1 Psychiatry Centre of Excellence for Drug Discovery, 2 Neurology Centre of Excellence for Drug Discovery and 3 Computational, Analytical and Structural Sciences, GlaxoSmithKline, Third Avenue, Harlow, EssexCM19 5AW, UK and 4 Comparative Genomics, GlaxoSmithKline, King of Prussia, PA, USA
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Abstract |
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Keywords: apoptosis/receptor/signalling/TNFR1
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Introduction |
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Death domains were first described in two members of the tumor necrosis factor receptor (TNFR) superfamily, Fas and TNFR1, due to the characterized role of these receptors in apoptosis and also because of similarity to a pro-apoptotic Drosophila protein, Reaper (White et al., 1994). It is apparent, however, that this similarity is limited; the vertebrate death domain is longer and the three-dimensional structure of Reaper is likely to be significantly different to published structures for mammalian death domains (Liang and Fesik, 1997
). Yeast two-hybrid analyses have identified several intracellular adaptor proteins that also contain death domains and in many cases these associate directly via homotypic interaction with death receptors (Chinnaiyan et al., 1995
; Hsu et al., 1995
). Death domains have also been described in non-apoptotic proteins and, in the case of netrin receptors, may play a role in axonal guidance (Hofmann and Tschopp, 1995
).
In addition to some primary sequence conservation, the three-dimensional structure is also conserved in two other protein interaction domains involved in apoptotic signal transduction: the caspase recruitment domain (CARD) and death effector domain (DED) (Weber and Vincenz, 2001). Based on solution structures for some of these adaptors, it has been proposed that they interact via surface localized acidic and basic regions (Chou et al., 1998
). In addition, mutagenesis studies of Fas (Huang et al., 1996
) and TNF-R1 (Tartaglia et al., 1993
) death domains have implicated a charged surface region, formed by helices 2 and 3, that interacts with FADD or TRADD, respectively. In contrast, alanine-scanning mutagenesis of the TRADD death domain indicated that key residues are distributed across the surface and that specific subdomains are not associated with the roles of homotrimerization, TNFR1 binding, cell killing or NF
B activation (Park and Baichwal, 1996
).
The molecular interaction between the death receptor, TR3 and the adaptor TRADD is interesting because of the putative role of these signalling components in neurodegenerative disease (Harrison et al., 2000; Newman et al., 2000
). TR3 is a 417 amino acid (aa) type 1 membrane receptor. Its structure is similar to those of other death receptors that have three or four extracellular NGF domains with cysteine repeats and a C-terminal intracellular death domain (Ashkenazi and Dixit, 1998
). The TR3 death domain comprises 97 aa and is most homologous (48%) with the death domain of TNF-R1 (Chinnaiyan et al., 1996
). TRADD is a 312 aa intracellular adapter that contains a C-terminal death domain and is present in many cell types (Hsu et al., 1995
). The TR3 signal cascade is similar to that of TNFR1 (Chinnaiyan et al., 1996
; Kitson et al., 1996
; Marsters et al., 1996
; Bodmer et al., 1997
; Screaton et al., 1997
). Ligand binding induces receptor trimerization and the resultant receptor oligomer forms a scaffold for attachment of the adaptor proteins TRADD and FADD via sequential death domain interactions. The zymogen procaspase 8 is also recruited to the complex (via DED interaction with FADD) where it self-cleaves and initiates the caspase cascade (Ashkenazi and Dixit, 1998
). TRADD represents the earliest signal bifurcation point in the apoptotic and NF
B pathways.
This study investigated the homotypic interaction between the DDs of TR3 and TRADD. Initially, a three-dimensional model of the TR3 DD was generated, based on the NMR structures of Fas and p75. This model was used to choose candidate residues for alanine-scanning mutagenesis. The effect of these mutations on the interaction with TRADD DD was analysed by directed yeast two-hybrid analysis and co-immunoprecipitation studies using mammalian cells.
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Materials and methods |
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A computer model of TR3 DD was produced using the Quanta program (Accelrys, San Diego, 1998). TR3 DD was modelled on the NMR structures of the DDs of Fas (Huang et al., 1996) (PDB code 1DDF) and p75 (Liepinsh et al., 1997
)(PDB code 1NGR). A structural alignment of Fas and p75 DDs was generated and used as a template for TR3 DD (Figure 1
).
