Further insight into substrate recognition by USP7: structural and biochemical analysis of the HdmX and Hdm2 interactions with USP7.
Ubiquitin-specific protease 7 (USP7) catalyzes the deubiquitination of several substrate proteins including p53 and Hdm2. We have previously shown that USP7, and more specifically its amino-terminal domain (USP7-NTD), interacts with distinct regions on p53 and Hdm2 containing P/AxxS motifs. The ability of USP7 to also deubiquitinate and control the turnover of HdmX was recently demonstrated. We utilized a combination of biochemistry and structural biology to identify which domain of USP7 interacts with HdmX as well as to identify regions of HdmX that interact with USP7. We showed that USP7-NTD recognized two of six P/AxxS motifs of HdmX ((8)AQCS(11) and (398)AHSS(401)). The crystal structure of the USP7-NTD:HdmX(AHSS) complex was determined providing the molecular basis of complex formation between USP7-NTD and the HdmX(AHSS) peptide. The HdmX peptide interacted within the same residues of USP7-NTD as previously demonstrated with p53, Hdm2, and EBNA1 peptides. We also identified an additional site on Hdm2 ((397)PSTS(400)) that interacts with USP7-NTD and determined the crystal structure of this complex. Finally, analysis of USP7-interacting peptides on filter arrays confirmed the importance of the serine residue at the fourth position for the USP7-NTD interaction and showed that phosphorylation of serines within the binding sequence prevents this interaction. These results lead to a better understanding of the mechanism of substrate recognition by USP7-NTD.
Sarkari
Feroz
F
Department of Molecular Genetics, University of Toronto, 1 King's College Circle, Toronto, Ontario, Canada.
La Delfa
Anthony
A
Arrowsmith
Cheryl H
CH
Frappier
Lori
L
Sheng
Yi
Y
Saridakis
Vivian
V
D000595:Amino Acid Sequence; D001665:Binding Sites; D018797:Cell Cycle Proteins; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D025941:Protein Interaction Mapping; D020836:Protein Structure, Quaternary; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000378:metabolism*; D051736:Proto-Oncogene Proteins c-mdm2 / Q000737:chemistry* / Q000378:metabolism*; D016415:Sequence Alignment; D012694:Serine / Q000378:metabolism; D043222:Ubiquitin Thiolesterase / Q000737:chemistry* / Q000378:metabolism*; D000076084:Ubiquitin-Specific Peptidase 7
D018797:Cell Cycle Proteins; C482775:MDM4 protein, human; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D012694:Serine; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; C464569:USP7 protein, human; D043222:Ubiquitin Thiolesterase; D000076084:Ubiquitin-Specific Peptidase 7