The Solution Structure of FADD Death Domain
STRUCTURAL BASIS OF DEATH DOMAIN INTERACTIONS OF Fas AND
FADD*
Eui-Jun
Jeong
§¶,
SookHee
Bang
¶
,
Tae Ho
Lee**,
Young In
Park§,
Woong-Seop
Sim
, and
Key-Sun
Kim

From the
Structural Biology Center, Korea Institute
of Science and Technology, Seoul, 130-650, the § Graduate
School of Biotechnology and
Department of Biology, Korea
University, Seoul, 136-701, and ** Department of Biology, Yonsei
University, Seoul, 120-749, Korea
 |
ABSTRACT |
A signal of Fas-mediated apoptosis is transferred
through an adaptor protein Fas-associated death domain protein (FADD)
by interactions between the death domains of Fas and FADD. To
understand the signal transduction mechanism of Fas-mediated apoptosis,
we solved the solution structure of a murine FADD death domain. It consists of six helices arranged in a similar fold to the other death
domains. The interactions between the death domains of Fas and FADD
analyzed by site-directed mutagenesis indicate that charged residues in
helices
2 and
3 are involved in death domain interactions, and
the interacting helices appear to interact in anti-parallel pattern,
2 of FADD with
3 of Fas and vice versa.
 |
INTRODUCTION |
Activation of Fas receptor (called also CD95 or APO-1) with either
Fas ligand or anti-Fas antibody induces receptor clustering. This
recruits the adaptor molecule
FADD1/MORT1 and procaspase-8
to the Fas receptor through the homotypic interactions of death domains
(DDs) and death effector domains (DEDs), respectively, leading to
proteolytic activation of caspase-8 (1-5). The activation of caspase-8
initiates a cascade of caspases and leads to cell death. The clustering
of Fas receptor, FADD, and procaspase-8, termed death-inducing
signaling complex, is essential for Fas-mediated apoptosis and
caspase-8 activation (6) (7). In the death-inducing signaling complex
formation, FADD mediates signals from Fas receptor to procaspase-8 with
its C-terminal DD and N-terminal DED. FADD also participates in
signaling other members of the TNFR family. FADD binds to
TNFR1-associated death domain protein (TRADD), which interacts with the
stimulated TNFR1 in TNF-mediated apoptosis (8). Several viral and
cellular procaspase-8-like proteins, FLIPs (FLICE inhibitory proteins), also bind to FADD and modulate Fas-induced apoptosis (9-14). Among the
Fas-binding proteins, FADD is the only protein found in the death-inducing signaling complex (6) and has shown to be essential in vivo by use of FADD-deficient cells that are completely
resistant to Fas-mediated apoptosis (15, 16). FADD is also implicated in embryo development (15), T-cell proliferation (16), and TNF-induced
activation of acid sphingomyelinase (17). The downstream signal
transduction of Fas- or TNF-mediated apoptosis is blocked by the
N-terminal-truncated FADD that lacks death effector domain (18).
FADD exists in the cytoplasm of normal cells, but it does not induce
cell death except at a high concentration (1). This suggests that the
signal transduction of FADD be triggered by interactions of death
domains of Fas and FADD, possibly converting FADD into a form capable
of recruiting procaspase-8. However, the mechanism of a signal
transduction by FADD is not yet clear. To understand the mechanism of
FADD-mediated signal transduction, we determined the solution structure
of a murine FADD (2) death domain (FADD-DD), carried out site-directed
mutageneses, and analyzed the effect of mutagenesis on the binding
affinity of FADD-DD for Fas-DD to map an interaction site of death
domains. Thus far, the structures of Fas-DD (19), FADD-DED (20), and
caspase recruitment domain of RAIDD (21) has been determined and known to have similar global folds. It has been suggested that the
interactions between DDs or caspase recruitment domains are
electrostatic (19) (21), whereas those between DEDs are hydrophobic
(20). But the mode of interactions has not been clear because the
information about the counter-interacting domain has not been
available. By determining structure of FADD-DD, we now are able to
propose a model for the death domain interactions of Fas and FADD based on the structures of Fas-DD (19), FADD-DD, and mutagensis experiments.
