Solution NMR Investigation of the CD95/FADD Homotypic Death Domain Complex Suggests Lack of Engagement of the CD95 C Terminus

Structure

Article

Solution NMR Investigation of the CD95/FADDHomotypic Death Domain Complex SuggestsLack of Engagement of the CD95 C TerminusDiego Esposito,1 Andrew Sankar,1 Nina Morgner,2 Carol V. Robinson,2 Katrin Rittinger,1 and Paul C. Driscoll1,*1Division of Molecular Structure, MRC-National Institute for Medical Research, Mill Hill, London NW7 1AA, UK2Department of Chemistry, Physical and Theoretical Chemistry Laboratory, University of Oxford, Oxford OX1 3QZ, UK

*Correspondence: [email protected] 10.1016/j.str.2010.08.006

SUMMARY

We have addressed complex formation between thedeath domain (DD) of the death receptor CD95 (Fas/APO-1) with the DD of immediate adaptor proteinFADD using nuclear magnetic resonance (NMR)spectroscopy, mass spectrometry, and size-exclu-sion chromatography with in-line light scattering.We find complexation to be independent of theC-terminal 12 residues of CD95 and insensitive tomutation of residues that engage in the high-orderclustering of CD95-DD molecules in a recentlyreported crystal structure obtained at pH 4. Differen-tial NMR linewidths indicate that the C-terminalregion of the CD95 chains remains in a disorderedstate and 13C-methyl TROSY data are consistentwith a lack of high degree of symmetry for the com-plex. The overall molecular mass of the complex isinconsistent with that in the crystal structure, andthe complex dissociates at pH 4. We discuss thesefindings using sequence analysis of CD95 orthologsand the effect of FADD mutations on the interactionwith CD95.

INTRODUCTION

Ligation of death receptor (DR) CD95 (Fas/APO-1) by the

cognate CD95-ligand (CD95L/FasL) or specific classes of anti-

CD95 monoclonal antibodies (mAbs) can initiate apoptotic path-

ways that play a central role in the control of homeostasis and

lymphocyte populations in multicellular organisms (Krammer,

2000; Krueger et al., 2003; Nagata, 1997; Strasser et al., 2009).

The DR family is defined by the presence of a �90 amino acid

residue-long cytoplasmic domain, known as a death domain

(DD) (Ashkenazi and Dixit, 1998; Park et al., 2007a; Wilson

et al., 2009). The DD is the focus for the binding of adaptor

proteins during the signaling events that follow DR engagement,

illustrated most pertinently by CD95-DD mutations that lead

to type Ia autoimmune lymphoproliferative syndrome (ALPS)

(Bidere et al., 2006). CD95 is present on the plasma membrane

as a preassociated homo-oligomeric receptor (Papoff et al.,

1999; Siegel et al., 2000). CD95L (or agonistic mAb) administra-

1378 Structure 18, 1378–1390, October 13, 2010 ª2010 Elsevier Ltd

tion rapidly triggers reorganization of CD95multimers (Algeciras-

Schimnich et al., 2002; Kischkel et al., 1995; Medema et al.,

1997) and the recruitment to the receptor of the adaptor protein

FADD (Boldin et al., 1995; Chinnaiyan et al., 1995). FADD

comprises an N-terminal death effector domain (FADD-DED)

tethered to the C-terminal death domain (FADD-DD). Mutual

association of FADD with CD95 via their DDs leads to binding,

by interaction with FADD-DED, of the tandem DED domains of

the apical caspase-8 to form the death-inducing signaling

complex (DISC) (Kischkel et al., 1995;Medema et al., 1997). Cas-

pase-8 is then activated by transautoproteolysis, and the cata-

lytic subunits dissociate from the DISC to effect cleavage of

cytoplasmic apoptotic effector proteins (Krammer, 2000; Peter

et al., 2007; Thorburn, 2004; Wilson et al., 2009).

The homotypic CD95-DD/FADD-DD interaction is an essential

step for apoptotic CD95 signaling. Overexpression of the FADD-

DD on its own is sufficient to block DISC formation and cell death

indicating dominant-negative behavior (Newton et al., 1998,

2001), and the analysis of several CD95-DD mutations has

shown that the breakdown in signaling occurs as a result of

a failure to bind FADD (Huang et al., 1996; Martin et al., 1999).

Currently many aspects of the precise mechanism of DISC

assembly, architecture and evolution in time within the cell

remain unresolved. Experimental approaches to probe the

earliest events in CD95 signaling have adopted a number of

agonists including native soluble CD95L (sCD95L), affinity-

tagged variants of sCD95L that can be cross-linked by a

secondary reagent, membrane-bound CD95L (mCD95L), and

several anti-CD95 mAbs. A consensus is emerging that the

precise pattern of DISC behavior can vary from one agonist to

another (Beneteau et al., 2007; Chaigne-Delalande et al., 2009;

Holler et al., 2003; Huang et al., 1999; Legembre et al., 2003;

O’Reilly et al., 2009; Schneider et al., 1998). Early in the signaling

process CD95 is commonly found in microaggregates contain-

ing high molecular weight (�200 kD) SDS- and b-mercaptoetha-

nol-resistant CD95 oligomers (Algeciras-Schimnich et al., 2002;

Feig et al., 2007; Holler et al., 2003; Kischkel et al., 1995). These

and related observations (Lee et al., 2006; Siegel et al., 2004)

have led to the description of the CD95/FADD/caspase-8 DISC

as an apoptotic signaling ‘‘platform.’’

A fundamental question in CD95-signaling is how the receptor

can transit from the ‘‘resting’’ state, where it displays no interac-

tion with the other DISC components, to one in the presence of

agonist that binds FADD. In vitro DISC reconstitution has been

demonstrated using fusion proteins that effectively cross-link

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mailto:[email protected]

Figure 1. Structure of Hetero-Octamer Assembly of CD95 and FADD DDs in the Crystal Structure Protein Data Bank Code 3EZQ

(A and B) Orthogonal views of the 4+4 hetero-octameric assembly CD95 DD chains (blue) and FADD DD chains (green) representing 50% of the contents of the

asymmetric unit in 3EZQ (Scott et al., 2009).

(C) Superposition (over helices a1–a4) of a single CD95 DD chain (blue) from 3EZQ on the solution structure of isolated CD95-DD+CT (pink) obtained by NMR at

pH 4.0 (PDB code 1DDF) (Huang et al., 1996), highlighting the closed-to-open rearrangement of the C-terminal region to form stem and C-helices.

(D) Amino acid sequences and observed regular secondary structure elements for the CD95 DD in 3EZQ and 1DDF, and FADD DD in 3EZQ and the essentially

identical solution structure conformation (PDB code 13EY) (Berglund et al., 2000). Residues discussed in the text are depicted in color in the 3EZQ ribbon

diagrams and in the amino acid sequences: CD95 K263 (orange), mutation of which to Ala yields a more tractable DD construct (Huang et al., 1996); CD95

Q311 and L315 (magenta) involved in contacts between CD95-DD homodimers; and residues reported to be important for CD95:FADD interaction (red) including

sites of type Ia ALPS mutations in CD95 A257D and D260A (Martin et al., 1999; Vaishnaw et al., 1999) and FADD residues R113, R117, D123, K125, and R146

(Hill et al., 2004). Further depiction of interdomain contacts is provided in Figure S1.

