The RIP1/RIP3 Necrosome Forms a
Functional Amyloid Signaling Complex
Required for Programmed Necrosis
Jixi Li,1,6Thomas McQuade,2Ansgar B. Siemer,3,7Johanna Napetschnig,1Kenta Moriwaki,2Yu-Shan Hsiao,4
Ermelinda Damko,1David Moquin,2Thomas Walz,4,5Ann McDermott,3Francis Ka-Ming Chan,2and Hao Wu1,6,*
1Department of Biochemistry, Weill Cornell Medical College, New York, NY 10065, USA
2Department of Pathology, Program in Immunology and Virology, The University of Massachusetts Medical School, Worcester,
MA 01655, USA
3Department of Chemistry, Columbia University, 3000 Broadway, New York, NY 10027, USA
4Department of Cell Biology
5Howard Hughes Medical Institute
Harvard Medical School, Boston, MA 02115, USA
6Present address: Program in Cellular and Molecular Medicine, Immune Disease Institute, Children’s Hospital Boston and Department
of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA
7Present address: Department of Biochemistry and Molecular Biology, Zilkha Neurogenetic Institute, Keck School of Medicine of the
University of Southern California, Los Angeles, CA 90033, USA
RIP1 and RIP3 kinases are central players in TNF-
induced programmed necrosis. Here, we report
that the RIP homotypic interaction motifs (RHIMs)
of RIP1 and RIP3 mediate the assembly of heterodi-
meric filamentous structures. The fibrils exhibit clas-
sical characteristics of b-amyloids, as shown by
Thioflavin T (ThT) and Congo red (CR) binding,
circular dichroism, infrared spectroscopy, X-ray
diffraction, and solid-state NMR. Structured amyloid
cores are mapped in RIP1 and RIP3 that are
flanked by regions of mobility. The endogenous
RIP1/RIP3 complex isolated from necrotic cells
binds ThT, is ultrastable, and has a fibrillar core
structure, whereas necrosis is partially inhibited by
ThT, CR, and another amyloid dye, HBX. Mutations
in the RHIMs of RIP1 and RIP3 that are defective in
the interaction compromise cluster formation, kinase
activation, and programmed necrosis in vivo. The
current study provides insight into the structural
changes that occur when RIP kinases are triggered
the realm of amyloids to complex formation and
Recent studies have implicated the intracellular signaling kinase
RIP1 as a key switch of cell fate regulation. Depending on the
cellular context, RIP1 controls whether the pleiotropic cytokine
TNF induces NF-kB activation, apoptosis, or programmed
necrosis (Moquin and Chan, 2010). The E3 ligases cIAP1/2 and
LUBAC ubiquitinate RIP1in the TNFR1 signaling complex (Walc-
zak, 2011). Polyubiquitinated RIP1 then engages downstream
adaptors such as NEMO to activate IKK to promote NF-kB tran-
scriptional activity, leading to cell survival, proliferation, and
differentiation (Walczak, 2011). When RIP1 ubiquitination is
blocked by removal of the E3 ligases cIAP1 and cIAP2 through
genetic ablation, RNA interference (RNAi) knockdown, or inhib-
itor of apoptosis (IAP) antagonists, RIP1 forms a secondary
complex in the cytosol with Fas-associated death domain
(FADD) and caspase-8—termed the Ripoptosome—to initiate
apoptotic cell death (Feoktistova et al., 2011; Tenev et al.,
2011; Wang et al., 2008). Active caspase-8 within the Ripopto-
some cleaves and inactivates RIP1 (Chan et al., 2003; Lin
et al., 1999) and RIP3 (Feng et al., 2007). When caspases are in-
hibited bypharmacological inhibitors or under certain physiolog-
ical conditions such as viral infections, RIP1 and RIP3 form the
necrosome to initiate a third pathway known as programmed
necrosis or necroptosis (Cho et al., 2009; He et al., 2009; Zhang
et al., 2009).
The understanding of programmed necrosis is still unfolding.
