Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario , London, Ontario, Canada N6A 5C1.
Biochemistry. 2013 Oct 22;52(42):7369-76. doi: 10.1021/bi400780v. Epub 2013 Oct 9.
The ubiquitin signaling pathway consists of hundreds of enzymes that are tightly regulated for the maintenance of cell homeostasis. Parkin is an E3 ubiquitin ligase responsible for conjugating ubiquitin onto a substrate protein, which itself can be ubiquitinated. Ataxin-3 performs the opposing function as a deubiquitinating enzyme that can remove ubiquitin from parkin. In this work, we have identified the mechanism of interaction between the ubiquitin-like (Ubl) domain from parkin and three C-terminal ubiquitin-interacting motifs (UIMs) in ataxin-3. (1)H-(15)N heteronuclear single-quantum coherence titration experiments revealed that there are weak direct interactions between all three individual UIM regions of ataxin-3 and the Ubl domain. Each UIM utilizes the exposed β-grasp surface of the Ubl domain centered around the I44 patch that did not vary in the residues involved or the surface size as a function of the number of ataxin-3 UIMs involved. Further, the apparent dissociation constant for ataxin-3 decreased as a function of the number of UIM regions used in experiments. A global multisite fit of the nuclear magnetic resonance titration data, based on three identical binding ligands, resulted in a KD of 669 ± 62 μM for each site. Our observations support a multivalent ligand binding mechanism employed by the parkin Ubl domain to recruit multiple UIM regions in ataxin-3 and provide insight into how these two proteins function together in ubiquitination-deubiquitination pathways.
泛素信号通路由数百种酶组成，这些酶的活性受到严格调控，以维持细胞内环境的稳定。Parkin 是一种 E3 泛素连接酶，负责将泛素连接到靶蛋白上，而靶蛋白本身也可以被泛素化。Ataxin-3 作为一种去泛素化酶，具有相反的功能，可以从 Parkin 上移除泛素。在这项工作中，我们确定了 Parkin 泛素样（Ubl）结构域与 Ataxin-3 中三个 C 端泛素相互作用基序（UIM）之间的相互作用机制。（1）H-（15）N 异核单量子相干滴定实验表明，Ataxin-3 的三个 UIM 区域与 Ubl 结构域之间存在微弱的直接相互作用。每个 UIM 都利用 Ubl 结构域中围绕 I44 补丁的暴露β-抓取表面，该补丁在参与的残基或表面大小方面没有变化，而是作为参与的 Ataxin-3 UIM 数量的函数而变化。此外，随着参与实验的 UIM 区域数量的增加，Ataxin-3 的表观解离常数降低。基于三个相同结合配体的核磁共振滴定数据的全局多位点拟合导致每个位点的 KD 为 669±62μM。我们的观察结果支持 Parkin Ubl 结构域采用多价配体结合机制来招募 Ataxin-3 中的多个 UIM 区域，并提供了关于这两种蛋白质如何在泛素化-去泛素化途径中协同作用的见解。