 |
MATERIALS AND METHODS |
Sample Preparation--
Recombinant FADD was prepared from the
murine FADD gene subcloned into an expression vector pET3d
(Novagen) in Escherichia coli strain BL21 (DE3). When cell
growth is reached at the logarithmic phase at 37 °C, protein
expression was induced by adding 0.4 mM isopropyl-1-thio-
-D-galactopyranoside for 3 h. The
harvested cell paste was disrupted by a sonicator in a lysis buffer (50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM dithiothreitol, 10% glycerol, and 1 mM
phenylmethylsulfonyl fluoride), and ammonium sulfate up to 30% was
added to the soluble fraction of the cell extract. The precipitant was
collected by centrifugation and dialyzed overnight at pH 4.0 and
further purified by reverse phase high performance liquid
chromatography using a C8 Vydac column. Purified FADD was subjected to proteolytic digestion for 2 h at 15 °C by adding subtilisin of one-hundredth of FADD in phosphate buffer at pH 8.0. The
molecular weight and N-terminal amino acid sequence of the resistant
fragments were analyzed, and the corresponding DNA fragments were
subcloned into a pET15b expression vector. The C-terminal fragment of
FADD protein (FADD-DD) was expressed in E. coli strain BL21
(DE3) by inducing with 0.4 mM
isopropyl-1-thio-
-D-galactopyranoside for 3 h at
28 °C. The protein was purified by an affinity chromatography with
nickel nitrilotriacetic acid-agarose column (Qiagen). The polyhistidine
tag was removed by thrombin and further purified by gel filtration in
ammonium acetate buffer at pH 4.0. Purified protein that has an
N-terminal cloning artifact of Gly-Ser-His-Met was lyophilized and
stored at
20 °C. A uniformly 15N and/or
13C -labeled protein was prepared from cells grown in M9
minimal medium containing 1 g of
15NH4Cl/liter and/or 2.0 g of
[U-13C]glucose/liter. NMR samples were prepared by
dissolving about 10 mg of protein in 0.5 ml of 50 mM sodium
acetate buffer composed of either 90% H2O, 10%
2H2O, or 99.9% 2H2O,
and the pH was adjusted to 4.00 ± 0.05 (glass electrode, uncorrected) with concentrated NaO2H.
NMR Spectroscopy--
The heteronuclear NMR experiments were
carried out with a 15N and/or 13C-labeled
sample in 90% H2O, 10% 2H2O using
Varian UNITYplus 600 (in Advanced Analysis Center in KIST)
or Inova 500 (University of Alberta) spectrometers at 30 °C. The
protein concentration was about 2 mM. Once FADD-DD protein is dissolved in water for more than a week, NMR signal begins to lose
its intensity, indicating the tendency of aggregation. So all
experiments were carried out within a week after dissolution. Deuterium
exchange of amide protons in FADD-DD was initiated by dissolving
lyophilized sample in 2H2O, and two-dimensional
1H-15N HSQC spectra were recorded at 20 °C,
pH 4.0. After 1 h at 20 °C, the temperature was increased to
30 °C, and spectra were acquired. According to cross-peaks intensity
remaining in each spectrum, exchange rates were divided into four
groups. Two-dimensional 1H-13C constant
time-HSQC, three-dimensional 1H-15N NOESY-HSQC,
1H-15N TOCSY-HSQC (22), HNHA (23) were acquired
with the 13C- or 15N-labeled sample, and
13C-, 15N-edited NOESY (24), CBCA(CO)NH, HNCACB
(25), and 1H-15C HCCH-TOCSY (26)
were acquired with the 13C-, 15N-labeled
sample. NMR data were processed using a program NMRPipe (27).
Assignments and Distance Restraints--
Starting with
identifications of 15N and HN chemical shifts in
1H-15N HSQC, spin systems were partly
identified in 1H-15N TOCSY-HSQC, and the
sequential assignments of each amino acid were made using
1H-15N NOESY-HSQC. The 13C chemical
shifts of each amino acid were assigned in
1H-13C CT-HSQC and HCCH-TOCSY and verified by
HNCACB and CBCA(CO)NH. Stereospecific assignments of H
protons, and
methyl groups of Val and Leu were based on the intensity of HN-H
or
HN-H
cross-peaks in 1H-15N TOCSY-HSQC and
1H-15N NOESY-HSQC spectra (28) and NOE
intensity of the stereospecifically assigned H
protons to
-methyl
protons of Leu. NOE distance restraints were derived from
three-dimensional 1H-15N NOESY-HSQC,
13C-,15N-edited NOESY, and 1H NOESY
spectra in 2H2O, all with a mixing time of 75 or 150 ms. Only cross-peaks observed in 75 ms mixing time were used for
the initial structure calculation to exclude a spin diffusion artifact.
