Structural and Functional Studies of Ubiquitin Binding Domains

现在至少有16个已知的泛素结合域。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和A20锌F，促进自动素化，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，与Kd&gt；100微米。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。 酵母Vps27Hse1复合体和同源哺乳动物HrsSTAM复合体将泛素化的跨膜蛋白递送到ESCRT内体分选途径。Vps27是一种含有VHS、FYVE、UIM结构域、核心区和富含非结构化基序的C-末端的多模蛋白。HSE1包含VHS、UIM和SH3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域的蛋白质的范例。这些结构域的结构和相互作用已被孤立地表征。Vps27FYVE结构域是我们实验室以前结晶的，负责与内体脂质磷脂酰肌醇3-磷酸有中等亲和力的结合。单独的UIM结构域以非常低的亲和力结合泛素。Vps27Hse1复合体直接与泛素化的跨膜蛋白结合，并招募泛素连接酶和脱泛素酶。我们已经解决了在3.0埃分辨率下组装Vps27Hse1络合物的核心的晶体结构。该结构由两个相互缠绕的GAT结构域组成，每个结构域由一个亚基的两个螺旋和另一个亚基的两个螺旋组成。这两个GAT结构域通过一个反平行的螺旋线圈连接起来，形成一个90长的杠铃状结构。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转运功能所必需的。这种结构使Vps27和Hse1的结构域彼此靠近，它们招募泛素化的货物和去泛素化的酶。对Vps27Hse1复合体在膜上的粗粒度蒙特卡罗模拟(与Gerhard Hummer，NIDDK合作)显示了该复合体如何与脂质和泛素化的膜蛋白协同结合，并作为泛素化反应的支架。与膜和最多三个泛素化的货运蛋白的协同结合为功能识别提供了一种机制，尽管单个UIM：泛素相互作用的亲和力很低。

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.