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.
- Authors:
-
[4];
[1]
- Department of Biophysics and Biophysical Chemistry Johns Hopkins University School of Medicine Baltimore MD USA
- Division of Biochemistry and Oncode Institute Netherlands Cancer Institute Amsterdam The Netherlands
- Faculty of Medicine University of Ostrava Ostrava Czech Republic
- Medical Research Council Laboratory of Molecular Biology Cambridge UK
- Faculty of Medicine University of Ostrava Ostrava Czech Republic, Medical Research Council Laboratory of Molecular Biology Cambridge UK
- Publication Date:
- Research Org.:
- SLAC National Accelerator Laboratory (SLAC), Menlo Park, CA (United States). Stanford Synchrotron Radiation Lightsource (SSRL)
- Sponsoring Org.:
- USDOE Office of Science (SC), Basic Energy Sciences (BES); National Institute of General Medical Sciences (NIGMS); European Research Council (ERC); Medical Research Council; Michael J. Fox Foundation; Lister Institute for Preventive Medicine; University of Ostrava; Ministry of Education, Youth and Sports; USDOE Office of Science (SC), Biological and Environmental Research (BER); National Institutes of Health (NIH)
- OSTI Identifier:
- 1547096
- Alternate Identifier(s):
- OSTI ID: 1547097; OSTI ID: 1627932
- Grant/Contract Number:
- AC02-76SF00515; GM095822; GM109102
- Resource Type:
- Published Article
- Journal Name:
- EMBO Reports
- Additional Journal Information:
- Journal Name: EMBO Reports Journal Volume: 19 Journal Issue: 10; Journal ID: ISSN 1469-221X
- Publisher:
- European Molecular Biology Organization
- Country of Publication:
- Germany
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; biochemistry & molecular biology; cell biology; deubiquitinating; enzyme; polyubiquitin; ubiquitin binding
Citation Formats
Morrow, Marie E., Morgan, Michael T., Clerici, Marcello, Growkova, Katerina, Yan, Ming, Komander, David, Sixma, Titia K., Simicek, Michal, and Wolberger, Cynthia. Active site alanine mutations convert deubiquitinases into high-affinity ubiquitin-binding proteins. Germany: N. p., 2018.
Web. doi:10.15252/embr.201745680.
Morrow, Marie E., Morgan, Michael T., 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. Germany. https://doi.org/10.15252/embr.201745680
Morrow, Marie E., Morgan, Michael T., Clerici, Marcello, Growkova, Katerina, Yan, Ming, Komander, David, Sixma, Titia K., Simicek, Michal, and Wolberger, Cynthia. Mon .
"Active site alanine mutations convert deubiquitinases into high-affinity ubiquitin-binding proteins". Germany. https://doi.org/10.15252/embr.201745680.
@article{osti_1547096,
title = {Active site alanine mutations convert deubiquitinases into high-affinity ubiquitin-binding proteins},
author = {Morrow, Marie E. and Morgan, Michael T. and Clerici, Marcello and Growkova, Katerina and Yan, Ming and Komander, David and Sixma, Titia K. and Simicek, Michal and Wolberger, Cynthia},
abstractNote = {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.},
doi = {10.15252/embr.201745680},
journal = {EMBO Reports},
number = 10,
volume = 19,
place = {Germany},
year = {Mon Aug 27 00:00:00 EDT 2018},
month = {Mon Aug 27 00:00:00 EDT 2018}
}
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