STRUCTURAL BIOLOGY OF HUMAN UBIQUITIN E3 LIGASES AND INTERACTING FACTORS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 泛素化调节多种过程，包括DNA修复、信号转导、细胞周期进程、内吞作用和蛋白质降解等，并且与疾病有关。泛素化系统的底物特异性由E3蛋白连接酶进行，其属于两个主要家族：RING E3连接酶和HECT E3连接酶。HECT E3连接酶各自含有约350个氨基酸的C-末端催化结构域，其具有与泛素形成硫醇中间体的保守活性位点半胱氨酸，将其从E2连接酶转移至蛋白质底物。HECT E3的高度可变的N-末端区域与特异性蛋白质底物相互作用，而催化结构域与其同源E2相互作用并进行催化反应。已经在HECT结构域蛋白的N-末端观察到多种结构域（WW结构域、C2结构域、犰狳样重复序列、BH 3、乌巴等），并且这些结构域已经用于进一步分类这些蛋白。与HECT E3连接酶不同，RING E3连接酶不直接进行泛素向底物的转移，而是将活化的E2和其底物结合在一起，从而允许催化发生。 我们建议，以确定高分辨率的结构，人类E3连接酶及其复合物与同源E2，底物和调节因子，以进一步阐明其催化机制，特异性和调节。作为结构基因组学联盟的一部分，我们有能力使用高通量技术来表达和纯化蛋白质，结晶和表征这些晶体。然而，我们需要在APS可用的明亮的X射线，以收集弱衍射晶体的数据和收集重原子衍生物的异常数据。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Ubiquitylation regulates a variety of processes, including DNA repair, signal transduction, cell cycle progression, endocytosis and protein degradation, amongst others, and has been implicated in disease. The substrate specificity of the ubiquitylation system is carried out by the E3 protein ligases, which belong to two major families: the RING E3 ligases and the HECT E3 ligases. The HECT E3 ligases each contain an approximately 350 amino acid C-terminal catalytic domain with a conserved active site cysteine that forms a thiol-intermediate with ubiquitin, transferring it from an E2 ligase to the protein substrate. The highly variable N-terminal regions of the HECT E3s interact with specific protein substrates while the catalytic domain interacts with its cognate E2 and carries out the catalytic reaction. A variety of domains (WW domain, C2 domain, armadillo like-repeats, BH3, UBA, amongst others) have been observed in the N-terminus of the HECT domain proteins and these have been used to further classify these proteins. Unlike the HECT E3 ligases, the RING E3 ligases do not directly carry out the transfer of ubiquitin to substrate, but instead bring together the activated E2 and its substrate thus allowing catalysis to occur. We propose to determine the high-resolution structures of human E3 ligases and their complexes with cognate E2s, substrates and regulating factors in order to further elucidate their catalytic mechanisms, specificity and regulation. As part of the Structural Genomics Consortium, we have the capability to use high-throughput techniques to express and purify proteins, crystallize and characterize these crystals. However we require the brilliant X-rays available at the APS in order to collect data from weakly diffracting crystals and to collect anomalous data from heavy-atom derivatives.