Structure

NMR of CD95/FADD Death Domain Complex

CD95-DDs (Hughes et al., 2009; Roy et al., 2008). On the other

hand, it has been observed that the isolated DDs of CD95 and

FADD can bind each other directly to form a homotypic

protein-protein complex (Boldin et al., 1995; Ferguson et al.,

2007; Scott et al., 2009; Yang et al., 2005). Understanding the

three-dimensional structure of this complex has the potential

to inform the basis of DISC assembly, because it presumably

reflects a state of the intact CD95 receptor that is competent

to form interactions with the adaptor protein. In vitro, recombi-

nant CD95-DD tends to form soluble aggregates at neutral pH.

The nuclear magnetic resonance (NMR) structure of monomeric

wild-type CD95-DD was obtained at pH 4, and mutations of

certain exposed residues can mitigate against CD95-DD self-

association (Huang et al., 1996), although mostly with loss of

FADD-binding. We have previously reported that CD95-DD

tagged with a solubility enhancement tag (SET) allowed for

Structure 18, 1378–1

a preliminary NMR analysis of CD95-DD interactions with

FADD-DD (Ferguson et al., 2007). More recently, the crystal

structure was reported of the complex of wild-type CD95-DD

with FADD-DD prepared from mixed Escherichia coli lysates,

with crystal growth achieved at pH 4.0 (Scott et al., 2009). The

asymmetric unit contains two ‘‘4+4’’ hetero-octameric DD

assemblies (Figure 1), within which the CD95-DD chain is

dramatically reconfigured compared to the structure in solution

(Huang et al., 1996). The C-terminal part of the chain comprising

the canonical helices a5 and a6 rearranges to form an elongated

‘‘stem’’-helix in a a-a hairpin interaction with a ‘‘C’’-helix

formed from the extremeC-terminal residues that are disordered

in the free CD95-DD (Huang et al., 1996). The rearrangement

exposes the hydrophobic core of CD95-DD, and sequesters

FADD-DD with a normal DD conformation (Figure 1). The overall

complex comprises a tetramer of the CD95-DD:FADD-DD

390, October 13, 2010 ª2010 Elsevier Ltd All rights reserved 1379

Figure 2. GST Pull-Down Assay of CD95-DD:FADD-DD Interactions

Both the intact intracellular domain of CD95 (GST-CD95-IC, lane 2) and the

death domain lacking the extreme C-terminal tail of CD95 (GST-CD95-DD,

lane 4) bind to FADD-DD. The interaction is abolished on mutation of either

CD95 A257D (corresponding to a type Ia ALPS lesion, lane 6) or FADD

R117E (lane 9), as before (Ferguson et al., 2007; Hill et al., 2004; Martin

et al., 1999). Controls indicate that GST alone (lane 8) or the glutathione-modi-

fied beads (lane 10) do not bind FADD-DD. See also Figure S2.

Structure


heterodimers where contacts are only made between the CD95

stem/C-helix regions (Scott et al., 2009) (see Figure S1 available

online).

The crystal structure is notable in that it provides a dramatic

example of protein structure plasticity and can rationalize at least

a subset of the structure-activity data available for CD95-FADD

interactions. It is important to establish whether the model

for the formation of the core DISC is more widely applicable to

homologous death receptor interactions with FADD (Ashkenazi,

2008; Bodmer et al., 2000; Kischkel et al., 2000; Sprick et al.,

2002). We previously reported that high molecular weight com-

plexes of FADD-DD with a Gb1-CD95-DD construct lacking

the 12 C-terminal residues (Ferguson et al., 2007). In this report

we describe further biophysical measurements of soluble CD95-

DD:FADD-DD complexes under near physiological conditions.

We address the dependence of complex formation on the

C-terminal tail region of CD95, the sample pH and mutations in

CD95-DD and FADD-DD, and discuss the results with reference

to a sequence analysis of emergent CD95 orthologs. We con-

clude that in solution the complex formed between CD95 and

FADD DDs has characteristics significantly different from that

predicted by the current crystal structure.

RESULTS

Complexation of the DDs of CD95 and FADDThere are conflicting reports concerning the ability of FADD-DD

alone to interact with CD95 (Boldin et al., 1995; Ferguson et al.,

2007; Muppidi et al., 2006; Sandu et al., 2006; Yang et al.,

2005). In our hands, both the intact intracellular domain of

CD95 (CD95-IC, residues 194–335) and the CD95 DD (residues

218–323) tagged to glutathione S-transferase (GST) are able to

pull down recombinant FADD-DD (residues 89–192) (Figure 2).

The interaction is abrogated by introducing either the substitu-

tion A257D in CD95-DD or R117E in FADD-DD. CD95 A257D

corresponds to a type Ia ALPS lesion for which the globular DD

structure is maintained (Martin et al., 1999). Similarly FADD-DD

R117E is a stably folded variant of FADD-DD (Figure S2) with

reduced CD95 binding activity (Hill et al., 2004). The CD95-IC

protein pulls down FADD-DD at least as efficiently as the shorter

CD95-DD. Therefore, in this experiment, the extra CD95-IC resi-

dues outside the globular DD do not interfere in the interaction

with FADD-DD (Chinnaiyan et al., 1995). These results demon-

strate a direct interaction between CD95-DD and FADD-DD

that can be abrogated by point mutations on the surface of either

protein.

Wild-type CD95-DD has an intrinsic tendency to self-asso-

ciate at physiological pH (Huang et al., 1996; Scott et al.,

2009). The fusion of CD95-DD to the C terminus of a SET such

as GST or the B1 domain from streptococcal immunoglobulin-

binding protein G (Gb1) (Zhou et al., 2001) can significantly

enhance its tractability (Ferguson et al., 2007; Huth et al.,

1997). For the present analysis we sought to avoid the use of

an appended SET by exploiting the observation that the K263A

mutant of CD95-DD displays a markedly reduced tendency to

self-aggregation while retaining FADD binding (Ferguson et al.,

2007; Huang et al., 1996). It was previously reported that

a CD95 construct lacking the C-terminal 15 residues makes

a stronger interaction with FADD than the full length receptor,


leading to the declaration of an inhibitory role for this region

(Chinnaiyan et al., 1995). However, in the recently reported

crystal structure of the complex of copurified CD95 and FADD

DDs this C-terminal region seems to carry out a critical role in

supporting the ‘‘bridge’’ between two ‘‘opened’’ CD95 chains

(Scott et al., 2009) (Figure S1). To probe the role of the C-terminal

residues of CD95 in the interaction with FADD-DD using solution

NMR spectroscopy, we created CD95(K263A) DD constructs

that either omitted (CD95-DD) or included (CD95-DD+CT) the

extreme C-terminal tail (CT) region.

