Epstein-Barr virus-driven B cell lymphoma mediated by a unique LMP1-TRAF6 complex

The latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) drives viral B cell transformation and oncogenesis. LMP1's transforming activity depends on its cytoplasmic C-terminal activation region 2 (CTAR2), which induces NF-κB and JNK by engaging TNF receptor-associated factor 6 (TRAF6). The mechanism of TRAF6 interaction with LMP1 and its critical role for LMP1 signaling has remained elusive. Here we demonstrate that TRAF6 interacts directly with a novel viral TRAF6 binding motif within CTAR2. Structural modeling supported by NMR and functional studies provides insight into the molecular architecture of the LMP1-TRAF6 complex and reveals substantial differences to CD40-TRAF6 interaction. The direct recruitment of TRAF6 to LMP1 is essential for NF-κB activation and survival of LMP1-driven B cell lymphoma. Disruption of the LMP1-TRAF6 complex by inhibitory peptides interferes with proliferation of EBV-transformed B cells. We identify LMP1-TRAF6 as critical virus-host interface and validate this interaction as novel therapeutic target against EBV. NMR spectra were recorded at 298K using a Bruker Avance 600 spectrometer with QCI cryogenic probe and topspin v.3.2 software (Bruker BioSpin). Spectra were processed using NMRDraw v.8.7 of the NMRPipe software 82 and analysed with the CCPN Analysis software v.2.4.1 83 . NMR titrations were performed by recording 1 H, 15 N HSQC experiments. For titrations of TRAF6, samples of 90 µM 15 N-labeled TRAF6 in PBS pH 7.4, 10% D 2 O, 5 mM β-ME were used. Samples contained either no ligand (reference), or ve-fold excess of unlabeled LMP1 peptides (PSL) as indicated. Shifts were highlighted in the TRAF6-LMP1 model using PyMol software (Schrödinger LLC). TRAF6 residues were annotated on the basis of the previously published partial backbone chemical shift assignment of TRAF6 59

The sequence P 379 VQLSY 384 , located at the C-terminus of CTAR2, is responsible for NF-κB and JNK activation by CTAR2 ( Figure 1A) 37,39 . Although this sequence includes the putative TRAF1/2/3 binding motif PxQxS 40 , direct physical interaction of CTAR2 with TRAF molecules has never been demonstrated.
One study suggested that TRAF6 recruitment to CTAR2 might be indirect, possibly mediated by the transcription factor BS69 41 .
The TRAF protein family consists of seven members, TRAF1 to 7. Of these seven isoforms, TRAF1 to 6 share the so-called TRAF domain, which is located at the C-terminus of the TRAF molecule and is composed of a TRAF-N (or coiled-coil) domain, and a TRAF-C domain, the latter built of seven to eight anti-parallel β-strand folds 42,43 . Mediated by their TRAF domains, TRAF proteins form mushroom-like trimers, which can interact with receptors through TRAF-C 43−45 . The TRAF domain of TRAF6 is su cient to mediate TRAF6 interaction with the LMP1 complex 19 . TRAF6 lacking its TRAF domain is unable to rescue TRAF6 de ciency in LMP1 signaling, further supporting a role of this domain for interaction with LMP1 21 . The N-terminal RING nger of TRAF6 possesses E3 lysine 63 (K63)-linked ubiquitin ligase activity, which plays an important role in activation of LMP1 downstream signaling including TAK1 and IKKβ activation 12,46 . LMP1 mimics signals of the co-stimulatory receptor CD40 during B cell proliferation and can largely replace CD40 functions in vivo 7,[47][48][49] . TRAF6 de ciency affects B cell numbers driven by a conditional CD40-LMP1 fusion protein in the lymph nodes of mice 50 . However, a potential role of TRAF6 in LMP1dependent lymphoma has not been demonstrated. Although both, LMP1 and CD40, engage TRAF6 for signaling, the underlying molecular mechanisms seem to differ. CD40 carries two major TRAF binding sites, a TRAF1/2/3 binding site with the sequence P 250 VQET, and the TRAF6 interaction site Q 231 EPQ 235 EINF 51,52 . The TRAF binding sites of CD40 are largely redundant with respect to their functions in NF-κB and JNK activation in B cells 53 . At the molecular level, JNK signaling induced by LMP1 differs from CD40 with respect to the functions of IKKβ and TPL2 34,54 .
