USP7 Bound to Epstein-Barr Nuclear Antigen 1

Molecular Cell, Vol. 18, 25–36, April 1, 2005, Copyright ©2005 by Elsevier Inc. DOI 10.1016/j.molcel.2005.02.029

Structure of the p53 Binding Domain of HAUSP/USP7 Bound to Epstein-Barr Nuclear Antigen 1: Implications for EBV-Mediated Immortalization Vivian Saridakis,1,6 Yi Sheng,2,6 Feroz Sarkari,1 Melissa N. Holowaty,1 Kathy Shire,1 Tin Nguyen,1 Rongguang G. Zhang,5 Jack Liao,2 Weontae Lee,2 Aled M. Edwards,1,3,4 Cheryl H. Arrowsmith,2,3,4 and Lori Frappier1,* 1 Department of Medical Genetics and Microbiology 2 Ontario Cancer Institute and Department of Medical Biophysics 3 Banting and Best Department of Medical Research 4 Structural Genomics Consortium University of Toronto Toronto, Ontario M5S 1A8 Canada 5 Biosciences Division Structural Biology Center Argonne National Laboratory 9700 South Cass Avenue Argonne, Illinois 60439

Summary USP7/HAUSP is a key regulator of p53 and Mdm2 and is targeted by the Epstein-Barr nuclear antigen 1 (EBNA1) protein of Epstein-Barr virus (EBV). We have determined the crystal structure of the p53 binding domain of USP7 alone and bound to an EBNA1 peptide. This domain is an eight-stranded ␤ sandwich similar to the TRAF-C domains of TNF-receptor associated factors, although the mode of peptide binding differs significantly from previously observed TRAFpeptide interactions in the sequence (DPGEGPS) and the conformation of the bound peptide. NMR chemical shift analyses of USP7 bound by EBNA1 and p53 indicated that p53 binds the same pocket as EBNA1 but makes less extensive contacts with USP7. Functional studies indicated that EBNA1 binding to USP7 can protect cells from apoptotic challenge by lowering p53 levels. The data provide a structural and conceptual framework for understanding how EBNA1 might contribute to the survival of Epstein-Barr virusinfected cells. Introduction EBV infects more than 90% of people worldwide and efficiently immortalizes infected cells, predisposing the host to a variety of cancers. Cellular immortalization by EBV occurs as part of its latent infectious cycle and involves a few EBV proteins including LMP1, which mimics an activated tumor necrosis factor receptor, and EBNA2, which activates the transcription of several cellular and viral genes (Dolcetti and Masucci, 2003). Whereas cellular transformation by other DNA tumor viruses (e.g., adenovirus, SV40, and papillomavirus) has clearly been shown to involve targeting of the p53 tu*Correspondence: [email protected] 6 These authors contributed equally to this work.

mor suppressor protein, surprisingly none of the EBV proteins required for immortalization have been shown to act through p53. EBNA1 is the only EBV protein consistently expressed in all proliferating infected cells and plays several important roles in EBV latent infection, including the initiation of EBV DNA replication, the mitotic segregation of the EBV genomes, and transcriptional activation of other EBV latency proteins (Kieff and Rickinson, 2001). In addition, several pieces of evidence suggest that EBNA1 plays a direct role in cellular transformation by EBV. First, EBNA1 is expressed in all EBV-associated tumors and is the only viral protein expressed in some of these tumors. Second, transgenic mice expressing EBNA1 develop malignant B cell lymphomas (Wilson et al., 1996). Third, the expression of EBNA1 in Hodgkin cells enhances their ability to form tumors in nonobese diabetic-SCID mice (Kube et al., 1999). Fourth, EBV genomes lacking the EBNA1 gene are several thousand-fold less efficient at B cell immortalization than EBV genomes expressing EBNA1 (Hume et al., 2003). Fifth, interference with EBNA1 function in Burkitt’s lymphoma cells by overexpression of the EBNA1 DNA binding domain increased cell death, suggesting that EBNA1 normally provides a survival function for these cells (Kennedy et al., 2003). EBNA1 was recently shown to stably interact with the ubiquitin-specific protease called USP7 or HAUSP (Herpes virus Associated USP; [Holowaty et al., 2003b]), which was originally identified as a binding target of the ICP0 protein of herpes simplex virus (Everett et al., 1997). EBNA1 sequences mediating this interaction were mapped to within amino acids 395–450, just N-terminal to the DNA binding domain. An EBNA1 mutant lacking this sequence (⌬395–450) failed to bind USP7 but continued to bind other known cellular protein targets of EBNA1. Functional studies with ⌬395– 450 showed that USP7 binding was not required for the replication, segregation, or transcriptional activation functions of EBNA1 but may inhibit the ability of EBNA1 to activate replication (Holowaty et al., 2003b). The deletion of the USP7 binding sequence also had no detectable effect on EBNA1 turnover or cell surface presentation. The lack of requirement of the USP7 interaction for the known EBNA1 functions suggested that the significance of this interaction may lie in EBNA1induced changes to the cell. A link to the p53 pathway was revealed by Li et al. (2002b), who showed that USP7 bound and deubiquitinated p53. Overexpression of USP7 stabilized p53, resulting in p53-mediated growth repression and apoptosis, whereas decreased USP7 levels destabilized p53. However, the role of USP7 in p53 regulation was recently shown to be more complicated than originally thought, as ablation of USP7 expression resulted in p53 accumulation, as opposed to the expected destabilization of p53 (Cummins et al., 2004; Li et al., 2004). This effect has been shown to be the result of the ability of USP7 to stabilize Mdm2, a ubiquitin ligase that promotes the degradation of p53. Therefore USP7 appears

