Abstract
Herpes simplex virus-1 immediate-early protein ICP0 activates viral genes during early stages of infection, affects cellular levels of multiple host proteins and is crucial for effective lytic infection. Being a RING-type E3 ligase prone to auto-ubiquitination, ICP0 relies on human deubiquitinating enzyme USP7 for protection against 26S proteasomal mediated degradation. USP7 is involved in apoptosis, epigenetics, cell proliferation and is targeted by several herpesviruses. Several USP7 partners, including ICP0, GMPS, and UHRF1, interact through its C-terminal domain (CTD), which contains five ubiquitin-like (Ubl) structures. Despite the fact that USP7 has emerged as a drug target for cancer therapy, structural details of USP7 regulation and the molecular mechanism of interaction at its CTD have remained elusive. Here, we mapped the binding site between an ICP0 peptide and USP7 and determined the crystal structure of the first three Ubl domains bound to the ICP0 peptide, which showed that ICP0 binds to a loop on Ubl2. Sequences similar to the USP7-binding site in ICP0 were identified in GMPS and UHRF1 and shown to bind USP7-CTD through Ubl2. In addition, co-immunoprecipitation assays in human cells comparing binding to USP7 with and without a Ubl2 mutation, confirmed the importance of the Ubl2 binding pocket for binding ICP0, GMPS and UHRF1. Therefore we have identified a novel mechanism of USP7 recognition that is used by both viral and cellular proteins. Our structural information was used to generate a model of near full-length USP7, showing the relative position of the ICP0/GMPS/UHRF1 binding pocket and the structural basis by which it could regulate enzymatic activity.
Highlights
Ubiquitin specific protease 7 (USP7) catalyzes the deubiquitination of many cellular proteins involved in tumor suppression, neural stem cell maintenance, DNA damage and immune responses [1,2,3,4,5,6,7,8]
USP7 is a cellular protein that binds and stabilizes many proteins involved in multiple pathways that regulate oncogenesis and as such is recognized as a potential target for cancer therapy
One such protein is the ICP0 protein of herpes simplex virus 1, which must bind USP7 in order to manipulate the cell in ways that enable efficient viral infection
Summary
Ubiquitin specific protease 7 (USP7) catalyzes the deubiquitination of many cellular proteins involved in tumor suppression, neural stem cell maintenance, DNA damage and immune responses [1,2,3,4,5,6,7,8]. USP7 consists of an N-terminal TRAF-like domain (NTD), a catalytic domain (CAT) and five C-terminal ubiquitin-like domains (CTD). Affinity chromatography coupled with in vitro proteolysis revealed that a region within residues 560–870, which corresponds to Ubl123, interacts with ICP0 [14]. Ubl123 has been reported to interact with GMPS [15]; a metabolic enzyme involved in nucleotide biosynthesis with a second independent function as a USP7 modulator [17,18,19]. The ability of GMPS to activate ubiquitin cleavage by USP7 involves its interaction with USP7-CTD, which is thought to stabilize a compact USP7 conformation leading to ordering of active site residues and stimulation of catalytic activity [15]
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