杆菌纯化的复合物在体外是有活性的，并且已经证明了两种底物，肌酸激酶（CK）和组蛋白八聚体的泛素化。CRL的结构模型预测Ub和基底之间的距离超过60埃，并且仍然不知道Ub如何跨越如此长的距离转移。此外，neddylation已被证明可以调节CRL的泛素化活性。在该项目的第一部分中，泛素化测定将被用来定量比较泛素化和非泛素化连接酶转移到CK和组蛋白八聚体的速率。将测量Ub附着位点和多聚泛素化程度。在该项目的第二部分，氢/氘交换质谱（HDXMS）将用于探测内部和全局构象动力学，这些动力学驱动ASB 9-CRL进行Ub转移所需的长距离构象变化。在该项目的第三部分，冷冻电子显微镜（cryoEM）将用于获得完整ASB-CRL复合物和重要子复合物的高分辨率结构。将通过单分子FRET（smFRET）研究构象状态的相互转换的种群和速率。总之，研究结果将揭示ASB 9-CRL的内部动力学和全局动力学，这些动力学和全局动力学是实现蛋白质底物进行性泛素化所必需的。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。