PT  - JOURNAL ARTICLE
AU  - Marie Morrow
AU  - Michael Morgan
AU  - Marcello Clerici
AU  - Katerina Growkova
AU  - Ming Yan
AU  - David Komander
AU  - Michal Simicek
AU  - Titia Sixma
AU  - Cynthia Wolberger
TI  - Active site alanine substitutions can convert deubiquitinating enzymes into avid ubiquitin-binding domains
AID  - 10.1101/238154
DP  - 2017 Jan 01
TA  - bioRxiv
PG  - 238154
4099  - http://biorxiv.org/content/early/2017/12/22/238154.short
4100  - http://biorxiv.org/content/early/2017/12/22/238154.full
AB  - A common strategy for studying the biological role 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.