In the present study, we characterize the interaction between LMP1 and TRAF6 as novel virus-host interface, which is based on direct protein-protein interaction. We provide structural insight into the molecular architecture of the LMP1-TRAF6 complex, and demonstrate that the direct interaction of LMP1 and TRAF6 is critical for LMP1 function and the survival of EBV-transformed B cells. In summary, we reveal the molecular mechanism of TRAF6 engagement by LMP1 for signaling and lymphoma development.

TRAF6 interacts directly with P 379 VQLSY 384 of LMP1
We examined all TRAF proteins involved in LMP1 signaling regarding their potential to directly bind to the LMP1 signaling domain ( Figures 1A and 1B, Supplementary Figure 1A). The puri ed recombinant TRAF domains of TRAF1, 2, 3, and 5 interacted with P 204 QQAT of CTAR1 in pull-down assays with glutathione S-transferase (GST)-coupled LMP1 181−386 ( Figure 1B). Mutation of P 204 xQxT into A 204 xAxA abolished LMP1 binding of TRAF1, 2, and 5. Residual amounts of TRAF3 were recruited by the A 204 xAxA mutant, which can be explained by contacts of TRAF3 with LMP1 residues adjacent to the P 204 xQxT core motif 55 .
Investigating the interaction between LMP1 and TRAF6 we made the surprising observation that also recombinant His-TRAF6 310−522 , which includes the TRAF domain of TRAF6, was e ciently recruited by GST-LMP1 ( Figure 1B). In contrast to all other TRAF proteins tested, TRAF6 recruitment to LMP1 was not affected by mutation of CTAR1, but was eliminated by the exchange of tyrosine 384 into glycine. In accordance with this nding, Flag-TRAF6 wildtype only co-immunoprecipitated with HA-LMP1 from HEK293 cells if CTAR2 was intact (Supplementary Figure 1B). These experiments provided the rst evidence for a direct protein-protein interaction as the molecular basis of TRAF6 recruitment to LMP1.
To further substantiate this result and to narrow down the LMP1 sequences involved in LMP1-TRAF6 interaction, we tested the ability of His-TRAF6 310−522 to interact with immobilized LMP1-derived peptides, which incorporate CTAR1 or CTAR2 sequences ( Figure 1C and Supplementary Figure 1C). Recombinant His-TRAF2 311−501 was used as control. Speci city of TRAF interaction was con rmed by including peptides, which harbored alanine exchanges within the TRAF2-binding motifs of CD40 (P 250 VQET to A 250 VAEA, peptides 1 and 2, respectively), LMP1 (P 204 QQAT to A 204 QAAA, peptides 6 and 7), and the TRAF6 binding motif of CD40 (Q 231 EPQEINF to A 231 EAQAINF, peptides 1 and 3). CD40-derived amino acids 244-273 lacked the TRAF6 binding site (peptide 4). Additional mutation of the TRAF2-binding motif within peptide 4 resulted in peptide 5. We have shown previously that amino acids 371-386 of LMP1 are su cient to induce TRAF6-dependent CTAR2 signaling 30 . To determine whether these sixteen amino acids contain the complete TRAF6 binding site of LMP1, they were included as peptide 8. Within peptide 8, Y 384 and Y 385 , or the cryptic TRAF interaction motif P 379 xQxS were mutated (peptides 9 and 10, respectively). Further, CTAR2 amino acids 357-386 were spotted (peptide 11), in which P 379 xQxS was mutated (peptide 12).
Both TRAF2 and TRAF6 speci cally interacted with their designated binding sites within CD40, con rming accuracy of the peptide array ( Figure 1C). Further, TRAF2 bound to P 204 QQAT of CTAR1 (peptides 6 and 7), but not to CTAR2 (peptides 8 to 12), which excludes the possibility of direct TRAF2 interaction with the cryptic TRAF interaction motif of CTAR2. Notably however, TRAF6 was e ciently captured by the CTAR2 peptides 8 (16mer) and 11 (30mer), whereas it did not bind to CTAR1 (peptide 6). Mutation of Y 384 and Y 385 to AA (peptide 9) and P 379 xQxS to AxAxA (peptides 10 and 12) abolished direct TRAF6 binding to CTAR2 ( Figure 1C).