Molecular Cell 26

Table 1. X-Ray Data Collection, Structure Solution, and Refinement Parameters X-Ray Data

Native

Peak

EBNA1 Complex

Space group Resolution (Å) Unit cell axes (Å3) Molecules/AU Se sites (No.) Total observations (No.) Unique reflections (No.) Intensity (I/σ) Completeness (%) a Rsym b Figure of merit (%) MR RF-function MR monitor c Rwork Rfree Protein atoms (No.) Water molecules (No.) Sodium atoms (No.) Rmsd bonds (Å) Rmsd angles (°) Rmsd dihedrals (°) Rmsd improper (°) Thermal factor (Å2)

P32 1.9 102.6 × 102.6 × 45.2 3 — 103 005 40 510 20.8 (2.7) 96.7 (80.6) 0.049 (0.289) — —

P32 2.0 102.5 × 102.5 × 45.0 3 12 24 8482 70 663 16.8 (2.7) 98.8 (91.9) 0.070 (0.336) 32.3 —

P41 1.7 70.0 × 70.0 × 45.9 1 — 96 636 24 692 13.7 (1.8) 99.7 (97.7) 0.078 (0.534) — 0.099 0.48 0.191 0.225 1181 186 21 0.006 1.40 25.4 0.94 18.7

0.235 0.295 2473 90 — 0.009 1.57 27.4 0.97 22.8

Numbers in brackets refer to the highest resolution shell, 1.97–1.90 Å for the native data, 2.07–2.00 Å for the MAD data, and 1.76–1.70 Å for the EBNA1 complex data. Data were integrated and scaled by using HKL2000. a Rsym = S|I − |/SI where I is the observed intensity and is the average intensity from multiple observations of symmetry-related reflections. b Figure of Merit of Phasing = |SP(α)eια|/SP(α) where P(α) is the phase probability distribution and α is the phase angle. c R = S|Fobs − Fcalc|/|Fobs|.

to play multiple roles in regulating the p53-Mdm2 pathway. The USP7-p53 interaction occurs between the USP7 N-terminal domain (NTD), amino acids 53–208, and residues 357–382 of the C-terminal regulatory region of p53 (Hu et al., 2002). This USP7 domain, which is similar in sequence to a TRAF domain (Zapata et al., 2001), is also responsible for the interaction with EBNA1 (Holowaty et al., 2003a). The fact that EBNA1 and p53 bind the same domain of USP7 raises the possibility that EBNA1 affects the regulation of p53 by disrupting the interaction of USP7 with p53. Indeed, the 395–450 fragment of EBNA1 binds USP7 with 1 ␮M affinity, whereas the p53 regulatory fragment (with or without the tetramerization domain) binds with 10-fold lower affinity (Holowaty et al., 2003a). The EBNA1 peptide 395–450 also displaces the p53 peptide from the p53-USP7 complex. These results indicate that EBNA1 and p53 bind the same or overlapping sites in the USP7 NTD and suggest that EBNA1 could sequester USP7 from p53 in vivo, thereby destabilizing p53. In this paper, we provide structural and functional evidence supporting a connection between EBNA1 and p53. Results Structure of the USP7 NTD To gain insight into the molecular basis for the EBNA1 and p53 interactions with USP7, the structure of the USP7 NTD (residues 54–204), previously shown to bind p53 and EBNA1, was determined; first alone and then in complex with a peptide from EBNA1 (see Table 1 for

structure solution and refinement statistics). The structure of USP7 was determined by using single anomalous dispersion, and the model was refined to 2.0 Å resolution. USP7 NTD is composed of a single domain with approximate dimensions 51 × 30 × 30 Å3, with structural similarity and identical topology to the TRAF-C domain of tumor necrosis factor-receptor associated factors (TRAFs) 2, 3, and 6 (Li et al., 2002a; Ni et al., 2000; Park et al., 1999; Ye et al., 2002; Ye et al., 1999). Like all TRAF domains, USP7 forms an eightstranded, antiparallel β sandwich (Figure 1A). Strand β7 contains a β bulge, which is important in peptide binding and found in all TRAF domain structures. Structure comparison using DALI identified the TRAF domain of TRAF2 (PDB accession number 1D0A) as the closest structural neighbor of the USP7 NTD with a Z score of 9.4 and an rmsd of 2.8 Å over 93 Cα atoms (see Figure 1C for superposition of these domains). A structure-based sequence alignment for the TRAF domains of USP7, TRAF2, TRAF3, and TRAF6 is shown in Figure 1D. Mapping of USP7 Binding Site on EBNA1 An EBNA1 fragment containing amino acids 395–450 stably binds the USP7 NTD (Holowaty et al., 2003a). Because TRAF domains typically bind peptides of ten amino acids or less, we imagined that the EBNA1 sequence involved in USP7 binding could be further localized to a shorter amino acid sequence. Consensus binding motifs have been identified that are important for binding TRAFs 2, 3, and 6 (see below), prompting us to search for these motifs in the EBNA1 395–450

Structural Basis for EBNA1 and p53 Binding to USP7 27

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.

fragments. Because obvious matches to any of these motifs were not evident, the USP7 binding sequence in EBNA1 was localized experimentally. A series of EBNA1 peptides fused to GST (420–450, 421–435, 426–440, 431–445, and 436–450) were used to assess interactions with the USP7 NTD by GST pull-down experi-

ments. The results showed a stoichiometric interaction between USP7 and fusion proteins containing EBNA1 residues 420–450 and 436–450 but little or no interaction with fusion proteins containing EBNA1 residues 421–435, 426–440, and 431–445 (Figure 2A). The USP7 binding sequence of EBNA1 is therefore found within