Whereas it was originally thought to be associated with nonspe-
cific cellular damages, genetic experiments in mice clearly show
that caspase-8-mediated cleavage and inactivation of RIP1 and
RIP3 is critical for preventing extensive necrosis during embry-
onic development in order to ensure proper clonal expansion
of lymphocytes and to preventextensive necrosisand inflamma-
tion in skin and intestinal epithelium (Kaiser et al., 2011; Oberst
et al., 2011; Welz et al., 2011; Zhang et al., 2011). In addition to
caspase inhibition, assembly of the RIP1/RIP3 necrosome also
requires intact RIP1 and RIP3 kinase activity (Cho et al., 2009).
Recent studies identified MLKL, a kinase-like protein, as
a substrate of the RIP3 kinase (Sun et al., 2012; Zhao et al.,
Cell 150, 339–350, July 20, 2012 ª2012 Elsevier Inc. 339
within the necrosome is poorly understood. Both RIP1 and RIP3
also has a death domain (DD) at its C terminus for recruitment to
the TNF receptor signaling complex (Stanger et al., 1995; Sun
(Feoktistova et al., 2011; Tenev et al., 2011; Wang et al., 2008)
(Figure 1A). Unique segments of homologous sequences in
RIP1 and RIP3 (RIP homotypic interaction motifs, RHIMs)
(Figures 1A and 1B) were shown to mediate their interaction
necrosis (Cho et al., 2009). The RHIM is found in a growing
number of signaling adaptors with crucial functions in cell death
and innate immunity (Moquin and Chan, 2010). For instance,
macrophage necrosis induced through TLR-3/4 requires RHIM-
mediated interaction between the adaptor TRIF and RIP3 (He
et al., 2011). Similarly, RHIM-mediated interaction between the
fected with murine cytomegalovirus (Upton et al., 2012).
Here, we show that RIP1 and RIP3 form an amyloid structure
through their RHIMs and that this heterodimeric amyloid struc-
necrosis. Our results not only provide insights into the mecha-
nism of RIP1 and RIP3 kinase activation but also further expand
the realm of amyloid structures to normal physiological functions
beyond those associated with human diseases (Eisenberg and
Complex In Vitro and in Cells
(A) Domain organization of human RIP1 and RIP3.
(B) Sequence alignment of RIP1 and RIP3 around
the core RHIMs. Residues in RIP1 and RIP3 that
are conserved across different species (Figure S1)
are highlighted in red. Regions predicted to be in
b sheet conformations are shown. Residues as-
signed by solid-state NMR are marked with ‘‘x.’’
Summary of mutagenesis results are displayed,
with red indicating most defective mutants show-
ing complete or partial dissociation between RIP1
and RIP3, magenta indicating defective mutants
showing smaller complexes, orange indicating
between that of WT and defective mutants, and
green indicating nondefective mutants.
(C) Coexpressed full-length and truncated RIP1/
RIP3 complexes. Left, superimposed gel filtration
profiles. Right, SDS-PAGE of the fractions.
(D) EM images of the RIP1/RIP3 complex.
(E) Representative class averages of the RIP1/
See also Figure S1.
The RIP1/RIP3 Complex Forms
Filamentous Structures In Vitro
and in Cells
The exact boundaries of RHIMs are
unclear, but the sequence conservation
available online). We coexpressed RHIMs of RIP1 (residues
496–583) and His-tagged RIP3 (residues 388–518) (RIP1/
3-RHIM) using regions that were previously shown to be suffi-
cient for complex formation (Sun et al., 2002). The RIP1/
3-RHIM complex copurified from Ni-affinity chromatography
eluted around the void position of a Superdex 200 10/300 GL gel
filtration column, which is much larger than the expected molec-
ular mass of a heterodimer (Sunet al., 2002) (Figure 1C). We then
coexpressed full-length RIP1 and RIP3 (RIP1/3-FL) in insect
cells, which similarly coeluted around the void position of the
gel filtration column (Figure 1C).
We investigated why RIP1/3-RHIM and RIP1/3-FL could form
such large complexes. Secondary structure predictions sug-
gested that the region between the KD and the DD in RIP1
(?residues 300–560) and the region C-terminal to the KD in
RIP3 (?residues 300-end) are mostly unstructured random coils
(Rost et al., 2004). The only exceptions are short segments of
sequences around the I(V)QI(V)G motif, which show propensities
for b strands (Figure 1B). Because amyloids are fibrous protein
aggregates composed of cross-b cores, we asked whether
RHIMs mediate assembly of amyloid-like fibrils.
