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...

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.

The factors determining the pH dependence of the formation and decay of the O photointermediate of the bacteriorhodopsin (bR) photocycle were investigated in the wild-type (WT) pigment and in the mutants of Glu-194 and Glu-204, key residues of the proton release group (PRG) in bR. We have found that in the WT the rate constant of O --> bR transition decreases 30-fold upon decreasing the pH from 6 to 3 with a pKa of about 4.3. D2O slows the rise and decay of the O intermediate in the WT at pH 3.5 by a factor of 5.5. We suggest that the rate of the O --> bR transition (which reflects the rate of deprotonation of the primary proton acceptor Asp-85) at low pH is controlled by the deprotonation of the PRG. To test this hypothesis, we studied the E194D mutant. We show that the pKa of the PRG in the ground state of the E194D mutant, when Asp-85 is protonated, is increased by 1.2 pK units compared to that of the WT. We found a similar increase in the pKa of the rate constant of the O --> bR transition in E194D. This provides further evidence that the rate of the O --> bR transition is controlled by the PRG. In a further test, the E194Q mutation, which disables the PRG and slows proton release, almost completely eliminates the pH dependence of O decay at pHs below 6. A second phenomenon we investigated was that in the WT at neutral and alkaline pH the fraction of the O intermediate decreases with pKa 7.5. A similar pH dependence is observed in the mutants in which the PRG is disabled, E194Q and E204Q, suggesting that the decrease in the fraction of the O intermediate with pKa ca. 7.5 is not controlled by the PRG. We propose that the group with pKa 7.5 is Asp-96. The slowing of the reprotonation of Asp-96 at high pH is the cause of the decrease in the rate of the N --> O transition, leading to the decrease in the fraction of O.

Nuclear factor kappaB (NF-kappaB) activation in tumor necrosis factor, interleukin-1, and Toll-like receptor pathways requires Lys63-linked nondegradative polyubiquitination. A20 is a specific feedback inhibitor of NF-kappaB activation in these pathways that possesses dual ubiquitin-editing functions. While the N-terminal domain of A20 is a deubiquitinating enzyme (DUB) for Lys63-linked polyubiquitinated signaling mediators such as TRAF6 and RIP, its C-terminal domain is a ubiquitin ligase (E3) for Lys48-linked degradative polyubiquitination of the same substrates. To elucidate the molecular basis for the DUB activity of A20, we determined its crystal structure and performed a series of biochemical and cell biological studies. The structure reveals the potential catalytic mechanism of A20, which may be significantly different from papain-like cysteine proteases. Ubiquitin can be docked onto a conserved A20 surface; this interaction exhibits charge complementarity and no steric clash. Surprisingly, A20 does not have specificity for Lys63-linked polyubiquitin chains. Instead, it effectively removes Lys63-linked polyubiquitin chains from TRAF6 without dissembling the chains themselves. Our studies suggest that A20 does not act as a general DUB but has the specificity for particular polyubiquitinated substrates to assure its fidelity in regulating NF-kappaB activation in the tumor necrosis factor, interleukin-1, and Toll-like receptor pathways.

The pH-dependence of photocycle of archaerhodopsin 4 (AR4) was examined, and the underlying proton pumping mechanism investigated. AR4 is a retinal-containing membrane protein isolated from a strain of halobacteria from a Tibetan salt lake. It acts as a light-driven proton pump like bacteriorhodopsin (BR). However, AR4 exhibits an "abnormal" feature--the time sequence of proton release and uptake is reversed at neutral pH. We show here that the temporal sequence of AR4 reversed to "normal"--proton release preceding proton uptake--when the pH is increased above 8.6. We estimated the pK(a) of the proton release complex (PRC) in the M-intermediate to be approximately 8.4, much higher than 5.7 of wide-type BR. The pH-dependence of the rate constant of M-formation shows that the pK(a) of PRC in the initial state of AR4 is approximately 10.4, whereas it is 9.7 in BR. Thus in AR4, the chromophore photoisomerization and subsequent proton transport from the Schiff base to Asp-85 is coupled to a decrease in the pK(a) of PRC from 10.4 to 8.4, which is 2 pK units less than in BR (4 units). This weakened coupling accounts for the lack of early proton release at neutral pH and the reversed time sequence of proton release and uptake in AR4. Nevertheless the PRC in AR4 effectively facilitates deprotonation of primary proton acceptor and recovery of initial state at neutral pH. We found also that all pK(a)s of the key amino acid residues in AR4 were elevated compared to those of BR.

Tumor necrosis factor (TNF) receptor (TNFR) associated factor 6 (TRAF6) is a unique member of the TRAF family of adaptor proteins that is involved in both the TNF receptor superfamily and the interleukin-1 receptor (IL-1R)/Toll-like receptor (TLR) superfamily signal transduction pathways. The ability to mediate signals from both families of receptors implicates TRAF6 as an important regulator of a diverse range of physiological processes such as innate and adaptive immunity, bone metabolism, and the development of lymph nodes, mammary glands, skin, and the central nervous system. This chapter will highlight the structural and biochemical studies of TRAF6 in receptor interactions and discuss the potential for peptidomimetic drug application based on TRAF6 receptor binding motif.

Amino acid esters react with C60 both thermally and photochemically to give different products. Refluxing a mixture of C60 and glycine ethyl ester afforded C60(Me2CHNHCHCOOEt) 1, whereas irradiation of the same mixture produced C60(EtOOCCHNHCHCOOEt) 2b as the main product. Photochemical reactions between C60 and sarcosine esters yielded two pyrrolidine derivatives C60(CH2N(Me)CHCOOR) 3 and C60(ROOCCHNHCHCOOR) 2 (R = Me, Et, CH2Ph). Compound 2a is also prepared from the photochemical reaction between C60 and iminodiacetic methyl ester in high yield. These ester derivatives are difficult to hydrolyze in excess mineral acids. The fullerene dicarboxylic acid C60(HOOCCHNHCHCOOH) 5 is synthesized from the tert-butyl derivative C60(tBuOOCCHNHCHCOOtBu) 4. A possible radical reaction mechanism for the photochemical reactions is proposed which involves an unprecedented C−N bond breakage.

Aminopolycarboxylic esters react with C60 under photolysis to produce fullerene multicarboxylates. Irradiation of tetramethyl ethylenediaminetetraacetate (EDTA) with C60 yields the EDTA-containing fullerene monoadduct C60(MeOOCCH)2NCH2CH2N(CH2COOMe)2. In addition, several other C60 monoadducts are also isolated and characterized, including compounds due to EDTA fragmentation. Similar results are observed with pentamethyldimethylenetriaminepentaacetate (DTPA). When partially methylated nitrilotriacetic acid is irradiated with C60, decarboxylation occurs and organodihydrofullerene derivatives such as C60(H)(CH2N(CH2COOMe)2) are formed. Radical mechanisms are proposed for both types of photoreactions. The fullerene derivatives are characterized by their spectroscopic data. Photoreactions of C60 with other analogous molecules also support the conclusions.