We performed an alanine exchange mutagenesis scan from G 378 to Y 385 of LMP1 to precisely map the residues that are involved in TRAF6 binding. We developed a highly reliable mix-and-measure screening assay for the LMP1-TRAF6 interaction based upon the Perkin-Elmer AlphaScreen technology, by which the effects of mutations on this protein-protein interaction (PPI) can be detected and quanti ed directly ( Figure 1D). Light emission at 520-620 nm is directly proportional to the a nity of the two protein components of the assay. Each of the LMP1 amino acids P 379 , V 380 , Q 381 and Y 384 was essential for direct TRAF6 recruitment to the LMP1 signaling domain ( Figure 1E). Mutation of Y 385 had only minor impact on TRAF6 binding at the lowest TRAF6 concentration tested in the assay (100 nM), whereas the side chains of G 378 , L 382 and S 383 were dispensable for interaction. Of note, the resulting TRAF6 binding sequence P 379 VQxxY exactly matches the NF-κB-and JNK-inducing region of CTAR2 37,39 . This nding strongly suggested that the direct binding of TRAF6 to this sequence is in fact the molecular basis for CTAR2 signaling.
TRAF6 showed a weaker a nity for LMP1 as compared to CD40. The K D of His-TRAF6 310−522 interaction with GST-CD40 was 17.8 ± 4 nM in contrast to 77.1 ± 21.7 nM with GST-LMP1, determined by the AlphaScreen PPI assay ( Figure 1F). Con rming our previous data, mutation of LMP1 P 379 xQxxY into A 379 xAxxA abolished TRAF6 binding. Analogous mutation of the TRAF6 binding motif within CD40, which was included as control, resulted in a loss of TRAF6 interaction as well ( Figure 1F).
Alignment of the consensus TRAF6 interaction motif PxExxF/Y/D/E of cellular receptors 45,51 with the newly identi ed TRAF6 binding sequence of LMP1 revealed high similarity, with the exception of one striking difference at the central position P 0 ( Figure 1F). Cellular TRAF6-recruiting sequences carry a glutamic acid at P 0 45, 56-58 , whereas this position is occupied by glutamine in LMP1. Remarkably, glutamic acid at P 0 of the TRAF6 binding motif of CD40 cannot be replaced by any other amino acid, including glutamine, without losing a nity to TRAF6 56 . We tested the effect of converting the TRAF6 binding motif of LMP1 into the cellular consensus motif by Q 381 E mutation. The resulting LMP1 Q 381 E mutant is capable of binding TRAF6 with signi cantly enhanced a nity as compared to wildtype LMP1 ( Figure 1G, compare to GST-LMP1 wildtype of Figure 1F). Q 381 E mutation reduced the K D from 77.1 ± 21.7 nM to 8.1 ± 0.9 nM, which is even lower as the K D of TRAF6 interaction with CD40. This may suggest that additional interactions of TRAF6 with LMP1 beyond P 0 , which are absent in the CD40-TRAF6 complex, stabilize LMP1 interaction with TRAF6 and allow P 0 being occupied by glutamine.
Position P 3 of LMP1's TRAF6-binding motif is lled by Y 384 , which has a critical role in LMP1 signaling and viral cell transformation 8, 19,37,39 . In cellular TRAF6-interacting receptors this position can be occupied by an aromatic or acidic amino acid 45 . Accordingly, a permutation scan at P 3 of CD40 showed that TRAF6 binding to CD40 still occurs if F 238 is mutated into tyrosine or tryptophan 51 . To test variability at the P 3 position of LMP1, we introduced a Y 384 F mutation resembling P 3 of the TRAF6 binding motif of CD40. The Y 384 F exchange was not only tolerated by LMP1, but even improved the a nity of LMP1 to TRAF6 ( Figure 1G). As expected, Q 381 A and Y 384 A exchanges abolished TRAF6 binding ( Figure 1G).
Taken together, TRAF6 is directly recruited by the JNK-and NF-κB-inducing sequence P 379 VQLSY within CTAR2 and is, thus, the rst identi ed cellular factor whose binding site exactly matches the signalingactive site of CTAR2. In contrast to cellular receptors, this motif contains glutamine at the central P 0 position, likely facilitated by unique structural characteristics of the viral LMP1-TRAF6 complex.