We used electron microscopy (EM) to visualize the structures
of the RIP1/RIP3 complexes. Consistent with our hypothesis,
EM of negatively stained RIP1/3-RHIM revealed filamentous
structures (Figure 1D). The fibrils exhibit a similar width of
?11–12 nm but vary in length. Class averages show substantial
340 Cell 150, 339–350, July 20, 2012 ª2012 Elsevier Inc.
structural variability, but closer inspection suggests that the core
of the complex, ?8 nm, may be ordered and that the variability
may be mostly due to flexible extensions (Figure 1E). EM of
negatively stained RIP1/3-FL showed mostly aggregates. We
reasoned that the KDs and DD in full-length RIP1 and RIP3 might
mediate additional interactions and mask the central amyloidal
fibril architecture. Upon limited proteolysis to remove the flank-
ing domains by subtilisin, the same enzyme used in the nuclear
magnetic resonance (NMR) sample preparation (see section
were revealed for RIP1/3-FL, similar to RIP1/3-RHIM (Fig-
immunoprecipitated with anti-RIP1 antibody was treated with
subtilisin and negatively stained, it showed mostly short and
sometimes long filamentous structures under EM only upon
necrosis induction with TNF, zVAD-fmk, and the IAP antagonist
LBW242, but not before induction (Figures 1D and S1B).
The RIP1/RIP3 Complex Exhibits Classical
Characteristics of Amyloid Fibrils
Amyloids are classically characterized using aromatic, cross-
b binding dyes such as Thioflavin T (ThT) (LeVine, 1999) and
Congo red (CR) (Klunk et al., 1989). To determine whether the
RIP1/RIP3 fibrils are indeed amyloidal, we first characterized
purified recombinant RIP1/RIP3 complexes in vitro. We added
ThT to either RIP1/3-RHIM or RIP1/3-FL and measured its fluo-
rescence after excitation at 430 nm. In comparison to ThT alone,
RIP1/3-RHIM and RIP1/3-FL caused ThT to display an emission
(A) Fluorescence emission spectra of ThT in the
absence (green) and presence (magenta) of the
(B) Both the RIP1/RIP3-RHIM and the full-length
RIP1/RIP3 complex bind ThT.
(C) Absorption spectra of CR in the absence
(green) and presence of either RIP1/RIP3-RHIM
(magenta) or full-length RIP1/RIP3 complex (blue).
(D) Circular dichroism spectrum of the RIP1/RIP3-
(E) Superimposed Fourier transform infrared
spectra of RIP/RIP3-RHIM (magenta) and the
I539D mutant of RIP1 (cyan). Only the WT RIP1/
RIP3 complexes, not the RHIM mutant, showed
the amide I0maxima at 1,623 cm?1(dashed
vertical red line), which is characteristic of
(F) An X-ray diffraction image of partially aligned
RIP1/RIP3 fibrils. The arrows indicate equatorial
and meridional reflections at 9.4 A˚ and 4.7 A˚
See also Figure S2.
2. TheRIP1/RIP3Complex Is
peak at ?485 nm with concomitant in-
crease in fluorescence intensity (Figures
2A, 2B, and S2A). Similarly, CR showed
a characteristic red shift from an absorp-
tion maximum of ?470 nm to ?540 nm
upon addition of either RIP1/3-RHIM or
RIP1/3-FL (Figures 2C and S2B) (Klunk et al., 1989). Therefore,
both the ThT- and the CR-binding assays confirmed that the
RIP1/RIP3 complex is amyloidal.