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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.

amino acids 436–450, and residues between 445 and 450 are essential for the USP7 interaction. This information was used to guide EBNA1 peptide design for cocrystallization trials with USP7. Structure Overview of the USP7-EBNA1 Complex A peptide corresponding to EBNA1 amino acids 441– 450 was cocrystallized with the USP7 NTD. The structure of the complex was determined by using molecular replacement, and the model was refined to 1.7 Å resolution. The EBNA1 peptide (acetyl-DPGEGPSTGP-amide) was located in the Fo − Fc difference electron density map after the initial round of refinement with the protein model alone (Figure 3E). Excellent density allowed residues 441#–448# of the EBNA1 peptide to be built unambiguously. There was no visible density for the aminoterminal acetyl moiety and residues 449# and 450#. The EBNA1 peptide was in an extended conformation and bound to the edge of the β sandwich. The peptide formed main chain H bonds solely with strand β7 in an antiparallel manner, increasing the number of strands in one of the sheets from four to five (Figures 1A and 3F). Like the unbound USP7 NTD, the EBNA1 bound domain was very similar in structure to other TRAF domains (Figure 1C). No significant conformational changes were observed between the peptide-free and EBNA1 bound USP7 NTD. The rmsd was 0.4 Å over 102 Cα residues. Within the region of peptide binding, there were some minor changes. In the peptide complex, the side chain of Asp164 rotated to interact with the side chain hydroxyl of Ser447#, and the side chain of Trp165 rotated to interact with OD1 of Glu444#. Specifically, atom OD1 of Asp164 moved 3.3 Å toward the peptide, and the NE1 atom of Trp165 moved 1.4 Å toward the

peptide. There were also differences in the lattice contacts formed in crystals of the USP7 NTD in the presence and absence of EBNA1 peptide. In the absence of the peptide, the USP7 NTDs interacted through the peptide binding surface and, as a result, these crystals were disrupted by soaking in the EBNA1 peptide. In crystals generated by the USP7-EBNA1 complex, lattice contacts were not observed in the peptide binding region but were confined to the loops connecting the β strands (amino acids 77, 80, and 82 interacted with residues 178, 180, and 181). USP7-EBNA1 Interactions The EBNA1 peptide bound to a shallow depression on the surface of USP7 (Figure 1B). Upon binding to USP7, the EBNA1 peptide buried a total of 742.3 Å2 of surface area. A combination of hydrophobic and hydrophilic interactions was formed between EBNA1 and USP7 (Figure 3F). These interactions occur predominantly with strand β7; however, strand β6 and loop β3-β4 are also involved. H-bonds are formed between OE1 of EBNA1Glu444# and NE1 of Trp165 as well as between OG of EBNA1-Ser447# and OD1 of Asp164. These are the two interactions that are predicted to confer specificity to EBNA1 for USP7 as they are the only H-bonding interactions that occur between side chains of USP7 and EBNA1. The carbonyl group of EBNA1-Ser447# interacts with NH1 of Arg104 from loop β3-β4. OE2 of EBNA1-Glu444# interacts with the amide of Ser155, and OE1 of EBNA1-Glu444# interacts with the carbonyl of Arg153 on strand β6 through bridging water molecules. The carbonyl of EBNA1-Pro 442# interacts with the amide side chain of Asn169. There are also four main chain H bonds formed between EBNA1 and strand β7. Hy-

Structural Basis for EBNA1 and p53 Binding to USP7 29

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.

drophobic interactions occur between the aliphatic side chains of EBNA1-Glu444# and Phe167, EBNA1Ser447# and Phe118, as well as EBNA1-Gly445# and Trp165. Comparison of the Peptide Interactions of USP7 and Other TRAF Domains The EBNA1 peptide binds to a groove within the USP7 TRAF domain at a position similar to that of peptides

bound to other TRAF domains. However, the conformation of the EBNA1 peptide differs from that of all other TRAF bound peptides whose structures have been determined, as shown in comparison to TRAF3 and TRAF6 bound peptides (Figures 3A–3D). Peptides bound to TRAF2 and TRAF3 run from top to bottom of the TRAF domain in an extended conformation (Figure 3B) cutting across and largely perpendicular to strands β3, β4, β6, and β7 (Ni et al., 2000; Park et al., 1999; Ye