Size-Exclusion Chromatography-Multiangle LightScattering AnalysisWe analyzed the complexes formed by CD95-DD and CD95-

DD+CT with FADD-DD by analytical size-exclusion chromatog-

raphy (SEC) coupled to multiangle light scattering analysis

(SEC-MALS) (Figure 3). Samples were run over a concentration

range corresponding to 2–10 mg/ml total protein. We obtained

an essentially symmetric chromatographic light scattering profile

consistent with a monodisperse particle with elution volume

12.4 ml and 12.2 ml, respectively, in the range of 4 to 10 mg/ml.

The elution volume was slightly larger at 2 mg/ml, suggesting

some limited dissociation of the complex at the lowest concen-

tration tested. The absence of any significant readout at elution

volumes >13 ml indicates the absence of any substantial popu-

lation of intermediate heteromeric species, suggesting that the

complex formation displays a considerable degree of coopera-

tivity. When fractions from semipreparative gel filtration experi-

ments are analyzed by SDS-PAGE, the eluted proteins appear

to be present in approximately equal amounts (Figure S3) con-

sistent with amino acid analysis of the excised gel bands

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Figure 3. SEC-MALS Analysis of Isolated

CD95-DD:FADD-DD Complexes

Both CD95-DD (A) and CD95-DD+CT (B) form

high molecular weight assemblies with FADD-DD

that elute at consistent volumes over a range of

concentration. The predicted particle molecular

masses from the MALS analysis are: 105, 105,

105, 103, and 100 kD for CD95-DD:FADD-DD

and 111, 110, 109, 108, and 104 kD for CD95-

DD+CT at 10, 8, 6, 4, and 2 mg/ml total protein

concentration, respectively. See also Figure S3.

Structure


(CD95-DD:FADD-DD ratio �1.2; data not shown). The SEC-

MALS data clearly indicate that under chromatographic condi-

tions the two DDs form a complex with a molecular mass of

�105 kD for CD95-DD with FADD-DD and �110 kD for CD95-

DD+CT with FADD-DD. Given the predicted molecular masses

of the individual domains (CD95-DD, 12.0 kD; CD95-DD+CT,

13.4 kD; FADD-DD, 11.9 kD) these results are consistent with

the formation of a particle that comprises at least nine polypep-

tides. We note that for the DD constructs that we have employed

(that lack the flexible C-terminal tail and 6*His affinity tag present

in the FADD construct used for the crystal structure [Scott et al.,

2009]) the SEC-MALS-derived masses are substantially larger

than would be predicted for a 4:4 complex. Critically, these

data clearly show that FADD-DD and CD95-DD form a complex

with high molecular mass independent of the presence of the CT

region of CD95.

Mass SpectrometryPurified CD95-FADD DD complexes were subjected to capillary

nanoflow electrospray ionization-mass spectrometry (nanoESI-

MS). Under conditions designed to maintain noncovalent inter-

actions, this methodology provides a snapshot of the overall

molecular mass and subunit composition of macromolecular

assemblies (Hernandez and Robinson, 2007). The mass spectra

obtained show signals for monomeric FADD-DD and CD95-DD

or CD95-DD+CT species in the low mass-to-charge (m/z)

region, together with higher m/z ion-series attributable to the

complexes. The clearly dominant signals represent complex

species with an overall mass of 120.1 kD and 127.1 kD respec-

tively, matching a 10-chain, 5:5 stoichiometry based on the

internal mass references for the monomeric species (Figure 4).

The spectra also indicated the presence of minor components

with a higher mass, correspondingmainly to 5:6 and 6:5 species.

The 5:5 ion series persisted as the dominant signal over a range

of protein concentration from 0.6 to 12mg/ml, indicating specific

complex formation. Importantly, no ion series was observed

consistent with a stable 4:4 complex in solution. Thus FADD-

DD and CD95-DD or CD95-DD+CT form complexes in solution

that are robust to nanoESI-MS analysis, with predicted molec-

ular mass consistent with a 5:5 domain composition. Again,

the ability to form the complex was independent of the presence

of the CD95 CT region.


NMR Titration of 15N-Labeled CD95-DD Constructswith FADD-DDUsing a protocol based on that reported previously (McAlister

et al., 1996), we separately titrated 15N-labeled CD95-DD and

CD95-DD+CT with unlabeled FADD-DD at constant concentra-

tion of the CD95 protein (Figures 5A and 5B). In each case, as

the FADD-DD concentration is increased, the intensities of the

majority of CD95 cross-peaks in the 2D 15N-HSQC spectrum

monotonically decrease to a level below the detection limit,

leaving a small number of signals derived from regions of the

CD95 backbone or Asn, Gln, or Arg side chains that remain

highly flexible in the complex. We obtained sequence-specific

resonance assignments relevant to the conditions employed

in the titration experiments, permitting unambiguous identifica-

tion that the persistent CD95 cross-peaks belong to the extreme

N- and C-terminal regions of the protein, e.g., for CD95-

DD+CT backbone amide cross-peaks from E218-A221 at the

N-terminus together with S322–V335 and the side chain NH2

cross-peaks of N324, N326, N329, and Q332 and the R328

N3H cross-peak from the C terminus (Figure 5B). 15N-relaxation

measurements (Kay et al., 1989) performed on the isolated

CD95-DD+CT protein confirm the dynamic flexibility on the

ns-to-ps timescale of the backbone for these N- and C-terminal

residues before complexation (data not shown). The lack of any

detectable FADD-dependent chemical shift perturbation for

these peaks coupled with the maintenance of their limited 1HN

chemical shift dispersion in the spectrum of the complex indicate

that CD95 residues E218–A221 and S322–V335 remain in

a highly disordered state in the complex. A similar pattern of

results was obtained with the shorter 15N-CD95-DD construct;

cross-peaks remaining at the end of the titration derive from

the disordered N- (E218–A221) and C-terminal (S322–E323)

chain segments, respectively (Figure 5B).

NMR Titration of 15N-Labeled FADD-DDwith CD95-DD ConstructsWhen 15N-FADD-DD was titrated with unlabeled CD95-DD+CT

virtually all of the FADD-DD cross-peaks dramatically diminish

in intensity (Figures 5C and 5D). At the highest concentration

of CD95-DD+CT only a few cross-peaks from the N-terminal

and C-terminal regions remain; the end-point spectrum

comprises the cross-peaks for the backbone NH of M92 and


Figure 4. Nanoflow ESI-MS Analysis of Isolated CD95-DD:FADD-DD

Complexes

CD95-DD+CT (A) and CD95-DD (B) complexes with FADD-DD, exchanged

into ammonium acetate buffer, yield ESI-MS spectra with a dominant ion

series in each case (blue symbols) with overall molecular mass 127,152 ± 22 D

and 120,131 ± 40 D, respectively. The corresponding predicted masses for

a 5:5 heterodecamer, based on the experimentally determined masses of

the protomer components, are 127,140 D and 120,100 D. The spectra suggest

the presence of small amounts of particles with 6:5 (green symbols) and 5:6

(red symbols) FADD:CD95 stoichiometry.

Structure


the side chain NH2 protons of N102 (N terminus), and the back-

bone NHs of residues R189–A192, side chain NH2 protons of

Q187 and N188, and the R189 N3H (C terminus). Essentially

identical behavior was observed when a solution of 15N-labeled

FADD-DD was titrated with unlabeled CD95-DD (data not

shown).