Arginine 392 of TRAF6 discriminates between LMP1 and CD40 To examine whether LMP1 binds to the same region at the surface of TRAF6 as cellular receptors, we mutated amino acids within TRAF6 that are involved in interaction with P −2 , P 0 , or P 3 of CD40 and receptor activator of NF-κB (RANK, also known as TRANCE receptor) 45 Figure 2A). Hence, this pocket forms an essential interaction with LMP1, most probably with LMP1 residue P 379 , which occupies P −2 of the P 379 VQxxY motif. Mutation of K 469 into alanine had no effect on TRAF6 interaction with LMP1 or CD40. The side chain of K 469 likely forms non-essential charge-charge interactions with the main chain carboxylate of P 0 of CD40 45 .
The TRAF6 mutant R 392 A revealed a striking difference regarding LMP1 and CD40 binding. R 392 forms an amino-aromatic interaction with F 238 at P 3 of CD40 45 . However, mutation of R 392 into alanine had no impact on TRAF6 binding to CD40, whereas interaction with LMP1 was fully eliminated by this mutation (Figure 2A). R 392 thus discriminates between LMP1 and CD40. This result suggested a different molecular architecture of the LMP1-TRAF6 complex as compared to CD40-TRAF6.
To verify the relevance of our ndings on LMP1-TRAF6 interaction in vivo, we expressed Flag-tagged TRAF6 wildtype or the mutants R 392 A, K 469 A, F 471 A and Y 473 A together with HA-tagged LMP1 in HEK293 cells and performed co-immunoprecipitations of both proteins ( Figure 2B and Supplementary Figure 2A). Con rming our previous results, each of the mutations R 392 A, F 471 A or Y 473 A, abolished TRAF6 interaction with LMP1, whereas K 469 A mutation had no negative effect on the interaction between both proteins in HEK293 cells. Confocal immuno uorescence studies in HeLa cells further veri ed these results ( Figure 2C). Flag-TRAF6 wildtype and the K 469 A mutant co-located to a high extent with HA-LMP1 clusters, demonstrating their interaction with LMP1 in situ. In contrast, the TRAF6 mutants R 392 A, F 471 A and Y 473 A showed a strongly decreased co-localization with HA-LMP1, which was comparable to the LMP1Δ371-386 mutant lacking the TRAF6 interaction site ( Figure 2C and Supplementary Figure 2B). In the absence of LMP1, all TRAF6 mutants showed a similar cytoplasmic distribution as TRAF6 wildtype (Supplementary Figure 2C). In summary, these results demonstrated that binding of TRAF6 to LMP1 involves the same TRAF6 residues in the cellular context as in our interaction studies with recombinant proteins, which strongly argues for the same and direct mechanism of LMP1-TRAF6 complex formation in vivo as in vitro.
Direct binding of TRAF6 to LMP1 is required for CTAR2 signaling CTAR2 signaling is defective in TRAF6-de cient mouse embryonic broblasts (MEFs) and can be rescued by exogenous TRAF6 expression 19,27,28,30 . To demonstrate that direct interaction of LMP1 and TRAF6 is indeed the molecular basis for CTAR2 signaling, we tested the TRAF6 mutants that are defective in direct LMP1 binding for their potential to rescue CTAR2 signaling in NF-κB reporter assays in TRAF6-/-MEFs ( Figure 3A).  Figure 3A). In the absence of TRAF6, CTAR2 was unable to induce NF-κB reporter activity ( Figure 3A, see w/o). As expected, expression of wildtype TRAF6 or the TRAF6 mutants alone (co-transfection with inactive A 204 xAxA/∆371-386) induced NF-κB to similar levels (grey bars), demonstrating that all mutants fully retained their downstream signaling capacity. However, only TRAF6 wildtype, but none of the binding-defective mutants, was able to rescue CTAR2 signaling to NF-κB (green bars). This result showed that the direct interaction of TRAF6 with LMP1 is critical for activation of CTAR2-mediated NF-κB signaling.