Molecular Cell 30

et al., 1999). The path of TRAF6 bound peptides is similar to this, although it deviates from the TRAF2 peptide path by 40° and makes more extensive contacts with strand β7 (Li et al., 2002a; Ye et al., 2002) (Figure 3C). The most extensive β7 contacts have been seen with the CD40 peptide, which makes five main-chain H bonds with strand β7. EBNA1 peptide follows strand β7 even more extensively than CD40, making seven different contacts with six different β7 residues, and, as a result, bends relative to the other peptides at the position of Glu444# (Figures 3A and 3D). Another difference that distinguishes the EBNA1USP7 interaction from other peptide-TRAF interactions is the sequence of the bound EBNA1 peptide, which does not conform to the known TRAF binding sequence motifs. TRAF-peptide interactions can be classified into two groups based on their peptide specificity. TRAF6 binds the consensus sequence, PxExx (f/acidic) (Ye et al., 2002), whereas TRAFs 1, 2, 3, and 5 all bind the consensus sequence (P/S/A/T) × (Q/E)E and its variants (fSxEE, QEE, and PxQxxD) (Park et al., 1999; Ye et al., 1999). The TRAF1, TRAF2, TRAF3, and TRAF5 residues involved in peptide binding are absolutely conserved (including Arg393, Tyr395, Phe447, Ser453, Ser454, Ser455, and Ser467 according to TRAF2 numbering) despite the fact that these TRAF domains share only 52%–64% sequence identity (Wajant et al., 2001). The EBNA1 peptide bound by USP7 (PGEGPS) does not match any of the consensus sequences identified for binding other TRAF domains. Although the EBNA1 sequence contains a PxE motif, which is a found within some of the TRAF binding consensus sites, the structural contacts made by these EBNA1 residues are different than those of other TRAF bound peptides. In all previously solved TRAF domains, the Pro and Gln/Glu residues in the bound peptides are largely super imposable. However, the Pro and Glu residues of the EBNA1 peptide are out of register in comparison to the TRAF bound TANK and CD40 peptides (Figure 3D). The Cα of EBNA1-Glu444# is 7.1 Å away from the Cα of Gln182# from the TANK peptide and 7.4 Å away from the Cα of Glu235# from the CD40 peptide. The Cα of Pro442# from EBNA1 is 9.4 Å away from the Cα of Pro180# from the TANK peptide and 6.8 Å away from the Cα of Pro233# from the CD40 peptide. The functions of the PxE motifs are also different between EBNA1 and the other TRAF binding peptides. For example, in all TRAF2, TRAF3, and TRAF6 interactions, the Pro and Gln/Glu residues of the bound peptide make specific interactions with the TRAF domain and within the peptide itself (Li et al., 2002a; Li et al., 2003; McWhirter et al., 1999; Ni et al., 2000; Park et al., 1999; Ye et al., 2002). In EBNA1-USP7 interaction, the specific interactions are mediated by the Glu and Ser residues. Thus the PxE sequence in EBNA1 appears to be fortuitous. Consistent with the different peptide sequence bound by USP7, the ten USP7 residues that contact the EBNA1 peptide are not conserved in other TRAF domains (Figure 1D), with the exception of Phe118 and Gly166 (USP7 numbering), which make similar peptide interactions in all TRAF-peptide structures. Phe118 and Gly166 make aliphatic interactions with the appropriately positioned peptide residues and two main chain

H bonds with the peptide, respectively. USP7 does not contain the residues that mediate peptide interactions in TRAF2 and TRAF3 and are highly conserved in TRAFs 1, 2, 3, and 5 (Arg393, Tyr395, Phe447, Ser453, Ser454, Ser455, and Ser467) nor are residues at most of these positions used for EBNA1 peptide binding. Similarly, the residues in TRAF6 that are important for peptide binding (Arg392, Phe471, and Tyr473) are not conserved in USP7. Therefore, USP7 forms a new class of peptide binding TRAF domain. To further investigate the specificity of the USP7 TRAF domain, we tested binding to five different peptides known to bind TRAF2 or TRAF6; namely, the TRAF2 binding peptides QVPFSKEEC from TNF-R2 (Park et al., 1999), PQQATDDSS from LMP1 (Ye et al., 1999), and PVQETLH from hCD40 (Ye et al., 1999), and the TRAF6 binding peptides QMPTEDEY from TRANCE-R (Ye et al., 2002) and KQEPQEIDF from hCD40 (Ye et al., 2002). None of these peptides gave detectable binding to USP7 at any of the concentrations tested (up to 100 ␮M), whereas the cocrystallized EBNA1 10 mer peptide bound USP7 with a Kd of 0.86 ␮M in the same assay. Because EBNA1-USP7 sequence-specific contacts in our structure were mediated by Glu444# and Ser447# of EBNA1, we also generated the EBNA1 395–450 amino acid fragment with these two residues mutated to alanines in order to verify their importance for USP7 binding. In keeping with the structural information, mutation of these residues severely decreased binding to USP7 such that no binding was detected up to 100 ␮M, whereas the wild-type (wt) 395–450 fragment bound USP7 with a Kd of 0.97 ␮M (Figure 2B). Point mutation of the Glu444# residue alone only slightly decreased binding, giving a Kd of 1.36 ␮M, whereas no binding was detected when Ser447# was mutated to alanine (Figure 2B). Thus, Ser447 plays a major role in USP7 binding. Comparison of the Interaction of p53 and EBNA1 Peptides with USP7 by NMR EBNA1 and p53 bind to the TRAF domain of USP7 and compete for binding. To elucidate the molecular basis of p53 binding, we used NMR chemical shift mapping to compare the binding of EBNA1 and p53 peptides to USP7. 85% of the backbone resonances were assigned for the NTD of USP7 (amino acids 62–205), and NMR titration experiments were performed on uniformly 15Nlabeled USP7 in the presence of unlabeled p53 (amino acids 355–393) and unlabeled EBNA1 (amino acids 410–450). Upon binding EBNA1 peptide, the resonances in β7 of USP7 showed strong perturbations (Figure 4A), especially residues Trp165, Gly166, Phe167, Ser168, Asn169, Phe170, and Met171. The resonances for some residues in β3, β4, and β6 were also affected, including Met100, Met102, Phe 117, Phe118, and Ser155. The results from NMR mapping data agree well with the crystal structure of the USP7 NTD EBNA1 complex (Figure 4C). Phe117 and Phe118 of β4 form contacts with the backbone amide groups of the peptide. Though the backbone amides of Met100 and Met102 (β3) seem far away from the peptide, their side chains are in close proximity of the C terminus of the peptide. Shorter EBNA1 peptides (amino acids 420–450 and

Structural Basis for EBNA1 and p53 Binding to USP7 31

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 ⌬δcomp = [⌬δ2HN + (⌬δN/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 ⌬δppm 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.