Ile, Leu, and Val 13CH3-Methyl Transverse RelaxationOptimized Spectroscopy AnalysisWe analyzed the complexation of CD95 and FADD DD proteins

within the paradigm of [1H,13C]-methyl group labeling for Ile,

Leu, and Val (ILV) side chainswithin a uniformly deuterated back-

ground (Goto et al., 1999), exploiting ILV-13C-methyl transverse

relaxation optimized spectroscopy (13CH3-TROSY) (Tugarinov

et al., 2003). This approach allows for the recording of slowly

relaxing methyl group NMR signals from ILV residues within

multiprotein assemblies (Ruschak and Kay, 2010). We sepa-

rately prepared ILV-13CH3-labeled perdeuterated samples of


CD95-DD+CT and FADD-DD; the corresponding 13CH3-TROSY

spectra are presented in Figure 6. In each case, the cross-peaks

are well resolved as expected for a small globular protein.

Addition of a perdeuterated sample of the DD binding partner

dramatically changes the appearance of the spectrum. When2H-labeled FADD-DD is added to ILV-13CH3-CD95-DD+CT the

intensity of all the cross-peaks is significantly diminished consis-

tent with complex formation (Figure 6A). However, the effects of

complex formation differ markedly for two different sets of cross-

peaks within the spectrum. First, a small number cross-peaks

retains a normal ‘‘monolithic’’ line shape, with relatively high

peak intensity, and unperturbed chemical shifts relative to those

for the free protein. These peaks can be ‘‘selected’’ by plotting

the spectrum at a relatively high base contour level as in Figure 6.

Despite equivalent data processing parameters, these peaks are

marginally broader than in the free-state spectrum. The assign-

ments of these lesser-perturbed peaks correspond to the set

of ILV residues from the flexible N- (V220, I222) and C-terminal

(I318, I331, L334, V335) tail regions of CD95-DD+CT. Plotting

the spectrum at a lower contour level reveals the signals for the

remaining ILV-13CH3 groups. For each such cross-peak in the

free-state spectrum, there appears to have been a dispersion

of the intensity into a local cluster of broader, distinct but often

overlapping features. These effects are most clearly identified

for the more highly resolved peaks in the spectrum of the free

protein (Figures 7A and 7B). For example, the I314 d1CH3

cross-peak is clearly converted into a cluster of at least four

cross-peaks in the spectrum of the complex. The 1H upfield-

shifted dCH3 group cross-peak of L229 appears to be dispersed

into at least four components with differing intensities (one

feature is at the level of the noise). A similar pattern of effects is

observedwhen 2H-labeled CD95-DD+CT is added to ILV-13CH3-

FADD-DD. Thus only a very small number of the FADD-DD13C-methyl cross-peaks retains a monolithic appearance with

unperturbed chemical shifts and relatively high signal intensity

in comparison to the free protein (Figure 6B). These cross-peaks

(L97, L180, L186, and the LeuVal-dipeptide from the vector-

encodedN-terminal affinity tag) arise from residues in the flexible

N- and C-terminal regions of the FADD-DD construct (Berglund

et al., 2000). The other cross-peaks in the spectrum exhibit

the behavior witnessed for the second set of CD95-DD+CT

cross-peaks, namely dispersion into apparently local clusters

of much weaker features (e.g., I126 d1CH3 in Figure 7C and

the 1H-upfield L119 dCH3 in Figure 7D). In a similar experiment

in which ILV-13CH3-FADD-DDwas titrated with CD95-DD essen-

tially the same outcome was obtained (data not shown).

The 13CH3-TROSY results clearly indicate that on complex

formation by FADD-DD with either CD95-DD or CD95-DD+CT,

a particle is formed that has disordered N- and C-terminal tail

regions of both proteins in concordancewith the results obtained

with the 15N-labeling. The dispersal into clusters of weak peaks

for the methyl groups residing in the globular parts of the two

DDs suggests that different FADD-DD and CD95-DD/CD95-

DD+CT protomers occupy nonequivalent sites within the com-

plex, suggesting a degree of structural asymmetry. Alternatively,

the cross-peak clusters correspond to a range of distinct com-

plexes with variant mass and subunit composition. For each

resolved cross-peak cluster, one component exhibits 1H and13C chemical shifts essentially identical to that of the free protein

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Figure 5. 2D 15N,1H-NMR of the Interaction

between CD95-DD and FADD-DD

Detail of the 2D 15N,1H-HSQC NMR spectra of15N-labeled CD95-DD+CT in the absence (A) and

presence (B) of excess unlabeled FADD-DD,

and 15N-labeled FADD-DD in the absence (C)

and presence (D) of excess unlabeled CD95-

DD+CT. The resonance assignments for cross-

peaks visible in the spectra of the respective

complexes are indicated (B and D). In (B) the

R328 3NH cross-peak is aliased from outside the15N spectral window; similarly for the R189 3NH

cross-peak in (D). Although the majority of CD95-

DD+CT cross-peaks are absent in (B) the back-

bone and side chain cross-peaks for the flexible

N- and C-terminal tail regions are clearly visible.

Likewise in (D) only the cross-peaks for the N-

and C-terminal tail regions of FADD-DD are

retained. The inset in (B) shows a section of a

similar spectrum of 15N-labeled CD95-DD in the

presence of excess FADD-DD. See also Figure S4.

Structure


(Figure 7), although with distinctly broader 1H linewidth. These

peaks can be attributed either to a small population of free

protein chains in exchange with the oligomeric complex, or

to a subset of sites in the complex that exhibit unperturbed

chemical shifts relative to the free protein. The relative intensity

of this component varies from peak cluster to peak cluster but

is invariant to addition of excess partner protein indicating

saturation of binding. Under equivalent sample conditions in

the 15N-based experiments, we failed to detect signals from

free protein chains. We therefore attribute these cross-peaks

most likely to protein chains in the bound state that exhibit similar

to free state chemical shifts. Moreover, because multiple cross-

peaks can be resolved the exchange of DD protomers between

the nonequivalent sites must be slow on the timescale repre-

sented by the relatively small chemical shift differences between

the components in each cluster.

Effect of C-Helix Mutations in CD95-DD+CTIn the 3EZQ crystal structure, the hetero-octamer of CD95-DD

and FADD-DD domains includes pairwise reciprocal contacts

between CD95-DD dimers comprised mainly of a cluster of

four Q311 side chains (one from each of the polypeptides) sand-

wiched between two pairs of L315 side chains (Figure 1; Fig-

ure S1). We constructed Q311A and L315A mutants of CD95-

Structure 18, 1378–1390, October 13, 2010 ª

DD+CT. Both of these variants yield15N-HSQCNMR spectra indicating reten-

tion of a stable DD fold (Figures 8A and

8B), consistent with the side chains of

these two residues being highly exposed

in the structure of isolated CD95 DD

(Huang et al., 1996). For each mutant,

we observed complex formation in the

presence of FADD-DD evidenced by the

characteristic loss of cross-peak intensity

for the dispersed signals from the glob-

ular part of CD95-DD, and retention of

the cross-peaks from the flexible terminal

regions, exactly as for unmodified CD95-DD+CT (Figures 8A

and 8B).