To further con rm this result, we retrovirally transduced TRAF6-/-MEFs, which stably express NGFR-LMP1, with TRAF6 wildtype or the TRAF6 mutants R 392 A, F 471 A and Y 473 A. NGFR-LMP1 is a fusion construct of the extracellular and transmembrane domains of the p75 nerve growth factor (NGF) receptor (NGFR) with the intracellular signaling domain of LMP1 13,34 . Instant NGFR-LMP1 activity can be triggered at the cell surface by incubation of the cells with an α-NGFR primary antibody and subsequent crosslinking by a secondary antibody ( Figure 3B). Antibody crosslinking of NGFR-LMP1 caused a rapid degradation of IκBα, which is indicative for activation of the canonical NF-κB pathway in wildtype MEFs  Figure 3C). In contrast, the TRAF6 mutants R 392 A, F 471 A and Y 473 A, which are unable to directly bind to LMP1, were also ineffective in rescuing canonical NF-κB activation by CTAR2 ( Figure 3C). Taken together, our data demonstrated that CTAR2 only induces NF-κB if TRAF6 is directly recruited to CTAR2.

Molecular model of the LMP1-TRAF6 complex
Our experiments with the TRAF6 mutant proteins showed that LMP1 binds to the same PPI interface of TRAF6 as CD40 and other cellular receptors. To gain structural insights into the binding of TRAF6 to LMP1, we used Molecular Operating Environment to derive an in silico model of the LMP1-TRAF6 complex ( Figure 4A). The sequence alignment between LMP1 and TRAF6-binding receptor peptides (see Figure 1F) showed no indication for signi cant structural differences in the proximity of position P −2 between LMP1 and cellular receptors, because this position is always occupied by a proline. Accordingly, P 397 of LMP1 is located in the hydrophobic indentation formed primarily by TRAF6 residues M 450 , F 471 and Y 473 ( Figure 4A). In line with this nding, mutation of the TRAF6 residues F 471 and Y 473 abolished LMP1 binding (see Figure 2). Also in our LMP1-TRAF6 model, hydrogen bonds are formed between Q 381 at P 0 of LMP1 and the amide NH atoms of L 457 and A 458 of TRAF6 ( Figure 4B). Yet, due to the different charge of the side chains, the strength of Q 381 interaction with TRAF6 is weaker compared to E 235 of CD40 with TRAF6. Accordingly, the Q 381 E exchange signi cantly increases the a nity between LMP1 and TRAF6 (see Figure 1F).
Mutation of L 382 at P 1 to alanine does not impair LMP1-TRAF6 binding. This is consistent with the model, which indicates that hydrogen bonds at this position are formed by the peptide backbone with the TRAF6 residues G 470 and R 392 and are hence invariant to changes of the side chain ( Figure 4C). In addition, R 392 forms another hydrogen bond with S 383 at P 2 of the LMP1 main chain. These interactions explain the NMR spectroscopy reveals shifting of TRAF6 residues upon LMP1 binding To con rm the binding position of LMP1 at TRAF6 proposed by our biochemical and modeling data, we recorded NMR spectra of TRAF6 in its free form as well as bound to the LMP1 peptide G 378 PVQLSYYD ( Figure 5A). Addition of the peptide caused signi cant shifts as well as line broadening in some peaks in the TRAF6 spectra, a clear indication of binding. Based on a previously published partial backbone chemical shift assignment of TRAF6 59 , several TRAF6 residues of interest could be assigned to peaks in the recorded spectra. Of those TRAF6 residues previously tested for their functions in LMP1 binding (see Figures 2 and 3), F 471 and K 469 are highlighted in both spectra (R 392 and Y 473 have not been assigned by Moriya and colleagues 59 ). Upon addition of the LMP1 peptide, the peaks corresponding to these residues are broadened beyond detection, indicating that these residues contribute strongly to binding. Because mutation of K 469 had no effect on LMP1 interaction, this result supports a role of the K 469 backbone in LMP1 binding. Next, the chemical shift pattern caused by the LMP1 peptide was calculated and plotted onto the modeled LMP1-TRAF6 complex (see Figure 4). The overall assigned shift perturbations caused by the addition of LMP1 peptide are clustered around the TRAF6 PPI surface and con rmed LMP1 binding at this position ( Figure 5B).