441–450) were also used in these experiments and gave the same chemical shift perturbation pattern as the longer one, indicating that EBNA1 residues outside 441–450 do not contact USP7 (data not shown). The addition of the p53 peptide also induced chemical shift perturbations in the 1H-15N HSQC spectra of USP7. All chemical shift changes observed in the p53 complex were also observed in the EBNA1 complex. However, compared with EBNA1 peptides, p53-induced changes were smaller both in magnitude and in the number of affected residues (Figures 4B and 4D). In the p53 complex, strong chemical shift changes were observed in β7 but only for residues Trp165, Gly166, and Phe167, indicating a smaller binding surface and a lower binding affinity for p53 peptide. The chemical shift patterns in the EBNA1 complex suggest that EBNA1 extends its binding site further along β7 and forms more specific interaction with the USP7 NTD. Residues Phe117, Phe118, and Leu119 from β4 were affected in the p53 complex, suggesting that interactions of the USP7 NTD with the C terminus of the peptides are similar for both p53 and EBNA1 peptides. Chemical shift changes for the residues in β3 and β6 (Met100, Met102, and Ser155) that were affected in the EBNA1 complex were not observed in the p53 complex.

Functional Studies on the EBNA1-USP7 Interaction Our biochemical (Holowaty et al., 2003a) and structural studies on the EBNA1-USP7 interaction both indicate that EBNA1 binding to USP7 could disrupt the interaction of USP7 with p53, which would be predicted to destabilize p53. Therefore EBNA1 expression in human cells might prevent p53-induced cell cycle arrest or apoptosis and contribute to cell immortalization by EBV. To test this possibility, U2OS cells were cotransfected with a plasmid expressing dsRed (transfection marker) and either an empty plasmid or a plasmid expressing EBNA1 or the ⌬395–450 EBNA1 mutant. This EBNA1 mutant does not bind USP7 but has the same stability and other known protein interactions as wt EBNA1 (Holowaty et al., 2003b). The cells were then UV irradiated to induce apoptosis, which was detected by deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assay followed by FACS analysis. EBNA1 expression decreased the percentage of apoptotic cells after UV irradiation, whereasa the ⌬395–450 EBNA1 mutant had little effect (Figure 5A) despite being expressed at similar levels as EBNA1 (data not shown). Similar results were obtained when apoptosis was induced in H1299 p53 null cells by p53 overexpression from a transfected plasmid and detected by Annexin V

Molecular Cell 32

staining (Figure 5B). The percentage of transfected cells (as determined by the EGFP marker) that was Annexin-positive was compared with and without p53 overexpression in the presence and absence of EBNA1. EBNA1 reduced the percentage of apoptotic cells generated by p53 overexpression to just above the background level of Annexin V staining seen in the absence of p53, but little effect was seen with the ⌬395–450 EBNA1 mutant. Results in Figures 5A and 5B are representative experiments; EBNA1 effects are similar in repeat experiments, but absolute numbers of apoptotic cells vary between experiments. The results indicate that, at least under these experimental conditions, EBNA1 can protect cells from apoptotic challenge, and this effect is largely due to the USP7 binding region of EBNA1. The above results suggest that EBNA1 may lower p53 levels by disrupting the p53-USP7 interaction. To test this possibility, we transfected U2OS cells with an oriP plasmid expressing EBNA1, ⌬395–450, or no EBNA1 and grew the cells under selection for the plasmid for 2 weeks. At that point, EBNA1 and ⌬395–450 were found to be expressed in w60% of the cells at levels equivalent to those in the EBV-transformed Raji cells (data not shown). Aliquots of the cells were then analyzed for p53 expression by Western blotting before and after induction of p53 by UV irradiation (Figures 5C and 5D). Although the relative level of p53 in the cell lines expressing and not expressing EBNA1 varied prior to UV treatment (i.e., p53 levels in EBNA1-expressing cells were either equivalent to or lower than those lacking EBNA1 expression), the degree to which p53 levels increased upon UV induction was consistently less in EBNA1-expressing cells than in cells expressing ⌬395– 450 or not expressing EBNA1. Therefore, EBNA1 can interfere with the stabilization of p53 by USP7 in response to UV irradiation. EBNA1-USP7 Interaction in B cells The interaction between EBNA1 and USP7 to date has been studied in epithelial cells. This is biologically relevant because EBV infects epithelial cells and can promote their transformation as evidenced by the induction of nasopharyngeal carcinoma and other epithelial-based tumors. However, because B lymphocytes are a major site of EBV infection and persistence, we wanted to verify that the EBNA1-USP7 interaction could also occur in this cell background. To this end, we used the EBNA1 affinity column approach, which

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, ⌬395–450, 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, ⌬395–450, 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, ⌬395450, or no EBNA1. SD is indicated by the error bars.