We then used circular dichroism (CD) to estimate the
secondary structure content of RIP1/3-RHIM, which revealed
a prominent negative peak at ?210 nm (Figure 2D). Analysis of
the spectrum with DICHROWEB (Whitmore and Wallace, 2004)
indicated that the sample is a mixture of b sheet and random
coil structures. Fourier transform infrared spectroscopy (FTIR)
has been well established to distinguish cross-b amyloid fibrils
from native b sheet proteins in that the former shows amide I0
maxima between 1,610 cm?1and 1,630 cm?1in wavenumbers,
which is smaller than native b sheet proteins (Zandomeneghi
et al., 2004). This difference has been attributed to smaller
b sheet twist angles and larger numbers of b strands in amyloidal
aggregates (Zandomeneghi et al., 2004). Indeed, RIP1/3-RHIM
showed a prominent amide I0
1,623 cm?1(Figure 2E), which is consistent with the amyloidal
nature of the complex. The RIP1-RHIM mutant I539D, which is
no longer amyloidal (see section ‘‘Mutations of RIP1 and RIP3
Weaken Filament Formation’’), did not exhibit this characteristic
Amyloids are characterized by cross-b quaternary structures
in which the b sheets are parallel to the fiber axis, and
the extended b strands lie approximately perpendicular to the
axis. This arrangement produces characteristic diffraction
patterns (Sunde et al., 1997). We partially aligned the RIP1/
3-RHIM complex and obtained its diffraction pattern by using
absorbance maximum at
Cell 150, 339–350, July 20, 2012 ª2012 Elsevier Inc. 341
Cu radiation (Figure 2F). Orthogonal diffractions at 4.7 A˚and
9.4 A˚resolutions were observed, corresponding to the inter-b
strand spacing along the meridional axis (parallel to the fibril
axis) and the inter-b sheet stacking distance along the equatorial
direction (perpendicular to the fibril axis), respectively. These
data established unequivocally the cross-b amyloid core of the
The Amyloid Core of the RIP1/RIP3 Complex
Secondary structure prediction suggests that the amyloid core
sequences of RIP1 and RIP3 are much shorter than either the
full-length or the truncated coexpression construct we used for
obtaining the RIP1/RIP3 complex. To further map this interac-
tion, we generated a series of coexpression constructs tagging
either RIP1 or RIP3 with a His-MBP tag. His-tag pull-down
experiments of the coexpression constructs showed that the
interaction was retained, even when RIP1 was only 31 residues
(525–555) and RIP3 was only 19 residues (446–464) in length
(Figures 3A, S3A, and S3B).
To further elucidate the core size and the secondary structure
of the RIP1/RIP3 complex, we used solid-state NMR. Because
sample conditions may influence amyloid structures, we col-
different coexpressed constructs, the RIP1 (residues 496–583)/
RIP3 (residues 388–518) complex, its subtilisin-digested coun-
terpart, and the RIP1 (residues 496–583)/RIP3 (residues 446–
518) complex. The overlay of these spectra showed very strong
correspondence (Figure 3B), indicating that the size and the
structure of the RIP1/RIP3 amyloid core are robust with respect
to construct lengths and details of the preparation.
To obtain sequence-specific secondary structure information,
enhanced by amplitude modulation (DREAM) and dipolar-assis-
ted rotational resonance (DARR) spectra;15N-13C NCO, NCA,
NcaCB, and NcoCX 2D spectra; and NCACX and NCACB 3D
spectra on the subtilisin-digested complex at ?10?C (Figures
S3C, S3D). Using the mapped amyloid core sequences (Figures
1B and 3A) as targets, the majority of the peaks in the NCA spec-
trum were assigned, with 16 residues of RIP1 (T532–Y544 and
Y546–E548) and 6 consecutive residues of RIP3 (G457–D462)
(Figures 3C and 3D). Some of the residues were assigned with
high confidence due to the nondegenerate15N and13C peaks
13C-13C dipolar recoupling
Figure 3. The Amyloid Core of the RIP1/RIP3 Complex
(A) Mapping the interaction between RIP1 and RIP3 by using coexpression and His-tag pull-down. The shortest constructs that retained interaction are circled
(B) Overlay of 2D DARR13C-13C solid-state NMR spectra of the RIP1 (residues 496–583)/RIP3 (residues 388–518) complex (blue), its subtilisin-digested
counterpart (magenta), and the RIP1 (residues 496–583)/RIP3 (residues 446–518) complex (red).
(C) 2D15N-13C NCA solid-state NMR spectrum of subtilisin-digested RIP1/RIP3 complex. Site-specific assignments are indicated for RIP1 (black) and RIP3 (red).
(D) Plot of the difference in secondary chemical shift between Ca and Cb, indicative of secondary structures (only DdCa for Gly).
