Structural and Functional Studies of Ubiquitin Binding Domains

现在至少有16个已知的泛素结合结构域。已经确定了许多与单泛素的复合物的结构，揭示了泛素与多个表面的相互作用。已经对两个乌巴结构域的多聚泛素特异性的理解取得了进展，其结构的特征在于与Lys 48连接的双泛素复合。几个泛素结合结构域，包括UIM、CUE和A20 ZnF，促进autoubiquitination，其调节含有它们的蛋白质的活性。这些结构域中的至少一个，A20 ZnF，通过在依赖于A20 ZnF的泛素结合活性的过程中募集泛素：泛素缀合酶硫酯加合物而充当泛素连接酶。这些结构域的monoubiquitin结合相互作用的亲和力范围很广，但最常见的是弱的，Kd > 100 μ M。单个结构域和单泛素之间的弱相互作用通过几种机制被利用成生理学相关的高亲和力相互作用：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结构域，以及泛素结合和通过其他结构域与磷脂和小G蛋白结合之间的协同性。 酵母Vps 27 Hse 1复合物和同源哺乳动物HrsSTAM复合物将泛素化跨膜蛋白递送到ESCRT内体分选途径。 Vps 27是一种多模块蛋白，包含VHS、FYVE、UIM结构域、核心区和富含非结构化基序的C-末端。Hse 1包含VHS、UIM和SH 3结构域、核心区域和非结构化区域。这些蛋白质是含有多个低亲和力泛素结合结构域的蛋白质的范例。这些结构域的结构和相互作用的特点是在隔离。Vps 27 FYVE结构域，这是以前在我们的实验室结晶，是负责结合中等亲和力的内体脂质磷脂酰肌醇3-磷酸。单个UIM结构域以非常低的亲和力结合泛素。Vps 27 Hse 1复合物直接结合到泛素化的跨膜蛋白，并招募泛素连接酶和去泛素化酶。我们已经以3.0埃的分辨率解决了负责Vps 27 Hse 1复合物组装的核心的晶体结构。该结构由两个相互缠绕的GAT结构域组成，每个结构域由来自一个亚基的两个螺旋和来自另一个亚基的一个螺旋组成。两个GAT结构域通过反平行卷曲螺旋连接，形成90长的杠铃样结构。在与Juan Bonifacino（NICHD）的合作中，我们发现核心结构的完整性是有效的生理运输功能所必需的。这种结构将Vps 27和Hse 1的结构域放置在彼此靠近的位置，这些结构域募集泛素化货物和去泛素化酶。Vps 27 Hse 1复合物在膜上的粗粒度蒙特卡罗模拟（与Gerhard Hummer，NIDDK合作）显示了复合物如何与脂质和泛素化膜蛋白协同结合，并作为泛素化反应的支架。与膜和多达三个泛素化货物蛋白的合作结合提供了功能识别的机制，尽管单个UIM：泛素相互作用的亲和力低。

There are now at least sixteen known ubiquitin binding domains 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, 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 microM. 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 yeast Vps27Hse1 complex and homologous mammalian HrsSTAM complex deliver ubiquitinated transmembrane proteins to the ESCRT endosomal sorting pathway. Vps27 is a multimodular protein containing VHS, FYVE, UIM domains, a core region, and an unstructured motif-rich C-terminus. Hse1 contains VHS, UIM, and SH3 domains, a core region, and an unstructured region. These proteins are paradigms for proteins that contain multiple low-affinity ubiquitin binding domains. The structures and interactions of these domains have been characterized in isolation. The Vps27 FYVE domain, which was previously crystallized in our laboratory, is responsible for binding with moderate affinity to the endosomal lipid phosphatidylinositol 3-phosphate. The individual UIM domains bind ubiquitin with very low affinity. The Vps27Hse1 complex directly binds to ubiquitinated transmembrane proteins and recruits both ubiquitin ligases and deubiquitinating enzymes. We have solved the crystal structure of the core responsible for the assembly of the Vps27Hse1 complex at 3.0 Angstrom resolution. The structure consists of two intertwined GAT domains, each consisting of two helices from one subunit and one from the other. The two GAT domains are connected by an antiparallel coiled-coil to form a 90 -long barbell-like structure. In collaboration with Juan Bonifacino (NICHD) we found that the integrity of the core structure was required for efficient physiological trafficking function. This structure places the domains of Vps27 and Hse1 that recruit ubiquitinated cargo and deubiquitinating enzymes close to each other. Coarse-grained Monte Carlo simulations of the Vps27Hse1 complex on a membrane (collaboration with Gerhard Hummer, NIDDK) show how the complex binds cooperatively to lipids and ubiquitinated membrane proteins and acts as a scaffold for ubiquitination reactions. The cooperative binding to membranes and up to three ubiquitinated cargo proteins offers a mechanism for functional recognition, despite the low affinity of the individual UIM:ubiquitin interactions.