The molecular determinants for distinguishing between ubiquitin and NEDD8 by USP2

Ubiquitin (Ub) shares the highest sequence identity with neuronal-precursor-cell-expressed developmentally downregulated protein-8 (NEDD8) in the Ub-like protein family. However, different enzyme systems are precisely employed for targeting Ub and NEDD8 to specific substrates. The molecular determinants for distinguishing between Ub and NEDD8 by Ub-specific peptidases (USPs) remain poorly characterized. By replacing the non-conserved residues of Ub with their NEDD8 equivalents by mutagenesis, and vice versa, we observed that the Ub^4K, Ub^12E, and Ub^14E mutants partially and the Ub^{4K/12E/14E/72A} mutant completely prevented their hydrolysis by USP2. The NEDD8^4F and NEDD8^14T mutants were slightly hydrolyzed by USP2; however, the NEDD8^12T/14T/72R and NEDD8^{4F/12T/14T/72R} mutants were accessible for hydrolysis by USP2, suggesting that Ub and NEDD8 residues 4, 12, 14, and 72 serve as the molecular determinants for specific recognition by USP2. We also demonstrated that the level of inhibition caused by Ub mutants with multiple mutation sites was not purely additive when compared with the single mutation results. Furthermore, USP2 was determined to bind to the N-terminus of Ub to form a stable interaction, after which it binds with the C-terminus of Ub to ensure substrate specificity. The same results were also discovered when Ub, Ub^{4K/12E/14E/72A}, NEDD8, and NEDD8^{4F/12T/14T/72R} were incubated with USP21.