Active site alanine mutations convert deubiquitinases into high-affinity ubiquitin-binding proteins.

Marie E Morrow,Titia K Sixma,Marcello Clerici,Michal Simicek,Michael T Morgan,Cynthia Wolberger,David Komander,Katerina Growkova,Ming Yan

doi:10.15252/embr.201745680

Abstract

A common strategy for exploring the biological roles of deubiquitinating enzymes (DUBs) in different pathways is to study the effects of replacing the wild‐type DUB with a catalytically inactive mutant in cells. We report here that a commonly studied DUB mutation, in which the catalytic cysteine is replaced with alanine, can dramatically increase the affinity of some DUBs for ubiquitin. Overexpression of these tight‐binding mutants thus has the potential to sequester cellular pools of monoubiquitin and ubiquitin chains. As a result, cells expressing these mutants may display unpredictable dominant negative physiological effects that are not related to loss of DUB activity. The structure of the SAGA DUB module bound to free ubiquitin reveals the structural basis for the 30‐fold higher affinity of Ubp8C146A for ubiquitin. We show that an alternative option, substituting the active site cysteine with arginine, can inactivate DUBs while also decreasing the affinity for ubiquitin.

Highlights

Deubiquitinating enzymes (DUBs) play fundamental roles in ubiquitin signaling through their ability to remove ubiquitin from target proteins and disassemble polyubiquitin chains [1]
The human genome encodes more than 90 DUBs [2,3], which can be grouped into families based on their fold: ubiquitin-specific protease (USP), ubiquitin carboxyl-terminal hydrolase (UCH), ovarian tumor family (OTU), Machado–Joseph domain (MJD) family, and JAMM/MPN domain (JAMM), as well as the recently discovered MINDY and ZUFSP families [4,5,6]
Resulting changes in substrate ubiquitination or downstream signaling pathways in cells expressing the mutant DUB are generally assumed to be due to the absence of deubiquitinating activity, with the notable exceptions of OTUB1, which inhibits E2 enzymes by a mechanism independent of catalytic activity [15,16,17] and OTUD4, which serves as a scaffold for USP enzymes [18]

Summary

Introduction

Deubiquitinating enzymes (DUBs) play fundamental roles in ubiquitin signaling through their ability to remove ubiquitin from target proteins and disassemble polyubiquitin chains [1]. We report here that some active site cysteine-to-alanine substitutions can dramatically increase the affinity of DUBs for either free ubiquitin or polyubiquitin chains. We show that substituting the active site cysteine with arginine in representative USP and OTU DUBs inactivates the enzymes while disrupting binding to ubiquitin, generating an inert DUB.

Results

Conclusion