Effect of Low pHThe 3EZQ crystal structure was reportedly obtained under

conditions of pH �4. We exchanged the buffer of a sample of

the 15N-CD95-DD+CT:FADD-DD complex prepared at pH 6.2

to pH 4.0 and rerecorded the NMR spectrum (Figure 8D). Under

these conditions the majority of the backbone NH cross-peaks

are clearly visible with characteristics consistent with unbound15N-CD95-DD+CT. This result suggests that the complex formed

in solution is dissociated at the lower pH.

Sequence Conservation of the Extreme C-TerminalRegion among CD95 OrthologsGiven the evidence from the crystal structure of the scope for

dramatic remodeling of the C-terminal region of CD95, we con-

structed an in-depth sequence analysis (Figure 9) of putative

CD95 species orthologs to examine the degree of conservation

of the residues involved. Sequences annotated as CD95 were

derived from a variety of genomic database sources and an

alignment constructed by optimization of the sequence similarity

over the globular part (a1–a6) of the canonical DD structure.

In Figure 9, only the part of the alignment focusing on the

2010 Elsevier Ltd All rights reserved 1383

Figure 6. ILV-13CH3-TROSY NMR of CD95-

DD:FADD-DD Complexes

Overview of the Ile- (top) and Leu/Val- (bottom)

regions of the 13CH3-TROSY spectrum of (A)

ILV-13CH3-,2H-labeled CD95-DD+CT and (B)

ILV-13CH3-,2H-labeled FADD-DD in the absence

(black contours) and presence (red) of 2H-labeled

FADD-DD and 2H-labeled CD95-DD+CT, respec-

tively. The spectrum of each complex is character-

ized by massive intensity loss for the majority of

cross-peaks. The residue-specific assignments

of the signals that retain significant intensity, cor-

responding to methyl groups within the N- and

C-terminal regions of the respective polypeptide

chain, are indicated; in (B) asterisks indicate

cross-peaks arising from the vector-encoded

Leu-Val dipeptide in the affinity tag (Berglund

et al., 2000). Dashed-line boxes indicate the

regions highlighted at lower contour level in

Figure 7.

Structure


C-terminal region, starting at the completely conserved Trp

residue in the globular DD, is presented. Inspection of the align-

ment as a whole (not shown) indicates that the sequence diver-

gence among known CD95 DDs is relatively limited, consistent

1384 Structure 18, 1378–1390, October 13, 2010 ª2010 Elsevier Ltd All rights reserved

with the predicted orthology. Neverthe-

less it is apparent that sequence con-

servation is significantly higher among

residues in helices a1–a6 than in the

adjoining regions,evidenced inFigure9by

the more definitive consensus sequence

for a5–a6 than for the extreme C ter-

minus. Most notable in this respect is that several species (cat,

dog, frog, and zebrafish) lack the extreme C-terminal region,

with polypeptides terminated at the position corresponding to

S320 in human CD95.

Figure 7. Effect of Complex Formation on

Selected, Well-Resolved ILV-13CH3 Methyl

Cross-PeaksSelected regions, corresponding to boxed areas

in Figure 6, of the 13CH3-TROSY NMR spectra of

ILV-13CH3-,2H-labeled CD95-DD+CT (A and B)

and ILV-13CH3-,2H-labeled FADD-DD (C and D)

in the absence (black contours) and presence

(red) of 2H-labeled FADD-DD and 2H-labeled

CD95-DD+CT, respectively. The residue-specific

assignments of the cross-peaks in the free

CD95-DD+CT and FADD-DD spectra are indi-

cated. The base contour level used for the spectra

of the complexes is substantially lower than in

Figure 6, and close to the noise floor for the

L229 cross-peak (B) that, although well-resolved,

is relatively weak in both free- and bound-states.

Figure 8. Effect of CD95 Mutations Q311A

and L315A and Low pH on Complex Forma-

tion with FADD-DD

Two-dimensional 15N,1H-HSQC NMR spectra of15N-labeled CD95-DD+CT Q311A (A) and L315A

(B) mutants of in the absence (back) and presence

(red) of excess unlabeled FADD-DD. Comparison

of these data with Figure 5 show that neither

mutation impacts complex formation, evidenced

by loss of all signals bar those in the flexible ter-

minal regions, exactly as for unmodified CD95-

DD+CT (Figure 5B). See also Figure S1. (C) 2D15N,1H-HSQC NMR spectrum of 15N-labeled

CD95-DD+CT in the presence of excess FADD-

DD at pH 6.2 (Figure 5B) and (D) the correspond-

ing spectrum of the sample after buffer exchange

to pH 4.0. The increase in the number and inten-

sity of cross-peaks in (D), with dispersion essen-

tially identical to that of a sample of isolated

CD95-DD+CT (not shown), indicates dissociation

of the CD95-DD+CT protein from the high molec-

ular weight complex at the lower pH.

Structure


DISCUSSION

We have described the analysis of the complex species formed

between the DDs of CD95 and FADD using heteronuclear NMR

spectroscopy, coupled with SEC-MALS and MS methods.

We cast the following discussion of our observations in terms of

the formation of a high molecular weight complex Cn:Fm from its

constituent protomers ‘‘C’’ (CD95-DD or CD95-DD+CT) and ‘‘F’’

(FADD-DD); n and m are integers. The results of our attempt to

characterize the composition of the Cn:Fm object using SEC-

MALS (Figure 3) suggest a composition that is at least nine protein

chains for both FADD-DD:CD95-DD and FADD-DD:CD95-DD

+CT complexes. In combination with the SDS-PAGE and amino

acid analysis data indicating an approximately equal number

of FADD and CD95 polypeptides in the complex we conclude

that the particles detected in the SEC-MALS experiment corre-

spond to either C4:F5 or C5:F4. When the same samples are sub-

jected to native phase nanoESI-MS, the clearly dominant ion-

series observed for both FADD-DD/CD95-DD and FADD-DD/

CD95-DD+CT combinations corresponds to a C5:F5 particle

(Figure 4). It is unclear why there is not a closer match between

the molecular weights reported by SEC-MALS and ESI-MS. In

the mass spectra the appearance of single monomers under the

soft instrument conditions used to prevent collision-induced

Structure 18, 1378–1390, October 13, 2010 ª

dissociation of the complexes suggests

the potential for dynamic rearrangement

and subunit exchange. Therefore we

must conclude that under the nonequilib-

rium conditions of the SEC-MALS experi-

ment one of the subunits of the 5:5 com-

plex is dynamically dissociated to leave

5:4 species. Critically, we did not ever

detect the presence of a C4:F4 species

that would correspond to the hetero-

octamer of the crystal structure.