All NOE cross-peaks were assigned using a program PIPP. The NOE
intensity was converted into three groups of classes (1.8-2.7,
1.8-3.5, 1.8-5.0), and pseudo-atom corrections were made
appropriately (29).
Other Restraints--
The scalar coupling constant of the
-proton to the amide proton was obtained from the HNHA experiments.
The backbone torsion angle
was restrained to
85 to
25 for
3JHNH
< 5.5 Hz,
60 to
180 for
3JHNH
7-8 Hz,
70 to
170 for
3JHNH
8-9 Hz, and
90 to
150 for
3JHNH
> 9 Hz. The
torsion angles of
helix region were restrained to
70 to
10. The side chain torsion
angles
1 were restrained based on cross-peak intensities
deduced from 1H-15N HSQC-TOCSY and
13C-, 15N-edited NOESY spectra. Additional
backbone H-bond restraints were given where secondary structures were
indicated based on NOE connectivity. For each hydrogen bond, two
restraints (rNH-O, 1.7-2.3; rN-O, 2.5-3.3)
were used. Additionally, J-coupling constants (30), carbon chemical
shifts of C
and C
resonances (31), and a data base potential (32)
(33) were directly included in the simulated annealing protocol during refinement.
Mutations and Measurements of Binding Affinity--
At first,
mutation sites were selected based on the structure of Fas death domain
(19), but further mutations were made when an initial structure of
FADD-DD was calculated. The mutation sites are in helices
2 and
3
and the connecting loop between them. Charged residues in
2 and
3
were replaced by Ala, and hydrophobic residues in the connecting loop
were replaced by Asn similar to the lpr mutant of Fas. All
mutants were single amino acid substitutions and purified as wild type
FADD-DD described above. For the binding affinity measurements, human
Fas-DD (Gly192-Ser304) was expressed in
E. coli and purified. The purified Fas-DD was coupled to
sensor chip CM5 by amine coupling at pH 4.0 to get about 2,600 response
units. In the same way, FADD was attached to a CM5 chip to have about
6,000 response units to measure self-association between FADD-DD and
FADD. A flow of purified FADD-DD in HBS buffer (10 mM
HEPES, pH 7.4, containing 150 mM NaCl, 3.4 mM
EDTA, and 0.05% surfactant P20) at 5 different concentrations between
55 to 880 nM was maintained over the protein-coupled chip
for 2 min to record association at the flow rate of 20 µl/min. The
bound FADD-DD was dissociated by passing HBS buffer without FADD-DD for
the next 6 min at the same flow rate. Binding constants were obtained
by BIAevaluation software (Biocore AB) using obtained sensorgrams. The
degree of self-association was estimated based on the resonance signal
obtained for 2 min of association. All binding experiments were
performed at 25 °C. All binding experiments were carried out with
BIAcore 2000, and the N-terminal amino acid sequence of the purified
protein was determined at Korea Basic Science Institute (KBSI) in Seoul.
 |
RESULTS |
Structure of FADD-DD--
The structure of FADD-DD (residues
89-183) is well defined by 1,253 experimentally derived NOEs, 174 dihedral angle restraints, 50 hydrogen bonds, 84 coupling constants,
and 83 additional chemical shifts (Table
I). FADD-DD shown in Fig.
1 consists of 6 helices similar to other
death domains of Fas (19) and the p75 neurotrophin receptor (34).