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Cloning the TR3 cytoplasmic domain and TRADD death domain
The TR3 (nt 9581254; aa 320417) and TRADD (nt 651918; aa 218306) death domains were amplified by PCR under standard conditions using either TR3-specific primers (CCAAGAATTC TCC CCA GCC GGC TCG CCA GCC, CCAACTCGAG TCA CGG GCC GCG CTG CAG GCG) or TRADD-specific primers (GAA TTC GAC CAA CAG ACG TTC GCG, GAA TTC CTA ATC GGT CAG GCC CAG CAA GTC); restriction enzyme sites are underlined. The resulting products were cloned into pCRTOPO2.1 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions, thus creating pCRTR3 and pCRTRADD. The cloned sequences were confirmed by DNA sequence analysis. pCRTR3 was digested with EcoRI and the fragment containing the TR3 death domain was isolated and ligated into EcoRI-digested pJG4-5 (Ausubel et al., 1993) and pHybLex/Zeo (Invitrogen), thus creating pADTR3 and pDBDTR3, respectively. These plasmids contain TR3 fused to the lexA activation domain (AD) and a DNA-binding domain (DBD), respectively. The orientation of the TR3 fragments in the two-hybrid vectors was confirmed using XhoI. Mutations were created in pDBDTR3 by the method described below.
To create the DBDTRADD fusion, pCRTRADD was digested with EcoRI and the fragment containing the TRADD death domain was isolated and ligated into EcoRI-digested pEG202 (Ausubel et al., 1993), thus creating pDBDTRADD.
Site-directed mutagenesis of the TR3 death domain
Site-directed mutagenesis was carried out using the Quik Change Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer's instructions. In each case, two complementary unphosphorylated primers were synthesized containing the desired mutation (Table I). All mutations were confirmed by PCR sequencing.
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Two-hybrid analysis (Fields and Sternglanz, 1994) was performed using strains EGY48 (Gyuris et al., 1993
) and EGY48
R. EGY48
R is isogenic to EGY48, except it is MAT
and contains the lacZ reporter plasmid pSH18-34 (Ausubel et al., 1993
). Except where indicated (see Table II
), directed analysis was performed by transforming EGY48 with the DBD fusion plasmids, selecting 46 transformants and mating these transformants by replica plating to a lawn of EGY48
R containing the relevant AD fusion plasmids. This plate was incubated at 30°C for 24 h and then replica plated to minimal medium that allowed growth of only diploid cells that contained the AD fusion, DBD fusion and lacZ reporter plasmids. Qualitative ß-galactosidase assays (Duttweiler, 1996
) and quantitative ß-galactosidase assays (Ausubel et al., 1993
) were performed on the resulting diploids. Diploids were also examined for their ability to grow in the absence of leucine on medium containing either galactose, which induces expression of the AD fusion protein, or glucose, which does not.
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The mutations R352A, R357A, R372A, E358A, E360A, E362A, E365A and E367A were introduced into pcDNA3.1/His B containing the TR3 cytoplasmic domain using site-directed mutagenesis as described.
Wild-type or mutant pcDNA3.1/His B-TR3 DD were cotransfected into IMR32-NCA240 cells with pcDNA3.1-TRADD using lipofectamine (Invitrogen); 24 h after transfection the cells were harvested and total protein extract was prepared. Transfection efficiency was measured by transfecting cells with a green fluorescent protein (GFP)-containing vector and calculating the percentage of the total population that was fluorescent after 24 h. Immunoprecipitation was performed on 100 µg of the extract with 2 µg/ml anti-Xpress epitope (Invitrogen). The protein was resolved by SDSPAGE. Western blotting was performed using a 1:250 dilution of anti-TRADD (Transduction Laboratories) followed by incubation with peroxidase-conjugated secondary antibody (Jackson Laboratories). Bound antibody was detected using an Enhanced ChemiLuminescence kit (Amersham).