We adopted asymmetric isotope label-

ing and heteronuclear NMR spectros-

copy to probe the nature of the complexes formed between

FADD and CD95 DD proteins. Both 15N-labeling with standard1H,15N-HSQC spectroscopy and ILV-13CH3-labeling with 13CH3-

TROSYNMR reveal that signals from the flexible terminal regions

of the proteins retain a relatively narrow line width and unper-

turbed chemical shifts, consistent with these regions retaining

their flexible, disordered characteristics in the complex. On the

other hand, the cross-peaks from the more globular parts of

the DDs are apparently broadened beyond detection (for the15N-labeling) or are dispersed into multiple cross-peaks with

slightly perturbed chemical shifts (ILV-13CH3-labeling). The

cross-peaks from the terminal regions appear to be in formal

fast exchange between the free and complex states, such that

only a very limited contribution to the cross-peak line widths

arises from the slower overall tumbling. The identification of

the exchange regime for the other signals is less straightforward

in the case of the 15N-labeled samples where the chemical shifts

of the cross-peaks are not recovered even at the highest

concentration of the binding partner. The overall outcome of

the experiments, where the titration of one (unlabeled) protein

with another 15N-labeled protein leads to intensity reduction

rather than gradual increase in the residual signal linewidth or

perturbation of the chemical shift, is consistent with slow

exchange (Figure 5; Figure S4). The spectrum changes because

2010 Elsevier Ltd All rights reserved 1385

Figure 9. Sequence Comparison of the

C Terminus of CD95 Orthologs

Sequence alignment of selected CD95 orthologs,

focused on the C-terminal region of the chain

including (top) the canonical a5–a6 helices of the

isolated CD95 DD in solution, PDB code 1DDF

(Huang et al., 1996), and the stem-C-helix regions

in the crystal structure of the CD95-DD:FADD-DD

complex PDB code 3EZQ (Scott et al., 2009). For

1DDF red symbols indicate residues buried in

the core of the death domain. For 3EZQ symbols

indicate residues involved in the intramolecular

stem-C-helix side chain contacts (green), inter-

molecular interactions between protomers in

the major CD95-DD homodimer contact (blue),

and side chain interactions describing the CD95-

DD dimer-dimer interface within hetero-octamer

(orange) (Figure 1; Figure S1). The alignment was

manually optimized using the sequences corre-

sponding to the entire globular DD (not shown).

Dashes indicate gaps introduced to optimize the

alignment. The avian sequences extend signifi-

cantly beyond position 335 (indicated by ..). Very

highly conserved residues and strongly con-

served residues have a dark gray and a light

gray background respectively. Bottom: Con-

sensus sequence. Essentially invariable residues

are shown in bold uppercase, strongly conserved

residues in upper case; lower case letters indicate

residues that are relatively well conserved in the

extreme C-terminal region.

Structure


the signal intensity corresponding to the ‘‘free’’-form of the 15N-

labeled protein (e.g., C) is depleted by the presence of the unla-

beled partner protein F as a result of complex formation—the

separate C resonances from the Cn:Fm complex being too broad

for detection as a result of slow rotational diffusion, or exchange

broadening among multiple resonances for the complex.

Strategies for ILV-13CH3-group labeling (Goto et al., 1999) and

the exploitation of the relaxation-cancelling effects obtained in13CH3-TROSY NMR (Ollerenshaw et al., 2003; Tugarinov et al.,

2003) provide a useful means to probe the structural and

dynamic characteristics of high molecular mass protein assem-

blies (Ruschak and Kay, 2010). The overall outcome of this

approach applied to the complex formed between the DDs

of FADD and CD95 yields a picture broadly consistent with

that obtained by 15N-HSQC spectroscopy. Thus, the ILV-13CH3

cross-peaks from the disordered N- and C-terminal tail regions

of the two constituent domains in the complex retain a relatively

narrow intense signal with unperturbed chemical shifts, whereas

the cross-peaks from the ordered globular regions are massively

diminished in intensity (Figure 6). In contrast to the case for 15N-

labeling, the second group of cross-peaks is revealed at low

contour level: each signal appears to have been dispersed

into a cluster of often partially overlapping features (Figure 7).

The simplest interpretation of these results is that: (1) the N-

and C-terminal regions of the two distinct protein chains retain

a high degree of disorder in the complex; and (2) the globular

regions of the domains can occupy one of a number of microen-

vironments within the complex, each with a distinct set of influ-

ences on the methyl group chemical shifts. The latter might

also arise if the sample contains multiple complex species

each with a different overall subunit composition. However the


pattern of cross-peak intensities is not consistent with the ESI-

MS and SEC-MALS analysis that indicate the predominance of

a single major species. On the other hand, there is no straightfor-

ward method to accurately characterize the compositional

dispersion of the sample at the relatively elevated NMR concen-

tration. An alternative explanation is that the FADD-DD:CD95-

DD complex possesses a low degree of symmetry and the local

environment of the equivalent NMR spins in different chains

of the complex can appear subtly different. It is interesting to

note that the reported 3.2 A resolution crystal structure of the

hetero-dodecameric �157 kD PIDDosome core complex com-

prising the DDs from the proteins PIDD and RAIDD, that pos-

sesses a stoichiometry of 7:5 RAIDD-DD:PIDD-DD, is a particle

with intrinsic low symmetry (Park et al., 2007b). Thus, although

the individual PIDDosome DDs are in quasi-equivalent environ-

ments, there are eight unique asymmetric DD-DD interfaces

and none of the individual PIDD-DDs (or RAIDD-DDs) resides

in an identical arrangement of contacts to any other protomer.

Moreover, the recently reported structure of the 14-chain

Myddosome assembly displays a similar nonexact equivalence

of the contacts between component DDs (Lin et al., 2010).

It seems possible that the NMR data for the FADD-DD:CD95-

DD complex could be reporting a similar lack of equivalence

for the constituent domains. Preliminary 13CH3-TROSY data

obtained in our laboratory for the PIDDosome core complex

exhibit highly similar characteristics to those observed for

FADD-DD:CD95-DD+CT (data not shown). We are not aware

that such a degree of complexity has been reported in 13CH3-

TROSY spectra before. Increased multiplicity (doubling) of Ile-

d1CH3 signals in slow exchange has been observed in the

ligand-induced T / R allosteric equilibrium for the regulatory

All rights reserved

Structure


chains of the heterododecameric aspartate transcarbamoylase

(ATCase) (Velyvis et al., 2007) and, recently, the observation of

nonequivalent methionine methyl group signals within an other-

wise symmetric homoheptameric system was reported (Religa

et al., 2010). However, in neither case was the outcome indica-

tive of a loss of overall symmetry of the major globular part of the

protein structure. Indeed in the ATCase case, the spectra imply

that symmetry of the overall structure is maintained in the T- > R

transition (Velyvis et al., 2007), and in the latter case the ‘‘split’’

signals derive from a flexible appendage that can exchange

position either side of an ‘annulus’ of limited dimension (Religa

et al., 2010). A complete accounting for the complexity of13C-ILV-methyl TROSY spectra for the FADD-DD:CD95-DD+CT

complex warrants further investigation. Nevertheless it is safe

to conclude that the spectra are not consistent with a monodis-

perse particle with high symmetry.