(E)13C 1D NMR spectra of the RIP1 (residues 496–583)/RIP3 (residues 388–518) complex. At above 0?C (top), the line width is generally narrower, and the INEPT
pulse sequence, which is sensitive to relatively dynamic domains of the sample, gives an intense spectrum. At below 0?C (bottom), most of the dynamics are
arrested, leading to no signal in the INEPT and an intense and broad cross-polarization (CP) spectrum, which is sensitive to the static domains of the sample.
See also Figure S3.
342 Cell 150, 339–350, July 20, 2012 ª2012 Elsevier Inc.
in the three-dimensional (3D) spectra with additional support
from Ca-Ca-1 cross-peaks in the two-dimensional (2D) DARR
spectrum. All assignments are consistent with the RIP1/RIP3
core sequences. Negative and positive values of the difference
in secondary chemical shifts between Caand Cb(i.e., DdCa-
mations, respectively. Most of the residues in the amyloid core
are compatible with b sheet conformations, and the 4 central
RHIM residues show consecutive negative shift differences
(Figures 3D and S3E).
The spectra recorded with cross-polarization (CP)-based
solid-state NMR above 0?C can be explained by ?37 resolved
residues. Most of the residues outside the b-amyloid core were
unobserved and either too dynamic or disordered to be visible
in these spectra. Fast molecular dynamics can lead to partial
or complete averaging of dipolar spin couplings that are essen-
tial for CP to work. When1H-13C insensitive nuclei enhanced by
polarization transfer (INEPT)—a sequence that only works when
dipolar couplings are absent—was utilized, we observed intense
and narrow spectra (Figure 3E). This indicated that there are very
dynamic domains outside the amyloid core. This observation is
of EM images (Figure 1E). At lower temperatures, these
dynamics slowed down so that more domains in the sample
could be detected with CP, but not with INEPT (Figure 3E).
Cross-Polymerization of the RIP1/RIP3 Complex
The sequenceconservation atthe core of the RHIMs of RIP1and
RIP3 prompted us to ask whether RIP1 or RIP3 alone could also
form amyloid fibrils. Expression of His-Sumo-RIP1 (residues
496–583) or His-Sumo-RIP3 (residues 388–518) alone, followed
by cleavage of the respective His-Sumo tag, showed that RIP1-
RHIM or RIP3-RHIM eluted around the void position from a gel
filtration column (Figure 4A). The RIP1 and RIP3 CD spectra
showed similar secondary structures as the RIP1/RIP3 complex,
and ThT fluorescence and CR absorption confirmed their
amyloidal structures (Figure S4). EM of negatively stained RIP1
and RIP3 samples revealed that the homocomplexes are able
to form fibrils by themselves as well (Figure 4B). However, these
fibrils appeared to be less regular and shorter than those of the
RIP1/RIP3 heterocomplex (Figure 1D).
Because RIP1 and RIP3 can each form fibrils on their own, we
wondered whether the observed fibrils are similar to that of the
RIP1/RIP3 complex. To address this question, we performed
cross-fibril polymerization experiments to see whether the
amyloids of RIP1, RIP3, or the RIP1/RIP3 complex can enhance
the polymerization of each other. Because the purified recombi-
nant proteins are already fibrillar, we needed to first denature the
proteins. Consistent with the recognized high stability of amyloid
structures (Balbirnie et al., 2001), we had to use harsh condi-
tions: 8 M urea for RIP1 and 150 mM NaOH for the RIP1/RIP3
complex. When RIP1 was first denatured and then diluted
100-fold into the native ThT binding buffer to allow refolding, it
showed minimal enhancement of ThT fluorescence as a function
of time. When the same denatured RIP1 was subsequently
diluted to allow renaturation in the presence of 10% native
RIP1, RIP3, or the RIP1/RIP3 complex as polymerization seeds,
ThT fluorescence increased much more quickly (Figure 4C).
Figure 4. Cross-Polymerization and Mutagenesis of the RIP1/RIP3 Interaction
(A) Left, superimposed gel filtration profiles of the RHIM fragments of RIP1 (residues 496–583), RIP3 (residues 388–518), and the RIP1/RIP3 complex. Right,
SDS-PAGE of gel filtration fractions of RIP1 and RIP3.
(B) EM images of RIP1 and RIP3.
