The molecular determinants for distinguishing between ubiquitin and NEDD8 by USP2

Yung-Cheng Shin,Jou-Han Chen,Shih-Chung Chang

doi:10.1038/s41598-017-02322-x

Yung-Cheng Shin, Jou-Han Chen + Show 1 more

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https://doi.org/10.1038/s41598-017-02322-x

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Journal: Scientific Reports	Publication Date: May 23, 2017
Citations: 9	License type: open-access

Affiliation: National Taiwan University

Abstract

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 Ub4K, Ub12E, and Ub14E mutants partially and the Ub4K/12E/14E/72A mutant completely prevented their hydrolysis by USP2. The NEDD84F and NEDD814T mutants were slightly hydrolyzed by USP2; however, the NEDD812T/14T/72R and NEDD84F/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, Ub4K/12E/14E/72A, NEDD8, and NEDD84F/12T/14T/72R were incubated with USP21.

Highlights

Ubiquitin (Ub), a highly conserved 76-amino-acid polypeptide, is conjugated to target proteins through a process called ubiquitination mediated by the enzyme cascades comprising Ub-activating enzyme (E1), Ub-conjugating enzymes (E2), and Ub ligase (E3)
The key residues of Ub recognized by USP2 for Ub-specific binding
The key residues of Ub, which are proposed to be involved in a specific interaction with USP2, are aligned with the equivalent residues of neural-precursor-cell-expressed developmentally downregulated protein-8 (NEDD8) (Fig. 1)

Summary

Introduction

Ubiquitin (Ub), a highly conserved 76-amino-acid polypeptide, is conjugated to target proteins through a process called ubiquitination mediated by the enzyme cascades comprising Ub-activating enzyme (E1), Ub-conjugating enzymes (E2), and Ub ligase (E3). NEDD8 can be conjugated to target substrates in a process that is similar to ubiquitination, called neddylation, which relies on its own E1 and E2 enzymes[14,15,16]. We[32] and other groups[33, 34] have discovered that residues at positions 51 and 72 of NEDD8 served as the molecular determinants for specific substrate recognition by SENP8. The realignment of the active site of HAUSP can be induced by Ub binding, leading to the specific recognition and processing of Ub. residues at the HAUSP-Ubal binding interface make crucial contributions to the binding of Ubal, including several hydrogen bonds and van der Waals interactions, that primarily involve the N-terminal residues of Ubal as well as a few amino acids in the middle stretch and at the C-terminus

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