The molecular determinants for ubiquitin NEDD8 discrimination by human USP2

Abstract

Ubiquitin‐like proteins (Ubls) share very high sequence and structural similarity; but conjugation to a protein leads to distinct biological consequences. Ubls are initially synthesized as precursor forms for processing by Ubl‐specific proteases to expose the C‐terminal di‐glycine motif for conjugation to target proteins. Ubiquitin and NEDD8 share 80% sequence identity, however, each exclusive protease can specifically react with ubiquitin or NEDD8. This work is aimed to study the substrate recognition mechanism of USP (ubiquitin‐specific protease) protein family.The crystal structure of USP2‐ubiquitin binding complex showed that residues 4, 12, 14 and 72 of ubiquitin were the major interaction sites. It is also noted that these residues are not conserved in NEDD8. By mutating these sites to the corresponding residues in NEDD8, we found that F4K ubiquitin mutant markedly inhibited its hydrolysis by USP2, and T12E and T14E ubiquitin mutants also showed partial catalytic inhibition due to the steric hindrance and electric repulsion between the mutated residue and USP2. Moreover, mutation of residue 72 further inhibited the hydrolysis of T12E/T14E/R72A ubiquitin mutant than T12E/T14E ubiquitin mutant. Because the key amino acids for interacting with residues 4, 12, 14 and 72 of ubiquitin are conserved in the USP protein family, this study suggests a general recognition mechanism by which USP can specifically discriminate between ubiquitin and NEDD8.