Structural Basis for EBNA1 and p53 Binding to USP7 33

We also tested whether EBNA1 and USP7 would coimmunoprecipitate from EBV-infected B cells by using the Raji Burkitt’s lymphoma cell line. We found that a proportion of the USP7 from Raji cell lysates could be immunoprecipitated and that EBNA1 coimmunoprecipitated with USP7 (Figure 6B). Therefore, EBNA1 efficiently interacts with USP7 in B cells as it does in epithelial cells. Discussion

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).

we had originally used to profile epithelial cell protein interactions of EBNA1 (Holowaty et al., 2003b), to compare the EBNA1 interactions seen in HeLa and B cell (BL41) lysates. The specific protein interactions of EBNA1 that were defined with HeLa lysates are also seen in B cell lysates, although the relative efficiency of the interactions varies in the two lysates (Figure 6A). Notably, the interaction with USP7 is more prevalent in the B cell lysate than in the HeLa cell lysate.

USP7 plays an important role in regulating cell proliferation and apoptosis through p53 and Mdm2 interactions. We have determined the structure of the p53 binding domain of USP7 alone and bound to an EBNA1 peptide and found that it forms a TRAF domain. This is consistent with a previous prediction based on limited sequence homology between this region of USP7 and known TRAF domains (Zapata et al., 2001). The location and orientation of the EBNA1 peptide on USP7 is similar to other known peptide-TRAF interactions; however, the sequence of the bound EBNA1 peptide (PGEGPS), the specific USP7 residues contacted by the peptide, and the conformation of the bound peptide are all significantly different from other TRAF-peptide complexes. The USP7 TRAF domain is also unique in that it is a monomer. No oligomeric interactions between USP7 NTDs were observed in the crystal structure, consistent with our previous analysis of full-length USP7 by analytical centrifugation that indicated that USP7 is a monomer (Holowaty et al., 2003a). TRAF proteins that mediate signaling form homo- and heterotrimers that involve interactions between the loops in the TRAF domains as well as through a coiled coil found just N-terminal to the TRAF domain (Chung et al., 2002; Park et al., 1999). There is no evidence of such a coiled-coil domain in USP7. Although some interaction of the purified USP7 NTD with in vitro-translated TRAFs was reported (Zapata et al., 2001), it remains to be determined if USP7 can heterotrimerize with TRAF proteins in vivo. The only other TRAF domain whose structure has been determined is that of the Siah ubiquitin ligase, and this TRAF domain forms dimers (Polekhina et al., 2002). p53 binds the USP7 NTD through residues located between amino acids 357 and 382 but with a 10-fold lower affinity than EBNA1 (Holowaty et al., 2003a; Hu et al., 2002). This p53 region does not contain a match to the USP7 binding sequence of EBNA1 nor is the ExxS motif that is responsible for sequence-specific contacts between EBNA1 and USP7 evident in the USP7 binding region of p53. This indicates that the peptide binding pocket in the TRAF domain of USP7 can accommodate sequence variation and the exact p53 sequence (within the 357–382 fragment) that contacts USP7 will have to be determined experimentally. We are currently defining the p53 sequence bound by USP7, which should enable the generation of p53USP7 cocrystals suitable for structure determination. EBNA1 binds USP7 through amino acids 442–447. Alignments between EBNA1 homologs of EBV-like viruses that infected other primates (cercopithicine herpesvirus 15, cynomolgus EBV, and Herpesvirus papio)

Molecular Cell 34

showed that, this USP7 binding sequence (PGEGPS) is absolutely conserved in these viruses, whereas sequences in the 395–430 region are highly divergent. This suggests that EBNA1 residues 442–447 are functionally important for the virus and that USP7 interactions likely also occur with EBV-related viruses. Because USP7 binding to p53 results in the stabilization of p53 (Li et al., 2002b), our structural data on the EBNA1 and p53 interactions with USP7 predict that EBNA1 would interfere with the stabilization of p53 by blocking the p53-USP7 interaction. In keeping with the prediction, EBNA1, but not an EBNA1 mutant deficient in USP7 binding, was found to increase the survival of cells that were induced to undergo apoptosis either by DNA damage or p53 overexpression. Similar antiapoptotic effects of EBNA1 have been reported by Kennedy et al. (2003). In keeping with this antiapoptotic effect, EBNA1 was found to decrease the stabilization of p53 that occurs in response to UV-induced DNA damage, and this effect required the USP7 binding region of EBNA1. Not surprisingly, we have not seen reproducible effects of EBNA1 expression on p53 levels in rapidly growing cells where p53 is unstable and expressed at low levels. The apoptotic protection experiments presented here were performed in the presence of EBNA1 that was expressed at levels approximately 8-fold higher than in Raji cells, and it remains to be determined whether similar protection is conferred by EBNA1 in EBV-infected cells. However, this possibility is supported by the findings that (1) EBNA1 binds USP7 in EBV-infected cells and (2) EBNA1 interferes with the UV-induced stabilization of p53 when expressed at levels similar to those in EBV-infected cells. Overall, the data indicate that EBNA1 can indirectly destabilize p53 by binding USP7, which could be important for initial cell immortalization by EBV, continued proliferation and survival of latently infected cells, and/or malignant transformation. Experimental Procedures Expression and Purification of USP7 USP7 fragments coding for amino acids 54–205 and 62–205 were expressed in E. coli from pET15b plasmids and purified as described (Holowaty et al., 2003a). Expression of selenomethionine (Se-Met)-containing USP7 54–205 was conducted in BL21(DE3) Gold cells according to Doublie (1997) and purified as for native protein. For NMR measurements, uniformly labeled 15N, 13C/15N, and 13C/15N/2H USP7 (amino acids 62–205) were produced in M9 media with 15N ammonium chloride (0.8 g/l) and 13C glucose (2 g/l) as the sole nitrogen and/or carbon sources, respectively, and using deuterated water (90%) for 2H-labeled samples. Labeled USP7 62– 205 for NMR was prepared as in Holowaty et al. (2003a) except that buffers containing 20 mM sodium phosphate (pH 7.5), 250 mM NaCl, 2 mM DTT, and 90% H2O/10%D2O were used. The concentration of the purified proteins for NMR experiments ranged between 0.5 and 0.8 mM. Peptide Preparation The EBNA1 peptide crystallized with USP7 consisted of amino acids 441–450 (DPGEGPSTGP). This peptide was synthesized by Dalton Chemicals (Toronto, Canada) with both amino terminal acetylation and carboxy-terminal amidation to mimic the native peptides. Peptides containing known TRAF binding sequences used in USP7 binding assays were also synthesized by Dalton Chemicals. Human p53 (355–393) and EBNA1 (410–450) peptides used in NMR studies and the EBNA1 395–450 fragment (with or without point