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Results |
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The helices of the Fas and p75 death domains aligned well with each other, apart from the final (sixth) helix, which is shorter in the p75 structure (Figure 1). The alignment of the TR3 sequence to the other sequences was reasonable for helices 2, 3, 4 and 5 (Figure 2
). The putative helices 2, 3 and 4 of TR3 are similar to the Fas sequence and the fifth helix is reminiscent of the corresponding p75 helix. The coordinates were copied from these structures to give protein backbone coordinates for the helical regions of the TR3 model. Side-chain coordinates were used where there was a correspondence or constructed in CHARMm.
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The model predicts a compact (35x25 Å), highly folded structure of six amphipathic, antiparallel helices with charged, polar and hydrophobic patches on the surface. The structure is highly similar to known DDs (Figure 1) and also to the related CARD and DEDs (Weber and Vincenz, 2001
).
To identify residues potentially involved in proteinprotein interactions, amino acids predicted to be on the surface of the death domain were examined. Residues corresponding to key points of interaction reported in other DDs were selected for mutagenesis from helices 2 and 3. An analysis of solvent exposure for surface residues in the helical regions of the model was performed using the Quanta program. This identified 15 amino acids whose surfaces were probably >75% exposed to solvent. An additional 18 residues were predicted to be 5075% exposed to solvent (Figure 2). Furthermore, two residues, R381 and R383, were considered to be trapped in local conformational minima and had the potential to be solvent exposed. Glycines were not considered for mutagenesis because they mostly occurred in the loop regions between helices. Substitution of these with alanine, which is larger than glycine, may perturb or disrupt the folding of the death domain.
Because the model predicts >75% exposure to solvent, the following residues were considered of highest priority for alanine scanning mutagenesis: R346, V366, I368, R372, Q384, R399 and D403. Residues predicted to be 5075% exposed comprised a secondary set: E349, R357, E358, E362, E367, R370, D373 and K380.
Although some of the exposed residues included hydrophobic amino acids (e.g. isoleucine and valine), we focused on charged residues (see the list above) based on recent reports indicating that these play a predominant role in death domain interaction (Chou et al., 1998; Jeong et al., 1999
). Those mutated to alanine in this study were R357, E358, E362, E367 and R372 (Figure 3
). Residues R352, E360 and E356 were included as controls for non-surface-exposed charged residues. For additional controls, surface-exposed residues in helix 1, Q334, L335, V338 and M339 (Figure 4
), were mutated to alanine, since helix 1 was considered to be remote from the interacting helices. It was expected that the helix 1 mutations would have no effect on the interaction of TR3 based on domain interaction sites reported in previous studies.
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To determine if the residues discussed above are necessary for proteinprotein interaction, mutations in the TR3 death domain were examined by directed two-hybrid analysis. This was accomplished by creating AD fusions for each TR3 mutation and testing them against DBD fusions of the wild-type TRADD and TR3 death domains. An unrelated protein, MST1, and vectors containing no inserts were used as controls. The wild-type TR3 death domain interacted with TRADD as shown previously (Chinnaiyan et al., 1996; Kitson et al., 1996
) and also with itself, but not with the negative control, MST1 (Table II
). Likewise, all of the mutant versions of the TR3 death domain interacted with TRADD and the wild-type TR3 death domain, but not with MST1 (Table II
). In addition to the quantitative ß-galactosidase analysis, qualitative analysis was performed by testing each proteinprotein combination for its ability to support cell growth in the absence of leucine using either galactose or glucose as the carbon source (see Materials and methods). All strains that yielded >40 units of ß-galactosidase activity were able to grow in the absence of leucine on galactose, but not on glucose. Those strains that yielded <40 units were unable to grow on either medium (data not shown). Taken together, these results suggest that these residues are not necessary for TR3TRADD or TR3TR3 protein interaction.
Cotransfection studies followed by immunoprecipitation in IMR32-NCA240 cell extracts using native and mutated TR3 death domain confirmed the ability of all helix 2/3 mutants to interact with TRADD (Figure 5). Some variability was observed between experiments for each mutant. This most likely reflects sensitivity to immunoprecipitation conditions rather than an absolute difference between mutants in ability to precipitate TRADD.
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Discussion |
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Although it would have been desirable to introduce dual mutations for comparison, it seemed probable from our structural predictions that multiple mutations could alter the structure of the TR3 death domain. Since a previous study had shown that single mutations introduced into the FADD death domain did not cause structural change (Huang et al., 1996), we adopted a similar strategy and examined TR3 mutants that contained single amino acid changes.
