MyD88, IRAK4 and IRAK2 are critical signaling mediators of the TLR/IL1-R superfamily. Here we report the crystal structure of the MyD88: IRAK4: IRAK2 death domain (DD) complex, which surprisingly reveals a left-handed helical oligomer that consists of 6 MyD88, 4 IRAK4 and 4 IRAK2 DDs. Assembly of this helical signaling tower is hierarchical, in which MyD88 recruits IRAK4 and the MyD88: IRAK4 complex recruits the IRAK4 substrates IRAK2 or the related IRAK1. Formation of these Myddosome complexes brings the kinase domains of IRAKs into proximity for phosphorylation and activation. Composite binding sites are required for recruitment of the individual DDs in the complex, which are confirmed by mutagenesis and previously identified signaling mutations. Specificities in Myddosome formation are dictated by both molecular complementarity and correspondence of surface electrostatics. The MyD88: IRAK4: IRAK2 complex provides a template for Toll signaling in Drosophila and an elegant mechanism for versatile assembly and regulation of DD complexes in signal transduction.

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...

SUMMARY NEMO is the regulatory subunit of the IκB kinase (IKK) in NF-κB activation and its CC2-LZ region interacts with Lys63 (K63)-linked polyubiquitin to recruit IKK to receptor signaling complexes. In vitro, CC2-LZ also interacts with tandem diubiquitin. Here we report the crystal structure of CC2-LZ with two dimeric coiled coils representing CC2 and LZ, respectively. Surprisingly, mutagenesis and nuclear magnetic resonance experiments reveal that the binding sites for diubiquitins at LZ are composites of both chains and that each ubiquitin in diubiquitins interacts with symmetrical NEMO asymmetrically. For tandem diubiquitin, the first ubiquitin uses the conserved hydrophobic patch and the C-terminal tail while the second ubiquitin uses an adjacent surface patch. For K63-linked diubiquitin, the proximal ubiquitin uses its conserved hydrophobic patch while the distal ubiquitin mostly employs the C-terminal arm including the K63-linkage residue. These studies uncover the energetics and the common U-shaped geometry for mutual recognition of NEMO and diubiquitins.

In addition to caspase inhibition, X-linked inhibitor of apoptosis (XIAP) induces NF-kappaB and MAP kinase activation during TGF-b and BMP receptor signaling and upon overexpression. Here we show that the BIR1 domain of XIAP, which has no previously ascribed function, directly interacts with TAB1 to induce NF-kappaB activation. TAB1 is an upstream adaptor for the activation of the kinase TAK1, which in turn couples to the NF-kappaB pathway. We report the crystal structures of BIR1, TAB1, and the BIR1/TAB1 complex. The BIR1/TAB1 structure reveals a striking butterfly-shaped dimer and the detailed interaction between BIR1 and TAB1. Structure-based mutagenesis and knockdown of TAB1 show unambiguously that the BIR1/TAB1 interaction is crucial for XIAP-induced TAK1 and NF-kappaB activation. We show that although not interacting with BIR1, Smac, the antagonist for caspase inhibition by XIAP, also inhibits the XIAP/TAB1 interaction. Disruption of BIR1 dimerization abolishes XIAP-mediated NF-kappaB activation, implicating a proximity-induced mechanism for TAK1 activation.

Inhibitor of κB (IκB) kinase (IKK) phosphorylates IκB proteins leading to their degradation and liberation of nuclear factor κB (NF-κB) for gene transcription. Here we report the crystal structure of IKKβ in complex with an inhibitor at 3.6 Å resolution. The structure reveals a tri-modular architecture with the kinase domain (KD), a ubiquitin-like domain (ULD) and an elongated, α-helical scaffold/dimerization domain (SDD). Surprisingly, the predicted leucine zipper and helix-loop-helix motifs do not form these structures but are part of SDD. The ULD and SDD mediate a critical interaction with IκBα that restricts substrate specificity, and the ULD is also required for catalytic activity. The SDD mediates IKKβ dimerization, but dimerization per se is not important for maintaining IKKβ activity, and instead is required for IKKβ activation. Other IKK family members IKKα, TBK1 and IKKi may share the similar tri-modular architecture and function.