Structural and Functional Studies of Ubiquitin Binding Domains

泛素结合域的结构和功能研究 泛素化对蛋白质的共价修饰是蛋白质降解和质量控制、内吞作用、囊泡运输、细胞周期调控、应激反应、DNA修复、生长因子信号转导、转录、基因沉默等生物学领域的主要调控机制。一类特定的泛素结合域介导了蛋白质泛素化的大部分作用。该组织的已知成员迅速扩大，现在至少包括16个域。确定了许多与泛素形成的络合物的结构，揭示了它们与泛素上多个表面的相互作用。两个UBA结构域在理解多泛素特异性方面取得了进展，它们的结构已被表征为与Lys48连接的二泛素复合体。几个泛素结合域，包括UIM、CUE、VHS和A20锌F，促进自体素化，调节含有它们的蛋白质的活性。其中至少一个结构域，A20锌F，通过招募泛素：泛素结合酶硫酯加合物作为泛素连接酶，其过程依赖于A20锌F的泛素结合活性。这些结构域的单结合蛋白相互作用的亲和力跨越广泛的范围，但最常见的是弱的，Kd&gt；100 mm。通过泛素聚合、修饰多样性、泛素化蛋白和结合结构域蛋白的寡聚化、串联结合结构域、具有多个泛素结合位点的结合域以及泛素结合和通过其他结构域与磷脂和小G蛋白的结合之间的协同作用，单个结构域与单泛素之间的弱相互作用被调节为生理上相关的高亲和力相互作用。这个项目的长期目标是：1)确定参与分子识别的泛素及其结合域的结构特征；2)将结构特征与这些蛋白质在运输过程中的功能特性联系起来；3)了解单个结合域与泛素部分之间的低亲和力相互作用并参与生理识别事件的机制。 ESCRT领域仍然存在的主要问题之一是泛素化的货物是如何从ESCRT-0包被区域转移到腔内芽中的，以及去泛素化酶在这个过程中发挥了多大的积极作用。另一个关键目标是了解ESCRT上游受体泛素化的特异性。实验室目前的努力集中在这些目标上。

Structural and Functional Studies of Ubiquitin Binding Domains The covalent modification of proteins by ubiquitination is a major regulatory mechanism of protein degradation and quality control, endocytosis, vesicular trafficking, cell-cycle control, stress response, DNA repair, growth factor signaling, transcription, gene silencing, and other areas of biology. A class of specific ubiquitin binding domains mediates most of the effects of protein ubiquitination. The known membership of this group has expanded rapidly and now includes at least sixteen domains. The structures of many of the complexes with monoubiquitin have been determined, revealing interactions with multiple surfaces on ubiquitin. Inroads into understanding polyubiquitin specificity have been made for two UBA domains, whose structures have been characterized in complex with Lys48-linked diubiquitin. Several ubiquitin binding domains, including the UIM, CUE, VHS, and A20 ZnF, promote autoubiquitination, which regulates the activity of proteins that contain them. At least one of these domains, the A20 ZnF, acts as a ubiquitin ligase by recruiting a ubiquitin:ubiquitin conjugating enzyme thiolester adduct in a process that depends on the ubiquitin-binding activity of the A20 ZnF. The affinities of the monoubiquitin binding interactions of these domains span a wide range, but are most commonly weak, with Kd > 100 mM. The weak interactions between individual domains and monoubiquitin are leveraged into physiologically relevant high affinity interactions via several mechanisms: ubiquitin polymerization, modification multiplicity, oligomerization of ubiquitinated proteins and binding domain proteins, tandem binding domains, binding domains with multiple ubiquitin binding sites, and cooperativity between ubiquitin binding and binding through other domains to phospholipids and small G-proteins. The long terms goals of this project are to 1) determine the structural features of ubiquitin and its binding domains that are involved in molecular recognition; 2) correlate structural features with functional properties of these proteins in trafficking; and 3) understand the mechanisms whereby low-affinity interactions between individual binding domains and ubiquitin moieties and leveraged into physiological recognition events. One of the main questions remaining in the ESCRT field is how ubiquitinated cargo is handed off from ESCRT-0 coated domain into intralumenal buds, and to what extent deubiquitinating enzymes play an active role in this process. Another key goal is to understand the specificity of receptor ubiquitination upstream of the ESCRTs. Current efforts in the lab focus on these goals.