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.