Helices
1 and
2 are interlocked with helices
4 and
5, and
helices
3 and
6 are located on each side. Helix
6 is well
packed against the interlocked helices, but
3 is more loosely
associated. Hydrophobic residues from
1,
2,
4,
5, and
6
form the hydrophobic core of the protein, but
3 is rather isolated
from the others (Fig. 1). Most hydrophobic residues are well buried
except for a few residues in
1 and
6. Helix
2 has mostly
positively charged residues (Arg110, Lys113,
Arg114, Arg117, and Lys120) on the
surface, whereas
3 has many negatively charged residues (Glu123, Asp127, Glu130, and
Glu131). Helices
2 and
3 form contiguous exposed
charged surfaces with opposite polarity (Fig. 4). Helix
4 is
relatively long compared with other helices, with a bend in the middle,
and a 310 helical turn is found between
4 and
5.
Helices
1 and
6 have mostly negatively charged residues and a few
hydrophobic residues on the surface (Fig. 1).
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Table I
Summary of structural restraints derived from experimental measurements
and structural statistics for the 20 final structures
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Fig. 1.
Structure of FADD-DD. A,
stereoview of the backbone atoms(N, Ca, C') of 20 structures of
FADD-DD. The side chains of the hydrophobic residues are shown. The
root mean square deviation about the mean coordinate position for
residues 6-93 is 0.35 Å for the backbone atoms (N, Ca, C') and 0.89 Å for all heavy atoms. No distance restraints are violated more than
0.5 Å in any structures, and no torsion angle restraints are violated
more than 5°. Other structural statistics are summarized in Table I.
B, ribbon drawing of the averaged minimized NMR structure of
FADD-DD. C, hydrogen exchange rates of FADD-DD backbone
amide protons are color-coded. The slowest to fastest group is coded
from red to blue. The slowest exchanging protons
are distributed over helices 1
(Glu94-Val108), 2
(Asp111-Glu118), 4
(Leu137-Ala151), 5
(Val158-Thr167), and 6
(Leu172-Gln181). Helix 3
(Glu123-Lys132) does not have any slowest
exchanging protons. D, surface electrostatic potential is
color-coded (left). The negative surface is in
red (< 8kBT), and the positive surface is in
blue (>8kBT). The orientation is the same as in
FADD-DD of Fig. 4. Surfaces with exposed hydrophobic residues such as
Leu, Ile, Met, Val, Trp, Ala, and Phe is colored in sky blue
(right). The orientation is rotated by 180 ° from the
representation in the left. Ribbon drawing was generated by programs
MOLSCRIPT (40) and RASTER3D (41), and electrostatic potential surface
was generated by a program GRASP (42).
|
|
Flexibility of FADD-DD--
When hydrogen exchange rates of the
backbone NHs were measured and classified into four classes by their
exchange rates, each helix showed a different internal flexibility
(Figs. 1 and 3). Helix
3 is the most flexible of all, and
5 is
the least. Helix
5 in p75ICD (34) is also reported to be better
protected from exchange compared with other helices, indicating that
the organization of hydrophobic core of death domains is similar.
Residues involved in the hydrophobic core formation are distributed
over
1,
2,
4,
5, and
6, as are the slowly exchanging
protons. The backbone NHs of the residues at the beginning of the
helices, loop regions, and some exposed sides of the helices are
exchanged in 30 min at 20 °C. The
helix hydrogen bonds generally
found at the end of the helices (35) are observed in FADD-DD, and the
exchange rates of these hydrogen-bonded backbone NHs
(Val121 and Leu170) are protected; also,
backbone NHs that have possible side chain N-cap interactions
(Lys125, Glu139, Gly160) were
protected compared with the neighboring residues with unsatisfied NHs.
Binding Interactions between Fas-DD and FADD-DD--
Because the
global fold of FADD-DD is similar to that of Fas-DD and helices
2
and
3 of Fas-DD is known to be involved in FADD binding, we focused
on
2 and
3. Mutants were constructed in which the charged
residues in helices
2 and
3 were substituted by Ala, and
Leu119 and Val121 were replaced by Asn (Fig.
