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

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

doi:10.15252/embr.201745680

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

Journal Article · Mon Aug 27 00:00:00 EDT 2018 · EMBO Reports

DOI:https://doi.org/10.15252/embr.201745680· OSTI ID:1547096

Morrow, Marie E. ^[1]; Morgan, Michael T. ^[1]; Clerici, Marcello ^[2]; Growkova, Katerina ^[3]; Yan, Ming ^[1]; ^[4]; Sixma, Titia K. ^[2]; Simicek, Michal ^[5]; ^[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

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 Ubp8^C146A 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.

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Research Organization:: SLAC National Accelerator Laboratory, Menlo Park, CA (United States). Stanford Synchrotron Radiation Lightsource (SSRL)

Sponsoring Organization:: European Research Council (ERC); Lister Institute for Preventive Medicine; Medical Research Council; Michael J. Fox Foundation; Ministry of Education, Youth and Sports; National Institute of General Medical Sciences (NIGMS); National Institutes of Health (NIH); USDOE Office of Science (SC), Basic Energy Sciences (BES); USDOE Office of Science (SC), Biological and Environmental Research (BER); University of Ostrava

Grant/Contract Number:: AC02-76SF00515

OSTI ID:: 1547096

Alternate ID(s):: OSTI ID: 1627932
OSTI ID: 1547097

Journal Information:: EMBO Reports, Journal Name: EMBO Reports Journal Issue: 10 Vol. 19; ISSN 1469-221X

Publisher:: European Molecular Biology OrganizationCopyright Statement

Country of Publication:: Germany

Language:: English

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59 BASIC BIOLOGICAL SCIENCES
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Active site alanine mutations convert deubiquitinases into high-affinity ubiquitin-binding proteins

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