mutations in Glu444, Ser447, or both) were generated by expression of relevant sequences from pET15b (Novagen) and purification as described in Holowaty et al. (2003a). Resulting clones were sequence verified. Crystallization, Data Collection, and Structure Determination of Unbound USP7 NTD Crystals of native or Se-Met-enriched USP7 NTD (30 mg/ml) were obtained in 35% MPD, 0.2 M MgOAc, and 0.1 M MES, (pH 6.5). Complete native and MAD data sets from frozen crystals were collected at beamline 19ID at the Advanced Photon Source by using the SBC-3 CCD detector. Data collection statistics are presented in Table 1. The native and MAD data were merohedrally twinned with a twinning factor of 0.36 for the native data and 0.40 for the MAD data. The structure was determined by using single anomalous dispersion with the peak data. There were three USP7 molecules in the asymmetric unit and each was monomeric. The selenium substructure was located as described in Saridakis et al. (2004), and 73 out of 151 amino acids of molecule A were built automatically (residues 70–77, 85–100, 116–121, 128–158, and 191– 202). Models of molecules B and C were built manually. The final protein model of molecule A consists of 97 residues from 65–78, 85–104, 115–142, 150–173, and 188–203. The amino terminus and four loops are completely disordered. In molecule B, the following residues were modeled: 67–78, 85–104, 115–142, 150–175, and 189–204 and in molecule C, 67–74, 85–102, 115–142, 150–178, and 188–204. The rmsd between the different molecules ranges from 1.0 Å for molecules B and C to 1.3 Å for molecules A and C or B and C over the same number of Cα residues. The final models were refined to 2.0 Å with an Rcryst of 23.5 and an Rfree of 29.5 and contain 62 water molecules. All of the residues are in the best regions of the Ramachandran plot. Crystallization, Data Collection, and Structure Determination of EBNA1 Bound USP7 NTD USP7 NTD (100 mg/ml) was cocrystallized with a 10 mer EBNA1 peptide corresponding to EBNA1 amino acids 441–450 at 1.5-fold molar excess of peptide. Large clusters of rods appeared after 4 weeks at 4°C in the dark in three conditions containing 30% PEG 4000, 0.1 M Tris (pH 8.5), and either lithium sulfate, magnesium chloride, or sodium acetate. The structure was determined by using molecular replacement and was refined and rebuilt as described in Saridakis et al. (2004). The final model refined to an Rwork of 0.21 and an Rfree of 0.25. There are 186 water molecules and 21 sodium ions. All residues are in the most favored and additionally allowed regions of the Ramachandran plot. Residues 54–62 and 107–111 are disordered in the final model of the complex. A summary of data collection and refinement statistics is presented in Table 1. Generation of GST-EBNA1 Fusion Proteins and Use in USP7 Binding Assays Oligonucleotides encoding EBNA1 amino acids 421–435, 426–440, 431–445, and 436–450 were expressed as GST-fusions from GST2TK plasmid (Amersham) in BL21-CodonPlus E. coli (Stratagene) at 37°C for 3 hr. Proteins were purified on glutathione-sepharose (Amersham) by using standard methods then dialyzed against assay buffer (20 mM sodium phosphate [pH 7.5], 250 mM NaCl, 2 mM DTT, 1 mM Benzamidine, and 0.5 mM PMSF). For USP7 binding assays, purified USP7 NTD (amino acids 62–205) was incubated with GST or GST-EBNA1 fusion proteins in the assay buffer in a 1:1 molar ratio at 4°C for 1 hr. The mixture was passed through a 0.2 ml glutathione-sepharose column. After extensive washing with assay buffer, bound proteins were eluted with 20 mM reduced glutathione and detected by SDS-PAGE and Coomassie staining. Assays of USP7 Binding by Tryptophan Fluorescence EBNA1 and TRAF binding peptides were titrated with the purified USP7 NTD (62–205), and change in tryptophan fluorescence was measured as described in Holowaty et al. (2003a). NMR Measurements NMR spectra were acquired at 30°C on a Varian Inova-500 and 600 MHz spectrometers equipped with pulsed field gradient triple-