2). The affinity of FADD-DD and variant proteins was measured by surface plasma resonance using BIAcore system
(Pharmacia Biosensor AB). R110A, R113A, R117A, E118A, V121N, and E123A
mutations virtually abolished the binding affinity of FADD-DD to
Fas-DD, and R114A, L119N, and D127A mutations decreased the binding
affinity more than four orders of magnitude (Fig. 3). All mutations decreased the binding
affinity of FADD-DD but D111A, K120A, E130A, and E131A had marginal
effects. All mutations constructed also decreased self-association of
FADD. The association tendency can be divided into four classes based
on self-association between FADD-DD and FADD. As indicated in Fig. 3,
self-association is also significantly alleviated by the mutations in
helices
2 and
3, indicating that self-association and binding to
Fas-DD use similar surface. These results indicate that the charged
residues in
2 and
3 are involved in Fas interaction and
self-association, which is also shown in Fas-DD (19).

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Fig. 2.
Sequence alignments of death domains from
mouse (m) FADD, human (h) FADD, mouse
Fas, human Fas, human TNFR1, and human TRADD. Mutated residues in
2 and 3 are boxed. Hydrogen exchange rates are grouped
into four classes. The slowest to the fastest exchanging protons are
shown in filled circles, half-filled circles,
open circles, and no symbols. The residues of Fas-DD
involved in binding to FADD and self-association are
underlined (19). *, mutation (Asp > Tyr) was found in
a prepared sample.
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Fig. 3.
Relative binding affinities of FADD-DD and
its variants to Fas-DD. The binding affinity of each mutant is
compared with wild type (WT) FADD-DD. Each mutant is
constructed by replacing residues in helices 2 and 3 by either
Ala or Asn. The binding affinities are shown in equilibrium association
constants (Ka) at pH 7.4. The mutations shown with
no binding constants (R110A, K113A, R117A, E118A, V121N, and E123A)
reduced binding affinity of FADD-DD by more than 10,000-folds, and
affinity could not be estimated because of the weak binding.
Self-association was estimated based on the binding between FADD and
FADD-DD after 2 min of association in 10 mM HEPES buffer
containing 150 mM NaCl, 3.4 mM EDTA, and 0.05%
surfactant P20 at 25 °C. +, the self-association tendencies are
indicated from the highest (++++) to the lowest (+).
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|
 |
DISCUSSION |
The structure of FADD-DD (Fig. 1) indicates that its fold is
similar to other death-related proteins such as Fas-DD (19), FADD-DED
(20), and caspase recruitment domain of RAIDD (21). Helices
1,
2,
4,
5, and
6 form a hydrophobic core, and helix
3 is
somewhat isolated from the rest of the protein and is the most flexible
among the helices. Considering that
2 and
3 are involved in the
binding to Fas-DD,
3 is most likely to be involved in binding
modulation and adapter protein selectivity. The flexibility of
3
could be crucial in maximizing contacts between the interacting death
domains. The lpr mutation in Fas receptor induces the
complete loss of
3 and reduces the binding affinity of Fas-DD to
FADD (36), and the corresponding mutation (V121N) in FADD-DD also showed similar loss in binding affinity. This may indicate that the
role of
3 is conserved in death domains. FADD-DD structure has an
exposed hydrophobic surface at the N- and C-terminal helices (Fig. 1),
and the same pattern was reported in FADD-DED (20). Because the C
terminus of DED is connected to the N terminus of DD in an intact FADD,
the structure of FADD would have 12 antiparallel helices comprising 6 helices from each domain and a disordered C terminus comprising
residues 184-205. The interactions between DED and DD domains are
expected to be from the residues of
1 and
6 of each domain. The
partial digestion of FADD by subtilisin indicates that the C-terminal
region comprising residues 184-205 is the most susceptible to the
protease. Also the fragments that remained intact after 2 h of
digestion were the N-terminal 88 residues (residues 1-88) and the
C-terminal 95 residues (residues 89-183), indicating that two domains
are connected by a flexible loop.
Mutations of charged residues in
2 and
3 of FADD-DD indicate that
the major binding sites between FADD-DD and Fas-DD appear to reside in
2 and
3. Mutations of Fas-DD (19), TNFR-DD (37), and TRADD-DD
(38) indicated that
2 and
3 are important for functions, but
other regions are also implicated. However, among the residues on the
surface, charged residues in
2 and
3 are most likely candidates
for protein interactions. Other residues affecting the structural
integrity of death domains, such as lpr mutant, could have
effects on protein functions. The result of extensive mutation studies
in
2 and
3 of FADD-DD indicates that this region affects binding
affinity of Fas-DD for FADD-DD. As shown in Fig.
