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Kinetic analysis of multistep USP7 mechanism shows critical role for target protein in activity.
- Source :
-
Nature communications [Nat Commun] 2019 Jan 16; Vol. 10 (1), pp. 231. Date of Electronic Publication: 2019 Jan 16. - Publication Year :
- 2019
-
Abstract
- USP7 is a highly abundant deubiquitinating enzyme (DUB), involved in cellular processes including DNA damage response and apoptosis. USP7 has an unusual catalytic mechanism, where the low intrinsic activity of the catalytic domain (CD) increases when the C-terminal Ubl domains (Ubl45) fold onto the CD, allowing binding of the activating C-terminal tail near the catalytic site. Here we delineate how the target protein promotes the activation of USP7. Using NMR analysis and biochemistry we describe the order of activation steps, showing that ubiquitin binding is an instrumental step in USP7 activation. Using chemically synthesised p53-peptides we also demonstrate how the correct ubiquitinated substrate increases catalytic activity. We then used transient reaction kinetic modelling to define how the USP7 multistep mechanism is driven by target recognition. Our data show how this pleiotropic DUB can gain specificity for its cellular targets.
- Subjects :
- Carbon Isotopes chemistry
Catalytic Domain genetics
Enzyme Assays methods
Kinetics
Models, Chemical
Mutagenesis, Site-Directed
Nitrogen Isotopes chemistry
Nuclear Magnetic Resonance, Biomolecular methods
Peptides chemistry
Peptides metabolism
Protein Binding
Recombinant Proteins chemistry
Recombinant Proteins genetics
Recombinant Proteins isolation & purification
Recombinant Proteins metabolism
Substrate Specificity
Surface Plasmon Resonance
Tumor Suppressor Protein p53 chemistry
Ubiquitin chemistry
Ubiquitin-Specific Peptidase 7 chemistry
Ubiquitin-Specific Peptidase 7 genetics
Ubiquitin-Specific Peptidase 7 isolation & purification
Protein Processing, Post-Translational
Ubiquitin metabolism
Ubiquitin-Specific Peptidase 7 metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 2041-1723
- Volume :
- 10
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- Nature communications
- Publication Type :
- Academic Journal
- Accession number :
- 30651545
- Full Text :
- https://doi.org/10.1038/s41467-018-08231-5