E2 interaction and dimerization in the crystal structure of TRAF6

Abstract

TRAF6 mediates Lys63 (K63)-linked polyubiquitination for NF-κB activation via its N-terminal RING and zinc finger domains. Here we report the crystal structures of TRAF6 and its complex with the ubiquitin conjugating enzyme (E2) Ubc13. The RING and zinc fingers of TRAF6 assume a rigid, strikingly elongated structure. Interaction of TRAF6 with Ubc13 involves direct contacts of the RING and the preceding residues while the first zinc finger plays a structural role. Surprisingly, this region of TRAF6 is dimeric both in the crystal and in solution, different from the trimeric C-terminal TRAF domain. Structure-based mutagenesis reveals that TRAF6 dimerization is critical for polyubiquitin synthesis and auto-ubiquitination. Fluorescence energy transfer analysis shows that TRAF6 dimerization induces higher order oligomerization of full-length TRAF6. The mismatch of dimeric and trimeric symmetry may provide a mode of infinite oligomerization that facilitates liganddependent signal transduction of many immune receptors.Tumor necrosis factor (TNF) receptor associated factors (TRAFs) play important roles in intracellular signal transduction of many receptor families such as the TNF receptor superfamily, the IL-1 receptors (IL-1R), the Toll-like receptors (TLR), T-cell receptors (TCR) and B-cell receptors (BCR) 1,2 . Upon receptor activation, TRAFs are directly or indirectly recruited to the intracellular domains of these receptors. They subsequently engage other signaling proteins to activate the inhibitor of κB (IκB) kinase (IKK) and MAP kinases, leading ultimately to activation of transcription factors such as NF-κB and AP-1 to induce immune and inflammatory responses and confer protection from apoptosis.Most TRAFs contain an N-terminal domain with RING (really interesting gene) and a variable number of zinc fingers and a C-terminal TRAF domain that comprises a coiled coil domain and a conserved TRAF-C domain (Fig. 1a) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscriptrevealed that the TRAF domain forms a mushroom-shaped trimeric structure with the TRAF-C domain as the head for interaction with receptors and adaptor proteins and the coiled coil domain as the stalk for trimerization [3][4][5] . Remarkably, TRAF6 is uniquely pleiotropic in participating in the signal transduction of many receptor systems while TRAF2, TRAF3 and TRAF5 appear to signal only within the TNF receptor superfamily 5 .The downstream signaling mechanism of TRAFs was first revealed from biochemical and cellular studies of TRAF6 to show the involvement of K63-linked polyubiquitination [6][7][8] . Ubiquitination is one of the most prevalent post-translational modifications 9 . It is accomplished in three steps, 1) ATP-dependent attachment of ubiquitin (Ub) via a thioester bond to a Ub activating enzyme (E1), 2) transfer of Ub from E1 to the active site Cys of a Ub conjugating enzyme (E2), and 3) transfer of Ub from the E2 active site to Lys residues of substrates (including other molecules of Ub) with the aid of a Ub lig...

LRRC19 expressed in the kidney induces TRAF2/6-mediated signals to prevent infection by uropathogenic bacteria

“…TRAF6 protein may also interact with UBE2N/UBC13 (ref. 38) and UBE2V1/UEV1A 39 , which is required for IkB kinase (IKK) activation. Thus, there may be a network constructed of LRRC19, TLR4 and TNF-a-mediated signalling pathway (s) to effectively prevent and eliminate UPEC infection in kidney tissue.…”

Higher‐order oligomerization promotes localization of SPOP to liquid nuclear speckles

“…Higher‐order complexes, that is, oligomers in which the number of monomers in a complex is broadly distributed and can be large, have important functions in signal transduction and cell fate decisions (Credle et al , 2005; Korennykh et al , 2009; Yin et al , 2009; Li et al , 2012a; Banjade & Rosen, 2014; Lu et al , 2014; Xu et al , 2014). The inherent size heterogeneity of such higher‐order assemblies generates challenges for their structural and mechanistic characterization, but recent progress has provided insight into their ability to mediate signal amplification, filter noise, and regulate signaling in time and space (Li et al , 2012b; Wu, 2013).…”

“…The inherent size heterogeneity of such higher‐order assemblies generates challenges for their structural and mechanistic characterization, but recent progress has provided insight into their ability to mediate signal amplification, filter noise, and regulate signaling in time and space (Li et al , 2012b; Wu, 2013). Some of these assemblies form via nucleation‐driven polymerization and result in stable complexes, and we are starting to understand the structural basis for their proximity‐enhanced activation (Korennykh et al , 2009; Yin et al , 2009; Li et al , 2012a; Lu et al , 2014). Other assemblies depend on transient interactions and are more labile, generating oligomers with broad size distributions (Rivas et al , 2000; Errington et al , 2012; Canzio et al , 2013).…”

Ubiquitination of E3 ligases: self-regulation of the ubiquitin system via proteolytic and non-proteolytic mechanisms

“…Consistently, some self-ubiquitinating RING ligases such as SIAH1 and TRAF (TNF receptor-associated factor) 6, have been detected as homodimers, and dimerization was found to be essential for the self-ubiquitination of TRAF6. 14,15 In other cases, self-ubiquitination could not be catalyzed in trans, implying that it is possibly an intramolecular event. This was described, for example, for the self-ubiquitination of the HECT ligase Rsp5, 12 and the F-box protein Grr1p.…”

“…64 Dimerization of TRAF6 (through its C-terminus or N-terminal RING domain) is essential and sufficient to induce its own ubiquitination and subsequent activation of the JNK and NF-kB signaling pathways. 15,64,65 The self-generated Lys63-based ubiquitin chains appear to function as recruitment adaptors to attract other substrates to TRAF6. TAB2 (TAK1-binding protein 2) binds specifically to Lys63-linked ubiquitin chains, which might serve to recruit TAB2 to the self-ubiquitinated TRAF6.…”