Structural Basis for EBNA1 and p53 Binding to USP7 35

resonance probes or a Bruker 600 MHz spectrometer equipped with a triple resonance cryo probe. The backbone 1H, 15N, and 13C resonances were assigned by using TROSY-HNCACB, TROSYCBCA(CO)NH, HNCACB, HNCO, and 15N-NOESY experiments (Salzmann et al., 1998; Kay, 2001). Greater than 80% of the backbone atoms were assigned. The interaction of USP7 with EBNA1 and p53 peptides was monitored by using the 1H-15N HSQC experiment at 30°C in a buffer containing 20 mM sodium phosphate (pH 7.5), 250 mM NaCl. Briefly, 0.7 mM 15N-labeled USP7 (amino acids 62–205) was titrated with unlabeled p53 (residues 355–393) or EBNA1 (residues 395–450, 410–450, or 441–450) peptides up to a 10:1 peptide:USP7 molar ratio. The data shown were collected by using a 3:1 and a 5:1 molar ratio of EBNA1:USP7 and p53:USP7, respectively, and no further changes in chemical shifts were detected in the 1H-15N HSQC spectra with higher peptide ratios.

mM MgCl2, 1.26 M potassium acetate, and 75% glycerol), cells were further dounce homogenized and extracted on ice for 30 min. The lysate was clarified by centrifugation, dialysed overnight against 10 mM HEPES (pH 7.9), 150 mM NaCl then precleared by incubation with 100 ␮l bed volume of Protein A Sepharose for 15 min. USP7 was immunoprecipitated from 500 ␮l (2.4 mg) lysate with 0 (negative control), 2, and 10 ␮l BL851 USP7 antibody (Bethyl Laboratories) and 20 ␮l bed volume Protein A Sepharose. After 5 hr of mixing at 4°C, beads were washed three times in 10 mM HEPES (pH 7.9), 150 mM NaCl and eluted with 40 ␮l 1% SDS. 20 ␮l of lysate and eluates were analyzed by Western blot using EBNA1 OT1X monoclonal antibody. The blots were then stripped and reprobed with BL851 USP7 antibody.

Acknowledgments Apoptosis Assays The effect of EBNA1 expression on cell death was determined in the U2OS osteosarcoma line by TUNEL assay upon induction of p53 by UV irradiation. Cells were seeded at 50% confluency on 60 mm2 dishes in duplicate 18 hr prior to transfection with 3 ␮g of pc3oriP plasmid expressing either EBNA1, EBNA1⌬395–450, or no EBNA1 (Holowaty et al., 2003b) and with 250 ng of pDsRed1-N1 (BD Bioscience) as a transfection marker. 24 hr later, one set of transfections was subjected to UV irradiation in a Stratagene 1800 ultraviolet crosslinker at 50 × 100 ␮J/cm2. Cells were grown another 24 hr, then stained by using the In Situ Cell Death Detection Kit, Fluorescein (Roche Applied Sciences) according to the manufacturer. Cells were filtered through a 0.7 ␮m strainer cap (BD Bioscience) prior to analysis on a Beckman-Coulter EPICS Elite (Flow Cytometry Facility, University of Toronto). The effect of EBNA1 on p53-mediated apoptosis was determined in the H1299 p53 null osteosarcoma line by Annexin V staining. Cells were seeded as above, 18 hr prior to transfection with 2 ␮g of pc3oriP plasmid expressing either EBNA1, EBNA1⌬395–450, or no EBNA1, 1 ␮g of pcDNA3p53 (Leng et al., 2003), and 500 ng of pEGFP-C1 (BD Biosciences) as a transfection marker. 24 hr later, cells were harvested and stained by using Annexin V-APC (BD Biosciences) according to the manufacturer. Cells were fixed in 2% paraformaldehyde/PBS overnight, washed in PBS, then filtered and analyzed by flow cytometry as described above. Effect of EBNA1 on p53 Levels U2OS cells in 60 mm dishes were transfected with 2 ␮g of pc3oriP plasmid expressing either EBNA1, ⌬395–450, or no EBNA1 by using Lipofectamine 2000 (Invitrogen). 5 hr later, cells were replated in 15 cm dishes and grown in the presence of 0.4 mg/ml G418 to select for cells containing the pc3oriP plasmids. After 2 weeks, cells were either harvested or UV irradiated (as described above) and harvested 4 or 8 hr post-UV treatment. Cells were lysed in 9 M urea, 5 mM Tris-HCl (pH 6.8), and sonicated. 30 ␮g of total protein was analyzed by Western blotting using antibodies PAb1801 for p53 (Banks et al., 1986), Ab-1 for actin (Oncogene research products) and R4 serum for EBNA1 (Holowaty et al., 2003b). Blots were developed by using the ECL plus system (Amersham), and enhanced chemifluorescence was quantified by using a Typhoon 9400 scanner (Amersham) and ImageQuant 5.0 software. p53 levels were determined in relationship to the actin loading control. Assays of EBNA1-USP7 Interactions in B Cells EBNA1 affinity column assays were conducted by using whole-cell extracts from BL41 (at 10 mg/ml) and HeLa (at 14 mg/ml) cells, which were generated as described in Holowaty et al. (2003b). 400 ␮l of each lysate was applied under physiological salt conditions to a 40 ␮l column containing EBNA1⌬61–83 (Wu et al., 2002) at 1 mg/ml and, after washing, bound proteins were eluted in high salt and identified by MALDI-ToF mass spectrometry as previously described (Holowaty et al., 2003b). For coimmunoprecipitation experiments, 2.5 × 108 Raji (EBV-positive Burkitt’s lymphoma) cells were lysed by dounce homogenization in 10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.1% Triton X-1001, and Roche protease inhibitors using 1.33 mls buffer per mg cell pellet. After addition of an equal volume of extraction buffer (50 mM HEPES [pH 7.5], 1.5

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Accession Numbers The atomic coordinates for USP7 NTD and USP7 NTD bound to the EBNA1 peptide have been deposited in the Protein Data Bank with the accession numbers 1YZE and 1YY6, respectively.