Relationship to the Crystal Structureof CD95-DD:FADD-DDIt is important to discuss our observations in the context of

the recently reported 2.7 A resolution crystal structure of a

FADD:CD95 DD-DD complex, Protein Data Bank code 3EZQ

(Scott et al., 2009). Apparently critical to the formation of the

contacts between the opened CD95-DD subunits within the

hetero-octameric assembly is the formation of an intrachain

a-helical hairpin loop that brings together hydrophobic side

chains from the stem-helix (L306, I310, I313, I314) with residues

from a new C-helix (F327, I331, L334) derived from the extreme

CT region of CD95 (Figure 1; Figure S1); this is a region that is

clearly disordered in the isolated CD95 DD (Huang et al.,

1996). Along with H-bond contacts involving the terminal side

chain amino group of K299, several of these residues combine

with others (notably L303, A307, I318) to make reciprocal pair-

wise interactions between the CD95-DD stem/C-helix hairpins,

forming an antiparallel four-helix bundle bridge as the CD95-

DD dimer interface (Figure S1). We note that the crystal

comprises a FADD-DD construct with a 23-residue C-terminal

tail region (including a His6-affinity purification tag) that is not

resolved in the electron density and a CD95-DD construct

appended by a nonnative C-terminal Leu336-Glu337 dipeptide

that makes multiple interchain contacts with the FADD-DD

partner and the neighboring CD95-DD (Figure S1). The predicted

molecular mass of the overall eight-chain assembly in the crystal

is 108 kD; the ordered region lacking the FADD tail peptide would

have a mass �98 kD.

The combination of our previous results (Ferguson et al.,

2007) together with data presented here unambiguously demon-

strate that complex formation occurs in vitro for combinations

of CD95 and FADD DD constructs both in the presence and

absence of the twelve C-terminal CD95 residues. Critically our

NMR data demonstrate that residues S322–V335 retain a high

degree of flexibility in the complex formed by CD95-DD+CT

(Figure 5B and Figure 6A), indicating that this region does not

participate in the formation of the stem/C-helix hairpin predicted

by the crystal structure. Moreover, we find an overall mass for the

complex that is substantially higher than would be consistent

with the hetero-octameric assembly in the crystal (Figure 3 and

Figure 4). Also we observed that high molecular weight complex

formation persists in solution despite Ala-substitution of residues


Q311 and L315 (Figures 8A and 8B), side chains that appear to

be important for crystal contacts between CD95-DD homo-

dimers (Figure 1; Figure S1). The result for the L315A mutant is

consistent with a previous report that this mutation does not

impact CD95 association with FADD (Huang et al., 1996).

It is difficult to rationalize the lack of concordance between

the crystal structure and the solution NMR data with respect to

the role of the C-terminal tail region of CD95. We note that the

crystallization buffer pH (reported as pH 4) is significantly

different from physiological conditions. Curiously, in our hands,

we find that lowering the pH of an NMR sample of the FADD-

DD:CD95-DD+CT complex leads to dissociation (Figures 8C

and 8D). Moreover, the crystal structure indicates that the

CD95 DD has the potential to dimerize via the conformational

transition from a closed to an open form. We have attempted

to find evidence that the CD95-DD+CT protein has a low popu-

lation of a significantly different conformation to the closed form

in solution by recording 15N relaxation dispersion experiments

(Palmer, 2004) at both pH 7 and pH 4, but we found no evidence

for chemical exchange (data not shown). In addition, it seems

unlikely that the structure of CD95-DD+CT is opened in our

NMR experiments on the complex, because we do not detect

ordering of the CT-region corresponding to the C-helix, and

the chemical shift changes of the ILV-13CH3-group cross-peaks

appear rather small (Figure 7).

If the remarkable remodeling of the CD95-DD structure in

the 3EZQ crystal structure properly describes the platform for

DISC formation then it would be reasonable to expect that the

structural features of the open conformation be conserved

across closely homologous receptors. The sequence align-

ment of putative CD95 orthologs (Figure 9) suggests that the

C-terminal tail region of CD95 is not conserved to the extent

that one could confidently predict that the formation of the

stem-C-helix hairpin is a common feature of CD95 signaling.

Some species appear to lack the C-terminal 15 residues that

would constitute the C-helix, and others have insertions, dele-

tions or substitutions compared to human CD95 that are not

straightforwardly incorporated in a model of intra- and intermo-

lecular interactions indicated by the crystal structure (Figure 1;

Figure S1).

In addition to the sequence-structure correlates relating to

interactions between CD95 chains in the crystal structure, there

are additional aspects relevant to our own observations con-

cerning the interaction betweenCD95-DD and FADD-DD, partic-

ularly relating to the effects of mutations in the FADD-DD chain.

For example, in line with a similar report (Hill et al., 2004), we

found that mutation of FADD-DD R117E abrogates binding to

wild-type GST-CD95-DD in a pull-down assay (Figure 2). More-

over when 15N-labeled FADD-DD(R117E) (Figure S2) was titrated

with wild-type Gb1-tagged CD95-DD (Ferguson et al., 2007) the

interaction was strongly attenuated compared to wild-type

FADD-DD (data not shown). Other researchers have also found

that mutation of R117 in both human and mouse FADD has

significant impact on the CD95 interaction (Bang et al., 2000;

Hill et al., 2004; Hughes et al., 2009; Imtiyaz et al., 2005; Jeong

et al., 1999). In the 3EZQ structure the R117 side chain is

directed away from helix a2 on the surface of FADD-DD that is

almost diametrically opposed to the intermolecular interface

with the open CD95-DD (Figure 1). It was also reported that


Structure


mutation of FADD-DD residues R113E, D123R, K125A, and

R146A significantly impacted the ability of FADD to bind CD95-

DD in a GST-pull-down assay (Hill et al., 2004), although none

of these FADD-DD residues is close to the FADD-DD:CD95-DD

interface in the crystal structure (Figure 1). We have previously

reported that CD95(D260A), corresponding to a site of mutation

in type Ia ALPS (Martin et al., 1999; Vaishnaw et al., 1999), fails to

interact with FADD-DD as assessed by NMR (Ferguson et al.,

2007) consistent with earlier reports for the behavior of this

mutant (Huang et al., 1996). We obtained a similar result with

CD95-DD(A257D) that represents another well-characterized

ALPS mutant that fails to bind FADD-DD (Martin et al., 1999)

(data not shown). In the 3EZQ structure A257 and D260 are

directed away from the surface of the CD95-DD a3 helix and

a2-a3 linker respectively, well outside the FADD-DD contact

zone.

Concluding RemarksCollectively, the combination of our SEC-MALS, ESI-MS, and

NMR investigations of the complex formed between FADD-DD

and CD95-DD proteins coupled with the evidence from muta-

genesis studies, particularly for the impact of mutations in

FADD-DD, lack consistency with the picture of the interaction

between these two proteins that has been provided so far

by X-ray crystallography. Critically, our data show that the

C-terminal tail region of CD95 is dispensable for complex forma-

tion between the two DDs in solution under near physiological

conditions. The interpretation of the SEC-MALS data in terms

of a cooperatively formed 5:4 complex is compatible with the13CH3-TROSY evidence for slow exchange and nonequivalent

chemical shifts. The indication from the mass spectrometry

data of a possible 5:5 composition is reminiscent of the PIDDo-

some core complex that has, in part, the juxtaposition of a ring

of five PIDD-DDs against a ring of five RAIDD-DDs where each

of the chains occupies a unique site (Park et al., 2007b). Such

asymmetry for the FADD-DD:CD95-DD complex would be con-

sistent with the appearance of cross-peak clusters in the 13CH3-

TROSY spectra. From the biological perspective, it is trans-

parent that more work is required to properly understand at

a structural level the earliest events in the mechanism of DISC

formation and to resolve the lack of concordance engendered

by the current analysis with respect to the available high resolu-

tion data.