LMP1-driven B lymphomas are strictly dependent on TRAF6
CTAR2 provides critical signals for effective growth transformation of primary B cells by EBV 8, 60 . Because we showed that direct TRAF6 interaction with CTAR2 is required for CTAR2 signaling we next asked whether TRAF6 is necessary for proliferation and survival of LMP1-driven B cell lymphomas. To address this question, TRAF6 was targeted by an ex vivo CRISPR/Cas9 approach in the two LMP1dependent B cell lymphomas LMP1-CL 37 and 40 derived from the transgenic CD19-Cre;R26LMP1 stop :CD3ε KO mouse model 9, 61 . The effect of three different gRNAs targeting the gene of interest (GOI) TRAF6 on tumor cell survival was examined. gRNAs targeting LMP1 as positive or the intracellular adhesion molecule 1 (ICAM1) as negative controls were included in parallel transfections. Cell survival was monitored seven days post transfection as selection score of the gRNAs targeting the GOI versus a non-targeting (NT) gRNA directed against an irrelevant Rosa26 sequence ( Figure 6A and Methods).
The knockout of LMP1 resulted in a drastic reduction of survival of the LMP1-CL 37 and 40 lymphomas ( Figure 6B and 6C). This result was expected because both lymphomas had been selected for their dependence on LMP1 61 (see Methods). In contrast, targeting of ICAM1 did not affect lymphoma survival.
More interestingly, we found that inactivation of TRAF6 by CRISPR/Cas9 caused a massive negative effect on lymphoma survival, which was comparable to the effect of LMP1 targeting itself ( Figure 6B and 6C). Hence, both lymphomas are absolutely dependent on TRAF6, demonstrating a previously unappreciated critical role of TRAF6 in the survival of LMP1-dependent B lymphoma cells. These ndings further suggested that the direct interaction between LMP1 and TRAF6 is an important factor for lymphoma development and may serve as a novel therapeutic target for inhibitory molecules.  45 . Because LMP1 also interacts with this region at the TRAF6 surface, we reasoned that the RANK-derived peptide should be able to block TRAF6 interaction with LMP1. An alignment of the TRAF6 inhibitory peptide with CD40 and LMP1 sequences is shown in Figure 7A. We used a cell-penetrating version of this peptide, fused to the Antennapedia leader sequence, to inhibit TRAF6 interaction with LMP1. A peptide containing the leader sequence only served as negative control.
Indeed, the TRAF6 inhibitor peptide blocked interaction of TRAF6 and GST-LMP1 in AlphaScreen PPI assays with an IC 50 of 177 nM, while the control peptide was inactive ( Figure 7B). TRAF6 binding to GST-LMP1 wildtype and the A 379 xAxxA null mutant demonstrated the dynamic range of the assay and veri ed that LMP1-TRAF6 inhibition by the peptide was complete ( Figure 7B). As expected, the inhibitor peptide had no effect on the recruitment of TRAF2 to LMP1 ( Figure 7C). TRAF6 binding to CD40 was inhibited by the peptide as well, albeit with strongly reduced e ciency as compared to LMP1 ( Figure 7D).
Finally, we examined the TRAF6 inhibitor peptide for its effects on LCL viability. Two lymphoblastoid cell lines, LCL721 and HA-LCL3, were incubated for three days in the presence of the TRAF6 inhibitor peptide or the control peptide, respectively. The EBV-negative Burkitt's lymphoma cell line BL41 was included as negative control ( Figure 7E). The TRAF6 inhibitor peptide, but not the control peptide, caused a severe reduction of cell viability in both LMP1-dependent LCLs, whereas no such effect was seen in LMP1independent BL41 cells. This result corroborated our previous results regarding the relevance of TRAF6 function for the survival of EBV/LMP1-transformed cells. It further showed that the direct interaction of TRAF6 with LMP1 is essential for LMP1's pro-survival function and might therefore constitute a novel therapeutic target for inhibitors, for instance small molecule LMP1-TRAF6 PPI inhibitors.

