磷酸化调控泛素单体与Rad23A/Ubiquilin-1中泛素结合域互作的检测

泛素（ubiquitin，Ub）是一种广泛存在、高度保守的信号蛋白质，它能够特异性识别成千上万种靶蛋白，以非共价方式行使不同的功能，其中包含蛋白质降解．Ubiquilin-1（Ubql-1）和Rad23A作为两种蛋白降解的转运因子，都包含有与泛素结合的结构域，被称为泛素结合域（ubiquitin-associated domain，UBA）．2014年，泛素S65位磷酸化修饰的特异性激酶PINK1被发现，磷酸化使泛素在溶液中呈现舒展态与收缩态两种互相转换的构象．本文通过核磁共振（nuclear magnetic resonance，NMR）技术对UBA和磷酸化泛素之间的相互作用进行检测，观测磷酸化对UBA和泛素结合的影响．实验结果表明Rad23A-UBA2与Ubql-1 UBA都特异性的与磷酸化泛素的舒展态相互作用，但是磷酸化未改变泛素与UBA之间的亲和力．值得注意的是与Ubql-1 UBA相互作用时，磷酸化促进了泛素收缩态向舒展态的转换．

Regulation of Inter-Protein Interactions Between Ubiquitin and Ubiquitin-Associated Domains in Rad23A/Ubiquilin-1 by Phosphorylation

Ubiquitin is a conserved signaling protein. It is able to interact with thousands of target proteins specifically and perform its biological functions via non-covalent interactions. Ubiquilin-1 (Ubql-1) and Rad23A are two transport factors in the protein-degradation process, and both contain ubiquitin-associated domains (UBAs). S65 of the ubiquitin can be phosphorylated specifically by the kinase PINK1, and the phosphorylated ubiquitin has two interchangeable conformations in solution, the relaxed conformation and the retracted conformation. In this work, nuclear magnetic resonance (NMR) methods were used to characterize the regulation of interactions between the ubiquitin and the UBAs of Rad23A/Ubql-1 by phosphorylation. The results indicated that the UBAs selectively interacted with the relaxed conformation of the phosphorylated ubiquitin without affecting the affinity. When interacting with the UBA of Ubql-1, inter-protein binding appeared to be able to promote the transferring from the retracted conformation to the relaxed conformation.