Structural Insights Into The Activation Mechanism Of Ubiquitin-specific Protease 46 By WDR Proteins WDR48 And WDR20

Ubiquitination is a widespread form of post-translational modification(PTM)for proteins in a range of cellular processes,and plays a crucial role in the regulation of stability,localization and cellular functions of the substrate proteins.Ubiquitination is a reversible covalent modification.Ubiquitination of proteins is carried out by three types of enzymes,named as E1s(ubiquitin-activating enzymes),E2s(ubiquitin-conjugating enzymes)and E3s(ubiquitin-ligase enzymes).Removal of the covalently linked Ub from substrate proteins is carried out by a set of enzymes called deubiquitinating enzymes(DUBs).There are approximately 100 DUBs encoded in the human genome,which can be classified into seven families based on their distinct catalytic domains.The ubiquitin-specific proteases(USPs)constitute the largest family with about 56 members sharing a common catalytic domain,some of which can interact with WDR proteins.USP46 is a member of USPs family,and plays an important role in the development of nervous system and tumor progression.Biochemical and functional studies suggest that USP46 alone has no measurable enzymatic activity,while the WDR48 and WDR20 binding can substantially stimulate its enzymatic activity.Therefore,it is of important biological significance to solve the structure of USP46 in complex with its interacting partners WDR48 and WDR20,and thus to shed light on the molecular mechanism for the activation of USP46 by WDR proteins.In this work,we first obtained the USP46-WDR48-WDR20 ternary complex with high stability and homogeneity via recombinant expression in vitro,and obtained crystals of the ternary complex with high quality X-ray diffraction capability based on crystallization condition screening and optimization.Finally,we collected a 3.1 (?)resolution diffraction data,and solved the crystal structure of the USP46-WDR48-WDR20 complex using the molecular replacement method.Structural analysis indicates that WDR48 and WDR20 bind to the Fingers and Palm subdomains of USP46 via their WD40-repeat domains,respectively.In addition,WDR48 interacts with WDR20 through the side face of their WD40-repeat domains.Through biochemical and cellular assays,we identified the key residues responsible for the USP46-WDR20 interaction.The WDR48 and WDR20 binding essentially enhance the enzymatic activity of USP46 in vitro and also promote USP46-mediated deubiquitination of PHLPP1 in vivo.Through detailed structural comparison of the USP46-WDR48-WDR20 complex with reported USP46 or USP12-correlated structures,we found that the WDR48 binding on USP46 not only stabilizes the Fingers subdomain of USP46 but also destabilizes several loops surrounding the catalytic triad,and consequently stimulates the enzymatic activity of USP46.However,the WDR20 binding can restore these flexible loops into stable conformation and thus the WDR48 and WDR20 binding synergistically enhance the enzymatic activity of USP46.Intriguingly,we also found that another WDR protein DMWD,which share48% sequence identity with WDR20,can bind to USP46 via a similar mode as WDR20,and hence promotes USP46-mediated deubiquitination of PHLPP1 in vivo.Through statistics analysis of the reported structures of the USPs family members,we propose two potential substrate recognition models for USPs.Moreover,our Co-IP assays indicate that the substrate protein PHLPP1 of USP46 can directly interact with WDR48 via its PP2 C and C-terminal domains,and hence mediates the functional role of USP46 in Akt signaling pathway.ITC measurements also indicate that the region consisting of residues 140 to 162 of RAD51AP1 is essential for its direct interaction with WDR48,which is important for mediating the functional role of USP1 in homologous recombination.Our structural and functional data together reveal the molecular basis for the activation of USP46 by WDR48 and WDR20,and provide mechanistic insights into the regulation of the function for USPs by WDR proteins.Moreover,it will also provide useful information for designing potential inhibitors targeting at the interfaces of the USP46-WDR48-WDR20 complex.