TR3 death domain self-association
Directed yeast two-hybrid analysis shows that wild-type TR3 DD is capable of self-interaction. In addition, we have screened a human whole brain cDNA library and found that wild-type TR3 death domain interacts with AD plasmids that encode the C-terminus of TR3 (unpublished data). Previous two-hybrid analysis suggests that mutations L354A, L356A and D373A abolish TR3 DD interaction with TNFR1 and also homotypic interaction with itself (Kitson et al., 1996). These residues are located in the loop between helices 2 and 3. L354 and L356 are conserved in TNF-R1, DR4 and DR5. D373 is conserved in two other death domain proteins, DR4 and DR5, and is functionally conserved in TNF-R1. In this study, we mutated charged residues, R352 and E360, which are conserved between TNFR1, DR4 and DR5. This, however, had no effect on interaction with the wild-type DD.
Since TR3 DD and TRADD are thought to interact with the TR3 DD simultaneously (receptor trimers interact via the death domain), it is likely that they bind to different domain surfaces. Residues L354 and L356, which are required for TR3TR3 interaction, are located on the N-terminus of helix 3, whereas D373 is located at its C-terminus. It is possible that some (or all) of these mutations perturb self-interaction by disrupting the structure of the DD rather than directly altering the binding region. This is more likely to be the case for L354 and L356 as the TR3 DD model predicts that they are <50% exposed to the molecular surface
TR3 and TRADD death domain interaction
We have shown for the first time in a yeast two-hybrid system that wild-type TR3 DD and TRADD interact. This supports previously published data generated by overexpression and immunoprecipitation analysis (Chinnaiyan et al., 1996). Surprisingly, directed two-hybrid analyses between the wild-type TR3 death domain, the TR3 mutants and TRADD showed that only two of the mutations in the TR3 death domain (R352A and E358A) yielded less ß-galactosidase activity when paired with TRADD than the wild-type TR3 paired with TRADD.
None of the combinations, however, yielded ß-galactosidase activities approaching those of the negative controls and all of the combinations tested grew well on galactose-containing medium that lacked leucine. Based on these results, the three-dimensional model suggests either that these residues are not necessary for proteinprotein interaction or that mutation of a single residue does not appear to be sufficient to abrogate binding. It should be noted that although the two-hybrid system has identified point mutations that abrogate proteinprotein interactions (e.g. Inouye et al., 1997), the artificial nature of the system can force non-physiological proteinprotein interactions to occur (Vidal and Legrain, 1999
). However, this would seem unlikely in this case because the interactions were confirmed in a different system.
The results described above are in direct contrast to previously published data for TNF-R1 and Fas (Tartaglia et al., 1993; Huang et al., 1996
) in which the introduction of single mutations in comparable regions was shown to decrease interaction with FADD or to prevent TRADD-mediated cytotoxicity. We mutated TR3 DD residues that correspond to several residues that are required for FasFADD and TNFR1TRADD interaction. For both Fas and TNF-R1, mutation of the residue corresponding to R352 in TR3 led to decreased FasFADD interaction and TNF-R1 cytotoxicity. In addition, mutation of the Fas residues corresponding to E358 and E362 led to decreased binding of FADD. In contrast, none of these mutations were sufficient to abrogate the interaction of TR3 DD and TRADD. Similarly, the mutation of residues which are either identical (R352 and E360) or conserved (E358, E362, E365 and R372) in TNF-R1, DR4 and DR5 had no apparent effect on the interaction of TR3 DD and TRADD. It is unlikely that these residues are critical for maintaining the structure of TR3 DD as they are predicted to be on the surface of the death domain.
Together with previous studies (Tartaglia et al., 1993; Huang et al., 1996
; Chou et al., 1998
; Telliez et al., 2000
), these data indicate that despite close structural similarity the nature of the death domain interactions (and related domains) appears to be unique for each pair. The success of this type of protein module may reside not only in its ability to activate multiple pathways, but also in its molecular specificity, which is a likely result of evolutionary changes that have subtly altered the surface of the death domain.
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Notes |
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References |
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Received December 18, 2001; revised July 16, 2002; accepted July 26, 2002.