Discussion
With this study we nally answer the long-standing open question how the signaling-active sequence P 379 VQLSY of LMP1 and its signaling mediator TRAF6 are connected at the molecular level. We demonstrate a direct protein-protein interaction of LMP1 and TRAF6 that is critical for both, CTAR2 signaling and the survival of LMP1-transformed B cells. The viral TRAF6 binding motif PVQxxY is unique compared to known cellular TRAF6 binding motifs because it carries glutamine instead of glutamic acid at P 0 . Interestingly, it was shown previously that an exchange of glutamic acid to glutamine at P 0 is not tolerated in CD40 without losing a nity to TRAF6 56 . The stabilizing interactions of LMP1 P 1 to P 3 with R 392 of TRAF6 are enabled by the different spacial orientation of Y 384 as compared to F 238 of CD40, and may explain why the Q-E exchange at P 0 is tolerated by LMP1, but not CD40. However, LMP1 wildtype shows weaker a nity to TRAF6 than CD40, which might constitute an important mechanism of balancing the signaling strength of constitutively active LMP1 within limits that are supportive for cell survival. Highly elevated LMP1 signaling levels have been shown to induce cytostasis or even cell death 12 .
Our results explain the earlier observation that mutation of LMP1 Y 384 YD into F 384 FD preserves LMP1's capacity of mediating CTAR2-dependent NF-κB signaling, whereas mutation into I 384 D abolishes LMP1 activity 36 . The Y 384 F exchange at P 3 maintains a functional TRAF6 interaction motif, with a decent K D of TRAF6 interaction (see Figure 1G). In contrast, I 384 D mutation is expected to destroy the TRAF6 binding motif. Accordingly, F 384 FD, but not I 384 D, supports B cell transformation 36 , which further underscores the relevance of the TRAF6 interaction motif and, thus, TRAF6 for the transforming capacity of LMP1.
Although the presence of LMP1 and TRAF6 in one signaling complex has been observed previously, direct interaction of both molecules was not reported 19,31,35 . It was suggested that TRAF6 is recruited to CTAR2 by an indirect mechanism involving TRADD or BS69 19,41 . We now show that direct interaction of TRAF6 with P 379 VQLSY is independent of any further factor. However, this does not exclude the possibility that cellular factors such as TRADD or BS69 act as further stabilizers or modulators of the complex in vivo, dependent on the cellular context or the expression levels of the involved proteins.
TRAF6 is critical for both canonical NF-κB and JNK activation by CTAR2 19,27,31 . In contrast, TRADD is rather involved in CTAR2-induced NF-κB but not JNK signaling, whereas the situation for BS69 is vice versa 30 We clearly demonstrate that TRAF6 binds to CTAR2, but is unable to directly interact with CTAR1.
However, it has been reported previously that TRAF6 has a role in CTAR1 signaling 19,21,26 . In mouse B cells, TRAF6 co-precipitates with an inducible mCD40-LMP1 fusion protein, an interaction, which is dependent on CTAR1 in these cells 21    Cell culture, retroviral transduction, and NGFR-LMP1 activation were analysed by immunoblotting using α-6*His-tag antibody.

Immunoblotting
Immunoblotting was essentially performed as described 34 . Proteins were detected on nitrocellulose membranes (Bio-Rad, #1620115) using the following primary antibodies: α-6*His-tag (clone 4A4 or 2F12,  MTT cell viability assay  Figure 2A). For K D determination the data from each experiment was cleaned for hook effect (signal depletion by oversaturation of the beads) and analysed using the equation "one site -speci c binding with hill slope" (Figure 1F and 1G). IC 50 was determined using the "log(inhibitor) vs.
response -variable slope (four parameters)" equation ( Figure 7B and 7D). Paired t-test was used for matched samples (Figure 7B and 7D) and unpaired t-test for not unpaired samples ( Figure 7E).

ACKNOWLEDGMENTS
We thank Ichio Shimada of the University of Tokyo for generously sharing NMR assignment data of TRAF6. This work was supported by grant Ki 825    (B) Chemical shifts induced by LMP1 peptide binding at the surface of TRAF6 are highlighted in the in silico LMP1-TRAF6 structure described in Figure 4. TRAF6 residues showing strong shifts upon LMP1 binding (>0.05) are indicated in dark blue, residues showing weaker shifts (0.025 -0.05) in light blue.
Residues without shift upon LMP1 binding are indicated in dark gray. TRAF6 residues without an assignment to a speci c NMR peak are shown in light grey. For the latter residues no NMR-based conclusion is possible regarding their shifting upon LMP1 binding.