Oligomerization-primed coiled-coil domain interaction with Ubc13 confers processivity to TRAF6 ubiquitin ligase activity

Ubiquitin ligase TRAF6, together with ubiquitin-conjugating enzyme Ubc13/Uev1, catalyzes processive assembly of unanchored K63-linked polyubiquitin chains for TAK1 activation in the IL-1R/TLR pathways. However, what domain and how it functions to enable TRAF6’s processivity are largely uncharacterized. Here, we find TRAF6 coiled-coil (CC) domain is crucial to enable its processivity. The CC domain mediates TRAF6 oligomerization to ensure efficient long polyubiquitin chain assembly. Mutating or deleting the CC domain impairs TRAF6 oligomerization and processive polyubiquitin chain assembly. Fusion of the CC domain to the E3 ubiquitin ligase CHIP/STUB1 renders the latter capable of NF-κB activation. Moreover, the CC domain, after oligomerization, interacts with Ubc13/Ub~Ubc13, which further contributes to TRAF6 processivity. Point mutations within the CC domain that weaken TRAF6 interaction with Ubc13/Ub~Ubc13 diminish TRAF6 processivity. Our results reveal that the CC oligomerization primes its interaction with Ubc13/Ub~Ubc13 to confer processivity to TRAF6 ubiquitin ligase activity.

3 (b) The activities of ZnF3 and ZnF4 mutants but not the CC mutant were readily reversed by the addition of ubiquitin aldehyde (Ubal). In vitro IKK activation was performed with TRAF6 or the indicated deletion mutants in the absence or presence of Ubal. The products were resolved on 10% SDS-PAGE and immunoblotted with the indicated antibodies.
(c) TRAF6 deletion mutants ZnF3 and ZnF4 were able to synthesize comparable polyUb chains as TRAF6 WT did. As in Figure 3c, but the ZnF3 and ZnF4 were used.  (c) Rapid assembly of long polyUb chains. A two-step ubiquitination reaction was performed in which Ub, E1, Ubc13/Uev2 and ATP were pre-mixed for 5 minutes. Following addition of T6RZC, the reaction was incubated at 30 ℃ for the indicated duration of time. PolyUb chains were immunoblotted with an anti-Ub antibody.
(d) As in Figure 4g, but without TRAF6. The reaction products were immunoblotted with an anti-Ub antibody.

Supplementary Figure 4. TRAF6 interaction mediated by Coiled-coil domain is important for polyUb chain synthesis and TAK1-IKK-NF-B activation.
(a) Activation of NF-B by TRAF6 and the mutants M1, M2 and M3. As in Figure 2d, 3x-B-Luc reporter assays were performed with TRAF6 and the mutants to assess their ability to activate NF-B. The data are showed as the mean±s.d. of triplicate independent sets of experiments.
(b) Coiled-coil domain mediates TRAF6 self-association. Flag-tagged TRAF6, mutant M3 or T6RZ were co-transfected with Myc-tagged TRAF6 into HEK293T cells and subjected to immunoprecipitation with anti-Flag M2 beads. The immunoprecipitated complexes (upper panels) and cell lysates (down panels) were immunoblotted with the indicated antibodies. 6 (c) As in Figure 2b, in vitro ubiquitination assay was performed to measure the E3 ligase activities of TRAF6 and TRAF6-M3.
(d) In vitro TAK1 activation assay was performed to compare their TAK1 stimulatory activities between TRAF6 and TRAF6-M3.
(e) In vitro IKK activation assay was performed with TRAF6 and TRAF6-M3. In addition to the detection of IB and IB phosphorylation, the reaction products were also used to capture K63-linked polyubiquitin chains by GST-TAB2-UBD. The GST pull-down products were analyzed by immunoblotting with a K63-Ubn-specific antibody.

Supplementary Figure 5. Oligomerized Coiled-coil domain interacts with Ub~Ubc13 contributing to
processivity of TRAF6 E3 activity. KO cells and purified using anti-M2 beads. CC alone, Ubc13 alone, or mixture of CC and Ubc13 were subjected to size-exclusion chromatography using Superose 6. Fractions were detected using the indicated antibodies.
(d) TRAF6 mutants M4 and M5 don't affect TRAF6 self-association. Flag-tagged TRAF6, mutants M4 or M5 was co-transfected with Myc-tagged TRAF6 into HEK293T cells and subjected to immunoprecipitation with anti-Flag M2 beads. The immunoprecipitated complexes (upper panels) and cell lysates (down panels) were immunoblotted with the indicated antibodies.
(g) Impaired activation of NF-B by TRAF6 mutants M4 and M5. As in Figure 2d, 3x-B-Luc reporter assays were performed with TRAF6 and the mutants to assess their ability to activate NF-B. The data are showed as the mean±s.d. of triplicate independent sets of experiments.
(h) Impaired in vitro IKK activation by TRAF6 mutants M4 and M5. In vitro IKK activation assay was performed with TRAF6, TRAF6-M4 and TRAF6-M5. In addition to the detection of IB and IB phosphorylation, the reaction products were also used to capture K63-linked polyubiquitin chains by GST-TAB2-UBD. The GST pull-down products were analyzed by immunoblotting with a K63-Ubn-specific antibody.
Bound proteins were separated on SDS-PAGE for TRAF6 immunoblotting or on non-reducing SDS-PAGE for Ub~Ubc13 detection using Coomassie brilliant blue (CBB) staining.