EXPERIMENTAL PROCEDURES

Plasmid Construction and Mutagenesis

We use the human CD95 numbering 1-335 corresponding to the intact

polypeptide including the membrane targeting signal sequence, consistent

with that used for the crystal structure 3EZQ (Scott et al., 2009). DNA encoding

CD95 residues 218–323 (CD95-DD) and 218–335 (CD95-DD+CT) was sepa-

rately inserted into a modified pET-32b vector that encodes an N-terminal

His6-affinity tag residues followed by a tobacco etch virus (TEV) protease-

cleavable streptococcal protein G immunoglobulin-binding B1 (Gb1) domain

(Zhou et al., 2001). After TEV-proteolysis the purified CD95 polypeptides

contain the nonnative N-terminal dipeptide Gly-Ser. N-terminal glutathione

S-transferase (GST)-fused CD95-DD and CD95-intracellular domain (IC;

residues 194–335), were obtained by subcloning the corresponding CD95

PCR products into a pGEX-2T bacterial expression vector. Point mutant

CD95 and FADD constructs were created using QuikChange site-directed

mutagenesis.


Protein Expression and Purification

Expression and purification of FADD-DD (residues 93–192) was as described

(Berglund et al., 2000). CD95-DD and CD95-DD+CT proteins were obtained

by expression in E. coli Rosetta (DE3) cells at 22�C. The protein in the super-

natant was recovered by Ni-IMAC, TEV cleavage, and semipreparative SEC

with final elution into 20 mM MES pH 6.2, 100 mM NaCl, 1 mM EDTA, 3 mM

NaN3, and 0.5 mM TCEP (Buffer A).

Uniform 15N-isotope, 13C- and 2H-labeling were achieved using stan-

dard methods in M9 media. Uniform 2H-, Ile-d1[13CH3], Leu-d, Val-g

[13CH3,12CD3]-labeling of CD95 and FADD proteins for ILV 13CH3-TROSY

NMR was achieved in 99% D2O M9 medium containing [2H,12C]-glucose;

1 hr before induction, the medium was supplemented with 60 mg/l [meth-

yl-13CH3]-2H a-ketobutyric acid and 100 mg/l [dimethyl-13CH3,

12CD3]-2H

a-ketoisovaleric acid (Goto et al., 1999). Proteins were exchanged into Buffer

A in D2O before the NMR measurement.

GST Pull-Down

GST-tagged CD95-DD and CD95-IC constructs were expressed in Rosetta

(DE3) cells at 37�C. The cell lysate supernatant was incubated with gluta-

thione-Sepharose and the suspension washed twice with 50 mM HEPES,

pH 7.4, 50mMNaCl, 5mMEDTA, 10%glycerol, 0.1%Nonidet P-40. The slurry

was then mixed with FADD-DD and binding allowed to proceed for 2 hr at 4�C.After washing twicewith binding buffer, 10 ml resin was loaded on a SDS-PAGE

gel, which was run and stained with Coomassie Blue.

SEC-MALS

Analytical SEC-coupled MALS profiles were recorded at 16 angles using

aDAWN-HELEOS laser photometer (Wyatt Technology) and differential refrac-

tometer (Optilab rEX) equipped with a Peltier temperature-regulated flow

cell maintained at 25�C (Wyatt Technology). The protein concentration c of

the eluent was determined from the refractive index RI(n) change (dn/dc =

0.186); wavelength 658 nm. Samples (100 ml, flow rate 0.5 ml/min) were

applied to a Superdex 200 10/300 GL column (GE Healthcare) equilibrated

in Buffer A. Before injection on the column CD95:FADD complexes were iso-

lated by semipreparative S200 gel filtration and concentrated to 20 mg/ml in

Buffer A. The data were analyzed using ASTRA 5.1 as described (Ivins et al.,

2009).

Mass Spectrometry

NanoESI-MS experiments were carried out in the positive ionmode on aQStar

mass spectrometer (MDS Analytical Technologies) modified for optimized

performance at high masses (Chernushevich and Thomson, 2004; Sobott

et al., 2002) using nanoflow capillaries. The following experimental parameters

were used: ion spray voltage, 1.4 kV; declustering potential, 100 V; focusing

potential, 150 V; and collision energy, 20–40 V, MCP 2350 V. Data analysis

was done with Massign, a software package developed in house on LabVIEW

basis (Hernandez et al., 2009; Natan et al., 2009). Measurements were taken

for intact protein complexes prepared as for SEC-MALS analysis and

exchanged into 50mMammonium acetate using Biospin 6microspin columns

(Bio-Rad Laboratories). The results shown are representative of multiple inde-

pendent experiments.

NMR Spectroscopy

All spectra were acquired at 25�C on Varian INOVA or Bruker AVANCE spec-

trometers operating at 14.1 T, 16.5 T, and 18.8 T. Data were processed with

NMRPipe/NMRDraw (Delaglio et al., 1995) and analyzed with CCPN Analysis

(Vranken et al., 2005). Resonance assignments for CD95 protein constructs

were obtained using standard procedures. Titrations of a labeled protein

(0.5 mM) with its unlabeled partner (0–2.5 molar equivalents) were conducted

at constant concentration of the labeled component (McAlister et al., 1996).

Typical acquisition times for 2D WATERGATE-flipback 15N,1H-HSQC (Grze-

siek and Bax, 1993) data sets were �1 hr. Overall acquisition times for 13CH3-

TROSY (Tugarinov et al., 2003) experiments (18.8 T) were�5 hr, with relaxation

delay 1.3 s. To examine the effect of low pH on the 15N-CD95-DD+DT:FADD-

DD complex, the sample was exchanged into 20 mM sodium citrate, 1.0 mM

TCEP, pH 4.0.

All rights reserved

Structure


SUPPLEMENTAL INFORMATION

Supplemental Information includes four figures and can be found with this

article online at doi:10.1016/j.str.2010.08.006.

ACKNOWLEDGMENTS

The work in the P.C.D. group is supported by the MRC (file reference

U117574559). We are grateful to the staff of the MRC Biomedical NMR Centre

and to Acely Garza-Garcia for helpful discussions. N.M. and C.V.R. are sup-

ported by the European Union within the 7th Framework Program PROS-

PECTS.

Received: May 21, 2010

Revised: July 20, 2010

Accepted: August 13, 2010

Published: October 12, 2010

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Solution NMR Investigation of the CD95/FADD Homotypic Death Domain Complex Suggests Lack of Engagement of the CD95 C Terminus