Volume 18, Issue 1, Pages 25-36 (April 2005) Structure of the p53 Binding Domain of HAUSP/USP7 Bound to Epstein-Barr Nuclear Antigen 1  Vivian Saridakis, Yi Sheng, Feroz Sarkari, Melissa N. Holowaty, Kathy Shire, Tin Nguyen, Rongguang G. Zhang, Jack Liao, Weontae Lee, Aled M. Edwards, Cheryl H. Arrowsmith, Lori Frappier  Molecular Cell  Volume 18, Issue 1, Pages 25-36 (April 2005) DOI: 10.1016/j.molcel.2005.02.029 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 Crystal Structure of the USP7 NTD with EBNA1 Peptide (A) Ribbon representation of the crystal structure of the USP7 NTD bound by EBNA1 peptide (stick form). (B) Electrostatic surface representation of (A). (C) Superposition of the TRAF domains of USP7 (blue) with TRAF2 (silver). (D) Structure based sequence alignment between the TRAF domains of USP7, TRAF2, TRAF3, and TRAF6. Residues that are identical (asterisks) or conserved (dots) in all four sequences are indicated. Residues involved in EBNA1 binding are in bold. Molecular Cell 2005 18, 25-36DOI: (10.1016/j.molcel.2005.02.029) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 Interactions of EBNA1 Peptides with the USP7 NTD (A) An equimolar mixture of the USP7 NTD and a GST fusion protein containing the indicated EBNA1 peptide (L) was mixed with glutathione-sepharose. After washing, protein was eluted with glutathione (E). (B) Increasing amounts of EBNA1 peptides 395–450 with wild-type sequence (circles) or E444A (squares), S447A (triangles) or E444A/S447A (diamonds) mutations were incubated with the USP7 NTD and binding was quantified by change in tryptophan fluorescence. Molecular Cell 2005 18, 25-36DOI: (10.1016/j.molcel.2005.02.029) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 The Path and Contacts of the EBNA1 Peptide Bound to USP7 (A–C) Transparent surface representation of USP7 bound to EBNA1 peptide (A), TRAF3 bound to TANK peptide (177′-CSVPIQCTDKT-187′; PDB accession number 1KZZ) (B), and TRAF6 bound to CD40 peptide (230′-KQEPQEODF-238′; PDB accession number 1LB6) (C). (D) Comparison of the bound conformations of EBNA1 (red), TANK (green), and CD40 (blue) peptides in the identical orientation as above (generated by superimposing the TRAF domains of the three TRAF-peptide complexes). (E) Electron density of the EBNA1 peptide. The final EBNA1 model is shown in the Fo − Fc difference density obtained after the initial rounds of refinement in the absence of peptide. (F) Detailed interactions between USP7 (silver) and EBNA1 (charcoal) shown in stereo. The H bonds are indicated by dashed lines. Molecular Cell 2005 18, 25-36DOI: (10.1016/j.molcel.2005.02.029) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 Comparison of the Changes in NMR Resonance Frequencies of USP7 upon Binding EBNA1 and p53 Peptides Composite chemical shift changes versus residue number for the USP7 residues 62–205 in the presence of EBNA1 410–450 (A) and p53 355–393 (B). The values shown were calculated by using the equation Δδcomp = [Δδ2HN + (ΔδN/5)2]1/2. The approximate locations of the USP7 NTD secondary structure elements are shown on top with an arrow for β strands and a rod for α helices. Chemical shifts of 0.15 Δδppm or greater induced upon binding EBNA1 (C) or p53 (D) peptides are indicated by the colored residues on the surface representation of the USP7 TRAF domain from the cocrystal structure. Shifted amino acids from strands β3, β4, β6, and β7 are colored in cyan, yellow, purple, and green, respectively. In (C) the position of the EBNA1 peptide from the cocrystal structure is also shown. Molecular Cell 2005 18, 25-36DOI: (10.1016/j.molcel.2005.02.029) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 EBNA1 Expression Affects Cell Survival and p53 Levels through the USP7 Binding Region (A) U2OS cells were transfected with plasmids expressing dsRed and the EBNA1 proteins indicated, then analyzed by TUNEL assay and flow cytometry before and after UV irradiation. The percentage of transfected (dsRed positive) cells that became TUNEL positive after UV irradiation is shown. (B) H1299 cells were transfected with an EGFP expression plasmid, a p53 expression plasmid or empty plasmid, and a plasmid expressing EBNA1, Δ395–450, or no EBNA1. Cells were stained for Annexin V and FACS sorted. The percentage of transfected (EGFP positive) cells that stained with Annexin V are shown after subtraction of the background staining seen in the absence of p53 expression. (C) U2OS cells expressing EBNA1, Δ395–450, or no EBNA1 were lysed before (0), 4 hr after, or 8 hr after UV irradiation and analyzed for p53 and actin levels by Western blotting. (D) Combined data from three experiments showing relative levels of p53 after UV induction in U2OS cells expressing EBNA1, Δ395-450, or no EBNA1. SD is indicated by the error bars. Molecular Cell 2005 18, 25-36DOI: (10.1016/j.molcel.2005.02.029) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 6 EBNA1-USP7 Interaction in B Cells (A) Comparison of cellular proteins retained on an EBNA1 affinity column from B cell (BL41) and HeLa cell lysates. A negative control of BL41 lysates applied to a column lacking EBNA1 is also shown. Labeled protein bands were identified by MALDI-ToF mass spectrometry. (B) Coimmunoprecipitation of EBNA1 with USP7 from Raji cell lysates (L) using the indicated amount of USP7 antibody. Western blots were probed with antibodies against USP7 (top) and EBNA1 (bottom). Molecular Cell 2005 18, 25-36DOI: (10.1016/j.molcel.2005.02.029) Copyright © 2005 Elsevier Inc. Terms and Conditions