(C and D) Cross-seeding in the polymerization of denatured RIP1 and the denatured RIP1/RIP3 complex by using ThT binding assays, respectively.
See also Figure S4.
Cell 150, 339–350, July 20, 2012 ª2012 Elsevier Inc. 343
Similarly, denatured RIP1/RIP3 complex can be induced to poly-
merize much more efficiently in the presence of 10% native
seeds of RIP1, RIP3, or the RIP1/RIP3 complex (Figure 4D).
These experiments suggest that the fibrils of RIP1 and RIP3,
as well as the RIP1/RIP3 complex, share a similar structural
Mutations of RIP1 and RIP3 Weaken Filament Formation
To elucidate the key determinants in RIP1/RIP3 interaction, we
generated point mutations on residues spanning the core RHIMs
of RIP1 and RIP3. Residues 533–548 of RIP1 and residues
451–466 of RIP3 were mutated to Asp for possible maximal
disruptive effects, except that D461 of RIP3 was mutated to
Lys (Figure 1B). The RHIM consensus sequences were also
mutated to Pro (Figure S5). For RIP1 mutants, we used a coex-
pression construct of His-RIP1 (residues 496–583) and RIP3
(residues 388–518). For RIP3 mutants, a coexpression construct
containing an untagged wild-type (WT) RIP1 and a His-tagged
RIP3 was used.
All mutant His-RIP1 proteins could pull down coexpressed WT
RIP3 with Ni-affinity beads. However, when the copurified
samples were subjected to gel filtration chromatography, clear
differences appeared (Figures 1B and 5A). Whereas RIP1
mutants flanking the core RHIM did not show any defects in
Figure 5. Mutagenesis of RIP1 and RIP3
(A) Superimposed gel filtration profiles of complexes of mutant RIP1 and WT RIP3. Left, RIP1 mutants that dissociated from RIP3;right, RIP1 mutants that did not
dissociate from RIP3 and migrated near the void position.
(B) Superimposed gel filtration profiles of complexes of mutant RIP3 and WT RIP1. Left, complexes of mutant RIP3 and WT RIP1 that migrated later than the void
position; right, complexes of mutant RIP3 and WT RIP1 that migrated near the void position.
(C) ThT fluorescence of WT and mutant full-length RIP1 and RIP3.
(D) EM images of negatively stained WT and mutant full-length RIP1 and RIP3.
See also Figure S5.
344 Cell 150, 339–350, July 20, 2012 ª2012 Elsevier Inc.
RIP3 interaction or fibril formation, RIP1 mutants near the center
of the RHIM (I539D, I539P, Q540D, Q540P, I541D, I541P,
G542D, G542P, A543D, Y544D, and N545D) were the most
ciated from RIP3, as observed by the appearance of an addi-
tional RIP1 mutant peak at ?17 ml.
Similarly, all His-RIP3 point mutants could pull down coex-
pressed WT RIP1 with Ni-affinity beads. When these complexes
were subjected to gel filtration chromatography, a range of
effects occurred, including dissociation on gel filtration chroma-
tography and a change in elution positions (Figures 1B, 5B, S5C,
and S5D). Asp mutants on central RHIM residues of RIP3
‘‘457-GVQVGD-462’’ eluted at around 12–14 ml in comparison
with the void position of around 8 ml for the WT RIP1/RIP3
complex. S456D was also partially defective, as it eluted around
11 ml. The changes in the elution positions suggest that the
mutant complexes have lower apparent molecular masses
than the WT complex and exhibit weakened interaction between
and V460E) induced dissociation from WT RIP1 on gel filtration
chromatography (Figure S5C). Pro mutants on central RHIM
residues of RIP3 ‘‘458-VQVG-461’’ resulted in partial dissocia-
tion with coexpressed WT RIP1, and V460P and G461P were
the most defective. In contrast, mutations on more peripheral
residues ‘‘451-NIYNC-455’’ and ‘‘463-NNYL-466’’ migrated in
a manner similar to the WT complex.
