Fig 2: USP7-CTD interacts with GMPS and UHRF1 peptides.(A) Alignment and dissociation constants of ICP0, GMPS and UHRF1 peptides. (B) Location of the KxxxK motif (316DRTPRKRISKTLN328) within a disordered loop on the GMPS crystal structure (PDB ID 2VXO). (C) Coomassie stained gel of GST pull-down assays with WT or mutant USP7-CTD (D762A, D764A and D762A/D764A) and GST-GMPS peptide. Lane 1 USP7-CTD and GST-GMPS peptide load, lanes 2–5 are the eluted fractions with WT and mutant USP7-CTD. (D) Coomassie stained gel of GST pull-down assays with WT USP7-CTD and mutant GST-GMPS peptides. Lanes 1 (load) and 2 (elute) USP7-CTD and WT GST-GMPS, lanes 3–5 are the eluted fractions with GST-GMPS mutants (K321A, K325A and K321A/K325A). (E) Coomassie stained gel of GST pull-down assays with WT or mutant USP7-CTD (D762A, D762A/D764A and D762R/D764R) and GST-UHRF1 peptide. Lanes 1–4 are the loaded fractions with WT and mutant USP7-CTD. Lanes 5–8 are the eluted fractions with WT and mutant USP7-CTD. (F) Coomassie stained gel of GST pull-down assays with WT USP7-CTD and mutant GST-UHRF1 peptides. Lanes 1–4 are the loaded fractions with WT and mutant (K644A, K648A and K644A/K648A) GST-UHRF1. Lanes 5–8 are the eluted fractions with WT and mutant (K644A, K648A and K644A/K648A) GST-UHRF1. In all instances, approximately 1–2% of the input and 2.5% of the eluate is loaded on the gels.

Fig 3: The D762R/D764R (MRGR) mutation disrupts ICP0, GMPS and UHRF1 binding to USP7 in human cells.(A) 293T cells were transfected with plasmid expressing myc-tagged WT or D762R/D764R (MRGR) USP7 and ICP0 or with WT USP7 or ICP0 expression plasmids alone. USP7 was precipitated with anti-myc antibody and recovered proteins were detected by Western blotting with antibodies against myc and ICP0. (B) 293T cells were transfected with plasmid expressing myc-tagged WT or D762R/D764R (MRGR) USP7 or with empty plasmid (None). USP7 was precipitated with anti-myc antibody and recovered proteins were detected by Western blotting with antibodies against myc, GMPS or UHRF1.