Kinetic analysis of multistep USP7 mechanism shows critical role for target protein in activity

Robbert Q. Kim, Paul P. Geurink, Monique P. C. Mulder, Alexander Fish, Reggy Ekkebus, Farid El Oualid, Willem J. Dijk, Duco Dalen, Huib Ovaa (), Hugo Ingen () and Titia K. Sixma ()
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Robbert Q. Kim: Division of Biochemistry and Oncode Institute, Netherlands Cancer Institute
Paul P. Geurink: Division of Cell Biology II, Netherlands Cancer Institute
Monique P. C. Mulder: Division of Cell Biology II, Netherlands Cancer Institute
Alexander Fish: Division of Biochemistry and Oncode Institute, Netherlands Cancer Institute
Reggy Ekkebus: Division of Cell Biology II, Netherlands Cancer Institute
Farid El Oualid: UbiQ Bio BV, Science Park 408
Willem J. Dijk: Division of Biochemistry and Oncode Institute, Netherlands Cancer Institute
Duco Dalen: Division of Cell Biology II, Netherlands Cancer Institute
Huib Ovaa: Division of Cell Biology II, Netherlands Cancer Institute
Hugo Ingen: Leiden University
Titia K. Sixma: Division of Biochemistry and Oncode Institute, Netherlands Cancer Institute

Nature Communications, 2019, vol. 10, issue 1, 1-16

Abstract: 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.

Date: 2019
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DOI: 10.1038/s41467-018-08231-5

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