Fig 1.
Ubl123 interacts with ICP0 residues 615–629.
(A) ICP0 domain structure and overlapping peptide sequences. (B) USP7 domain structure and residues of the fragments. (C) Coomassie stained gel of GST pull-down assays between ICP0 peptides and USP7-FL. Lanes 1–8 are the eluted fractions (2.5% on gel). Lane 9 contains a sample of the load (USP7-FL and GST-ICP0 peptide, 1% on gel). Lanes 10 (elute) and 11 (load) are the GST only controls. Lane 12 is USP7-FL alone. (D) Coomassie stained gel of GST pull-down assays between ICP0 peptides and USP7-CTD. Lanes 1–8 are the eluted fractions (2.5% on gel). Lane 9 contains a sample of the load (USP7-CTD and GST-ICP0 peptide, 1% on gel). Lanes 10 (elute) and 11 (load) are the GST only controls. Lane 12 is USP7-CTD alone. (E) Coomassie stained gel of GST pull-down assays between ICP0 residues 615–629 and various USP7 domains. Lanes 1–7 are the eluted fractions with USP7-FL (15%), USP7-NTD (2.5%), Ubl12 (2.5%), Ubl123 (2.5%), Ubl345 (10%), Ubl45 (2.5%) and USP7-CTD (2.5%). Lanes 8–14 are the GST only controls. (F) Coomassie stained gel of loads used in (E). Approximately 1% of the input is loaded on the gel.
Table 1.
Crystallographic statistics.
Fig 2.
The crystal structure of the Ubl123-ICP0 peptide complex.
(A) Interaction between Ubl123 and ICP0 peptide. Ubl123 is shown in cartoon representation and the ICP0 peptide in stick representation. (B) Electrostatic surface representation with acidic regions shown in red and basic regions in blue. (C) Front view of the ICP0 binding site. Interactions between Ub123 and ICP0 are highlighted by dashed lines. (D) Surface representation of the binding pocket with stick model of ICP0. (E) Side view of the ICP0 binding site. Interactions between Ub123 and ICP0 are highlighted by dashed lines.
Fig 3.
Ubl123 E759, D762 and D764 are essential for interaction with ICP0.
(A) Electrostatic surface comparison of the Ubl domains. Ubiquitin and the five Ubls are shown in the same orientation, a black ribbon indicates the loop region ahead of the β4-strand. (B) Structure based sequence alignment of the ICP0 binding region involving key residues D762 and D764 in Ubl2. Since the loop structure and length varies in the five Ubls, only the residues directly neighboring the structurally conserved β4-strand were included. (C) Sequence alignment of ICP0 proteins from human HSV-1 (P08393), human HSV-2 (P28284), chimpanzee HV (K9MG59), macaque HV-16 (X2FDL5), macaque HV-1 (Q7T400), baboon HV-2 (Q5Y0P4), rabbit HV-4 (J9QWJ6), marmoset HV-1 (E2IUH0), and kangaroo HV-1 (Q91CH9). (D) Coomassie stained gel of GST pull-down assays with mutant GST-ICP0 peptides. Lanes 1 (load) and 2 (elute) Ubl123 and GST-ICP0, lanes 3–11 are the eluted fractions with GST-ICP0 mutants. (E) The dissociation constants for the USP7 interaction with FITC-labeled ICP0 peptide. Average values with standard deviation for three or more experiments are shown. (F) GST pull-down assays with mutant Ubl123 (E759A, D762A, D764A and D762A/D764A) and GST-ICP0 peptide. Load (L) and eluate (E) represent the loaded and eluted fractions of each Ubl123 mutant. (G) GST pull-down assays with WT or mutant USP7-CTD (D762A, D764A and D762A/D764A) and GST-ICP0 peptide. Lane 1 USP7-CTD and GST-ICP0 load, lanes 2–5 are the eluted fractions with WT and mutant USP7-CTD. (H) GST pull-down assays with WT or D762R/D764R USP7-CTD and GST-ICP0 594–775. Lanes 1 and 2 are loaded (L) samples. Lanes 3 and 4 are the eluted (E) fractions with WT and D762R/D764R USP7-CTD. In all instances, approximately 1% of the input and 2.5% of the eluate is loaded on the gels.
Fig 4.
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 5.
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.
Fig 6.
Two conformational states of Ubl123.
(A) Extended conformation as observed in Se-Ubl123 (this work) and native USP7-CTD (PDB ID 2YLM). (B) Compact conformation observed for native Ubl123 (this work). (C) Details of contact formation between Ubl1 and Ubl3. (D) Details of contact formation between Ubl1 and Ubl3. (E) Details of the Ubl2-Ubl3 hinge. In F, the extended conformation is partially given for direct comparison; note that S814 is now occupying the former position of the backbone nitrogen atom of N815. A pink arrow indicates the shift from the extended to the compact conformation. The entire Ubl3 domain appears to rotate against Ubl12, the pivot point being between residue H792 and R793. Dashed lines indicate hydrogen bonds and salt-bridges, distances are given in Å. (F) Details of the Ubl2-Ubl3 hinge. In F, the extended conformation is partially given for direct comparison; note that S814 is now occupying the former position of the backbone nitrogen atom of N815. A pink arrow indicates the shift from the extended to the compact conformation. The entire Ubl3 domain appears to rotate against Ubl12, the pivot point being between residue H792 and R793. Dashed lines indicate hydrogen bonds and salt-bridges, distances are given in Å.
Fig 7.
Three-dimensional model of full-length USP7 suggests back folding of Ubl45 onto the CAT domain.
(A) Model generation: I. The crystal structure containing both NTD and CAT domain (blue/yellow, PDB ID 2F1Z) is superposed onto the spacer helix of Ubl123 (salmon, this work); II. The crystal structure of CTD (cyan, PDB ID 2YLM) is superposed onto Ubl1 and Ubl2 of Ubl123; III. The same crystal structure of CTD (magenta, omitting Ubl12 for clarity) is superposed onto Ubl3 of Ubl123 to generate an alternate compact conformation. Separate structures of the NTD containing EBNA1-peptide (red, PDB ID 1YY6) and the CAT domain containing ubiquitin-aldehyde (dark green, PDB ID 1NBF) are superposed onto the corresponding domains. (B) USP7 model in cartoon representation. Domains are shown in the same color-coding as in A. The Ubl2-Ubl3 hinge causes two conformations; a black arrow indicates the corresponding shift. (C) Side-view with Ubl5 and the catalytic domain in the foreground.