4, the charge distribution in
2 and
3 of FADD-DD and Fas-DD are similar, indicating that interactions
between the two proteins are anti-parallel. Helix
2 of Fas-DD
interacts with
3 of FADD-DD and vice versa.

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Fig. 4.
The structure of proposed interaction sites
of FADD-DD (left) and Fas-DD (19)
(right). Positively charged residues are colored
in blue, negatively charged residues are colored in
red, and two hydrophobic residues (Leu119,
Val121) are colored in sky blue. Only residues
of FADD-DD studied by mutations are labeled. Residues with marginal
effect on binding to Fas-DD are labeled in green.
Val121 is the corresponding residue at the position of
lpr mutant in Fas. Helices 2 and 3 in FADD-DD and
Fas-DD have the same charge distribution, suggesting that interaction
between two death domains is in antiparallel pattern.
|
|
In Fas-induced apoptosis, Fas receptor is trimerized upon stimulation
and then recruits FADD. It is not known yet whether Fas receptor trimer
is needed for creating a binding site for FADD, or trimerization is a
means to expose the extra binding surface of Fas receptor to FADD. Our
experiments showed that FADD-DD binds to Fas-DD with a dissociation
constant of about 200 nM. Considering that FADD and Fas
used in experiments are mouse and human origin, respectively, and the
fact that Fas-DD binds better to an intact FADD (39) than FADD-DD
alone, Fas receptor monomer seems to be capable of recruiting FADD.
This indicates that the trimerization of Fas receptor is required to
expose a binding surface rather than creating a binding surface. The
whole binding surface of Fas-DD may not be available in monomer either
by interacting with membrane or other factors. When induced by ligand,
the whole binding surface of Fas-DD would be exposed by conformational
change and recruit FADD. Once the death domain of Fas binds to FADD, the induced conformational change (3) would convert FADD into a high
affinity form for procaspase-8, triggering recruitment and activation
of caspase-8. The mode of conformational change is not clear, but
domain movement of DD and DED of FADD is most likely. However, the low
affinity binding between DDs or DEDs may occur in a normal cell, so
overexpression of proteins containing these domains can lead to cell
death. In fact, the difference in affinity between the low affinity and
the high affinity form of Fas receptor or FADD may not be that high.
The 10-fold difference in binding affinity is equivalent to about 1.3 kcal/mol at the physiological temperature, indicating that a small
conformational change can easily switch from the low affinity to high
affinity form.
In conclusion, FADD death domain consists of six antiparallel helices
similar to other known death-related domains. Helices
2 and
3 of
death domain constitute a major binding surface and appear to interact
antiparallel with the death domain of Fas receptor. We think that the
ligand-induced exposure of the binding site of Fas receptor and FADD is
crucial to Fas-mediated apoptosis.
 |
ACKNOWLEDGEMENT |
We thank Professor B. Sykes for letting us use
the NMR in his laboratory, Drs. S. Gagné and K. B. Lee for
the help with NMR experiments, Drs. K. Rajarathnam and C. Woodward for
critical reading and comments, Dr. D. Garrett for the PIPP program, Dr. L. Kay for pulse sequences, and Dr. G. M. Clore for the data base potential library.
 |
FOOTNOTES |
*
This work is supported by Biotech-2000 and KIST-2000 program
(to K.-S. K.) from the Ministry of Science and Technology (MOST) of
Korea.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The atomic coordinates and structure factors (codes 1fad and
r1fadmr) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.
¶
These authors contributed equally to this work.

To whom correspondence should be addressed. Tel.:
82-2-958-5934; Fax: 82-2-958-5939; E-mail: keysun{at}kist.re.kr.
 |
ABBREVIATIONS |
The abbreviations used are:
FADD, Fas-associated
death domain protein;
DD, death domain;
DED, death effector domain;
TNFR, tumor necrosis factor receptor;
TRADD, TNFR1-associated death
domain protein;
RAIDD, RIP-associated ICH-1/CED-3-homologous protein
with a death domain;
NOE, nuclear Overhauser effect;
NOESY, NOE
enhancement spectoscopy;
HSQC, heteronuclear single-quantum coherence;
TOCSY, total correlation spectroscopy.
 |
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