Similar to the results for the RHIM constructs of RIP1 and
RIP3, mutations on full-length RIP1 and RIP3 also weakened or
disrupted filament formation. ThT staining of insect cell-
expressed recombinant RIP1-FL mutant I539D and RIP3-FL
mutant V460P showed that both proteins exhibited close to
background levels of ThT fluorescence in comparison to the
WT proteins (Figure 5C). Upon subtilisin digestion, EM of WT
RIP1-FL and RIP3-FL showed filamentous structures, though
they were apparently less regular than the RIP1/RIP3 complex
(Figures 5D and 1D). In contrast, the RIP1-FL mutant I539D
and the RIP3-FL mutant V460P were mostly degraded by subtil-
isin and showed only residual aggregates (Figure 5D).
Amyloidal Nature and Ultrastability of the Endogenous
To determine whether the fibrils of endogenous RIP1/RIP3
complex isolated from necrotic cells that we observed under
EM(Figure1D)areamyloidal innature, wefirstused ThTbinding.
Because the amount of endogenous complexes isolated from
cells was much lower than that obtained from recombinant
proteins, we measured ThT fluorescence by using a more sensi-
tive instrument. HT-29 cells stimulated with TNF, zVAD-fmk,
and LBW242 underwent RIP1/RIP3-dependent programmed
necrosis (He et al., 2009). The RIP1-containing complex isolated
from necrotic cells, but not from control cells, exhibited in-
Given that the recombinant RIP1/RIP3 complex is ultrastable
and requires 150 mM NaOH to be denatured, we tested the
stability of the endogenous complex isolated from necrotic cells.
We lysed cells with buffers containing 4 M urea, 8 M urea, or
150 mM NaOH. Thirty minutes after lysis, we diluted the lysates
10-fold with regular nondenaturing lysis buffer. Immunoprecipi-
tation with anti-RIP3 antibody showed that 4 M urea or 8 M
urea did not disrupt the interaction between RIP3 and RIP1,
whereas 150 mM NaOH did (Figure 6B). This result is consistent
with thestabilityof therecombinant complexandwith therecog-
nized stability of amyloidal structures in general (Balbirnie et al.,
2001). In contrast, the interaction between polyubiquitinated
RIP1 and TNFR1, which is crucial for TNF-induced NF-kB acti-
vation, was completely abolished in all conditions, including
4 M urea (Figure 6C).
Classical b-amyloid-binding dyes often inhibit amyloid oligo-
merization (Sa ´nchez et al., 2003). Pretreatment of HT-29 cells
with CR (Figure 6D), ThT, or another b-amyloid-binding com-
pound, 2-(2-hydroxyphenyl)-benzoxazole (HBX) (Alavez et al.,
2011), inhibited necrosis in a dose-dependent manner (Fig-
ure 6E). The inhibition by ThT and HBX is specific for necrosis
Clustering of RIP1 and RIP3 into punctate-like structures is
a distinct feature of necrosis (Figures S6B–S6D). This was
confirmed by immunogold EM (Figure 6F). When HeLa cells,
which do not express endogenous RIP3, were transfected with
RIP3-mCherry and stimulated with TNF, zVAD-fmk, and
LBW242 to induce necrosis, they showed complete overlap of
ThT staining with RIP3-mCherry puncta (Figure 6G). By contrast,
ThT signal was undetectable in untreated cells (Figure 6G).
Collectively, these results definitively show that RIP1 and RIP3
form amyloidal complexes during induction of programmed
RHIM Residues of RIP1 and RIP3 Are Crucial for Cluster
Formation, Kinase Activation, and Programmed
To determine the functional role of RHIMs in the assembly of the
amyloidal RIP1/RIP3 complex in vivo, we introduced selected
mutations within and flanking the RHIMs that were shown to
cause disruption of filament formation and mutual interaction
in vitro (Figure 1B). For RIP3, we stimulated HeLa cells trans-
fected with WT and mutant RIP3-yellow fluorescent protein
(YFP) with TNF, zVAD-fmk, and LBW242. As expected, WT
RIP3-transfected cells underwent cell death, whereas the
RHIM AAAA mutant was highly protected. Importantly, RIP3
mutants V458P, V460P, and G457D protected cells from TNF-
induced necrosis, whereas the mutant N464D that did not
show significant defects in vitro behaved most similar to the
WT (Figure 7A). In addition, mutants V458P and V460P
completely abolished puncta formation in response to necrosis
stimulation, whereas G457D partially disrupted RIP3 clustering
(Figure 7B). On the other hand, N464D still formed puncta (Fig-
ure 7B). Expression of RIP3-YFP in 293T cells showed that the
mutations V458P and V460P severely compromised RIP3 kinase
activation by using myelin basic protein (MBP) as the substrate
sion, we stably reconstituted RIP3?/?fibroblasts with WT and
mutant RIP3. In agreement with results in 293T cells, WT RIP3
showed robust kinase activity upon induction of necrosis,
whereas the V458P, V460P, and G457D mutants were dramati-
cally impaired in kinase activation (Figure 7C). Notably, there is
a close correlation between in vitro and in vivo experiments.
