Molecular Understanding of USP7 Substrate Recognition and C-Terminal Activation

Abstract

The deubiquitinating enzyme USP7 has a pivotal role in regulating the stability of proteins involved in fundamental cellular processes of normal biology and disease. Despite the importance of USP7, the mechanisms underlying substrate recognition and catalytic activation are poorly understood. Here we present structural, biochemical, and biophysical analyses elucidating the molecular mechanism by which the C-terminal 19 amino acids of USP7 (residues 1084-1102) enhance the ubiquitin cleavage activity of the deubiquitinase (DUB) domain. Our data demonstrate that the C-terminal peptide binds the activation cleft in the catalytic domain and stabilizes the catalytically competent conformation of USP7. Additional structures of longer fragments of USP7, as well as solution studies, provide insight into full-length USP7, the role of the UBL domains, and demonstrate that both substrate recognition and deubiquitinase activity are highly regulated by the catalytic and noncatalytic domains of USP7, a feature that could be essential for the proper function of multi-domain DUBs.

The structure of the deubiquitinase USP15 reveals a misaligned catalytic triad and an open ubiquitin-binding channel

“…The only other USP shown to have a misaligned catalytic triad in a structure to date that requires significant conformational rearrangements for activation is the catalytic domain of USP7 (USP7-CD) ( 30 , 38 ). However, in contrast to USP15, the USP7-CD only displays limited activity, and the C-terminal UBLs and C terminus are required for full catalytic competency, whereby they interact with the SL to promote conversion of the catalytic loop to an α-helical conformation and catalytic triad alignment ( 46 – 48 ). The activity of USP15, on the other hand, is only minimally influenced by the presence of additional domains, as observed by us and others ( 24 ), but also has a misaligned catalytic triad, from which we infer a different mechanism of regulation.…”

Mechanisms of Deubiquitinase Specificity and Regulation

“…The last two of five HAUSP ubiquitin-like domains (HUBLs), together with a C-terminal peptide (CTP), enhance USP7 activity 100-fold by binding back to the so-called switching loop in the USP domain, which increases both k cat and K M (147). Structural work has illustrated how HUBLs and CTP regulate USP7 and stabilize a catalytically competent conformation (148,149). In addition, the first three HUBL domains (HUBL-123) are also important for GMP synthetase (GMPS)-dependent USP7 hyperactivation (147,149,150).…”

Targeting USP7-Mediated Deubiquitination of MDM2/MDMX-p53 Pathway for Cancer Therapy: Are We There Yet?

“…The USP7 protein consists of 1102 amino acids that are distributed in three major domains, including the N-terminal tumor necrosis factor receptor-associated factor (TRAF) domain (amino acids 53-206), a central catalytic domain (amino acids 208-560), and the C-terminal tandem ubiquitin-like (Ubl) domain (UBL1-5, amino acids 560-1102) (Figure 1A). Among these domains, TRAF is critical for the binding of USP7 to its substrates, including MDM2, MDMX, and p53 via P/AxxS motifs (Hu et al, 2002(Hu et al, , 2006Saridakis et al, 2005;Sheng et al, 2006;Sarkari et al, 2010;Rouge et al, 2016). It has also been found that the nuclear localization of USP7 is partially dependent on the TRAF domain through the production of USP7 domain deletion mutants (Fernandez-Montalvan et al, 2007;Tavana and Gu, 2017).…”