The role of dynamics in E3 ligase function

该项目将探索一个大型细胞机器的运动，该机器可以标记要破坏的蛋白质。这种标签是一种被称为泛素（Ub）的小蛋白质，它附着在被谴责的蛋白质上，将其靶向到蛋白酶体上，在那里它被降解。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300千顿，它们包含8到10个蛋白质亚基，它们共同作用将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运作，所以了解它们是如何工作的是必要的。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。两名研究生将全职参与该项目，他们将学习蛋白质生物化学和生物物理实验方法。高中学生将在夏季的几个月里在实验室工作。这些高中学生，其中一些来自贫困家庭，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-CUL5-RING E3s (ASB-CRL)，这是一大类E3连接。它们由18种锚蛋白和SOCS盒蛋白（ASBs）中的一种作为底物受体、长链蛋白B和C （ELOB/C）、cullin 5 （CUL5）和e2结合蛋白（RBX2）组成。每个ASB结合10-15个底物。asb - crl没有可用的结构。该项目旨在了解ASB9-CRL的结构、动态和功能。到目前为止，全长ASB9-CRL已经在大肠杆菌中进行了战略性共表达纯化。纯化的复合物在体外具有活性，并且证实了两种底物，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和衬底之间的距离超过60埃，但Ub如何在这么长的距离上转移仍然未知。此外，类化修饰已被证明可调节crl的泛素化活性。在项目的第一部分中，将使用泛素化分析来定量比较泛素化连接酶和非泛素化连接酶将ub转移到CK和组蛋白八聚体的速率。Ub的附着位点和多泛素化程度将被测量。在该项目的第二部分，氢/氘交换质谱（HDXMS）将用于探测驱动ASB9-CRL Ub转移所需的长距离构象变化的内部和全局构象动力学。在项目的第三部分，低温电子显微镜（cryoEM）将用于获得完整的ASB-CRL配合物和重要亚配合物的高分辨率结构。用单分子FRET （smFRET）研究构象态的居群和相互转换速率。总之，这些结果将揭示ASB9-CRL的内部动力学和全局动力学，这是实现蛋白质底物的过程泛素化所必需的。该奖项反映了美国国家科学基金会的法定使命，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。

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.