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

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

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
JournalEMBO Reports
DOIs
StateAccepted/In press - Jan 1 2018

Fingerprint

Ubiquitin
Alanine
Catalytic Domain
Carrier Proteins
Mutation
Cysteine
Arginine
Cells
Deubiquitinating Enzymes
Enzymes

Keywords

  • deubiquitinating enzyme
  • polyubiquitin
  • ubiquitin binding

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Genetics

Cite this

Active site alanine mutations convert deubiquitinases into high-affinity ubiquitin-binding proteins. / Morrow, Marie E.; Morgan, Michael; Clerici, Marcello; Growkova, Katerina; Yan, Ming; Komander, David; Sixma, Titia K.; Simicek, Michal; Wolberger, Cynthia.

In: EMBO Reports, 01.01.2018.

Research output: Contribution to journalArticle

Morrow, Marie E. ; Morgan, Michael ; Clerici, Marcello ; Growkova, Katerina ; Yan, Ming ; Komander, David ; Sixma, Titia K. ; Simicek, Michal ; Wolberger, Cynthia. / Active site alanine mutations convert deubiquitinases into high-affinity ubiquitin-binding proteins. In: EMBO Reports. 2018.
@article{b2f2ab0ea5314ee49adcb7add798336b,
title = "Active site alanine mutations convert deubiquitinases into high-affinity ubiquitin-binding proteins",
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.",
keywords = "deubiquitinating enzyme, polyubiquitin, ubiquitin binding",
author = "Morrow, {Marie E.} and Michael Morgan and Marcello Clerici and Katerina Growkova and Ming Yan and David Komander and Sixma, {Titia K.} and Michal Simicek and Cynthia Wolberger",
year = "2018",
month = "1",
day = "1",
doi = "10.15252/embr.201745680",
language = "English (US)",
journal = "EMBO Reports",
issn = "1469-221X",
publisher = "Nature Publishing Group",

}

TY - JOUR

T1 - Active site alanine mutations convert deubiquitinases into high-affinity ubiquitin-binding proteins

AU - Morrow, Marie E.

AU - Morgan, Michael

AU - Clerici, Marcello

AU - Growkova, Katerina

AU - Yan, Ming

AU - Komander, David

AU - Sixma, Titia K.

AU - Simicek, Michal

AU - Wolberger, Cynthia

PY - 2018/1/1

Y1 - 2018/1/1

N2 - 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.

AB - 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.

KW - deubiquitinating enzyme

KW - polyubiquitin

KW - ubiquitin binding

UR - http://www.scopus.com/inward/record.url?scp=85052479055&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85052479055&partnerID=8YFLogxK

U2 - 10.15252/embr.201745680

DO - 10.15252/embr.201745680

M3 - Article

JO - EMBO Reports

JF - EMBO Reports

SN - 1469-221X

ER -