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


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.

SUBMITTER: Kim RQ 

PROVIDER: S-EPMC6335408 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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

Kim Robbert Q RQ   Geurink Paul P PP   Mulder Monique P C MPC   Fish Alexander A   Ekkebus Reggy R   El Oualid Farid F   van Dijk Willem J WJ   van Dalen Duco D   Ovaa Huib H   van Ingen Hugo H   Sixma Titia K TK  

Nature communications 20190116 1


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 describ  ...[more]

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