Cell 150, 339–350, July 20, 2012 ª2012 Elsevier Inc. 345
For instance, V460P is the most defective in fibril formation and
RIP1 interaction in vitro (Figure S5D; note the weakest RIP3
band in the fibril fraction) and is the most defective in cell death
induction and kinase activation. Similarly, G457D is a less defec-
tive mutant in cell death induction and is also less impaired in
kinase activation and cluster formation (Figures 7A–7C).
To assess RIP1 mutational phenotypes in vivo, we transfected
RIP1?/?fibroblasts with WT and mutant RIP1-green fluorescent
protein (GFP). Cell death upon necrosis induction was enumer-
ated in the GFP+population by using flow cytometry. Like the
RHIM AAAA mutant, the RIP1 mutants I539P, I541P, and
N545P were much weaker in mediating necrosis than WT RIP1
(Figure 7D), recapitulating the in vitro interaction data. Similar
to evaluation of the kinase activity of RIP3 mutants, we ex-
pressed RIP1-GFP constructs in 293T cells. After immunopre-
cipitation with anti-GFP antibody, the immune complexes were
subjected to in vitro kinase assay using MBP as the substrate.
tive kinase activation (Figure 7E). We attempted to stably recon-
stitute RIP1?/?fibroblasts with WT and mutant RIP1; however,
because RIP1 is generally expressed at much lower levels than
RIP3, likely due to toxicity, we could not evaluate the kinase
activities in these cells. Similar defects in clustering in mutations
within the core RHIM residues were observed with truncated
RIP1 (residues 496–583) and RIP3 (residues 388–518) lacking
the KDs and DD (Figure S7B). Thus, these results define a critical
role for RHIM-mediated amyloidal RIP1/RIP3 fibrils in the activa-
tion of RIP1/RIP3 kinase activity and the induction of pro-
Amyloids are fibrous protein aggregates composed of cross-
b structures and associated with many neurodegenerative (Chiti
and Dobson, 2006) and infective prion diseases (Uptain and
tions, such as host interaction, hazard protection, and memory
storage (Chiti and Dobson, 2006). However, this aspect of the
function of amyloids is less defined, especially in mammals.
Here, weshow thatRIP1 and RIP3forma functional, hetero-olig-
omeric amyloidal signaling complex that mediates programmed
necrosis. The discovery of cross-b amyloid structures in protein
complexation and signal transduction provides new insights into
both the amyloid field and the signaling field.
How is the assembly of the RIP1/RIP3 necrosome regulated?
It has been shown that both RIP1 and RIP3 kinase activities are
required for complex formation and cell death (Cho et al., 2009).
Consistent with a role for amyloid assembly in RIP1 and RIP3
predominantly in NP-40 insoluble fractions (Figures S6C and
S6D). Interestingly, abnormal phosphorylation of tau and a-syn-
uclein by multiple kinases is known to be involved in the
Figure 6. RIP1 and RIP3 Form Amyloidal Clusters In Vivo during Programmed Necrosis
(A) Endogenous RIP1-containing complexes from necrotic HT-29 cells bind ThT. The right panel shows the specific pull-down of RIP3 by RIP1 upon TNF, zVAD-
fmk, and LBW242 stimulation (T+Z+L).
(B) RIP3 complexes isolated by immunoprecipitation from HT-29 cells treated with T+Z+L after lysis in regular lysis buffer or buffer containing the indicated
amount of urea or NaOH.
(C) TNFR1 complexes isolated by immunoprecipitation from MEFs treated with TNF after lysis in regular lysis buffer or buffer containing the indicated amount of
urea or NaOH.
(D and E) Amyloid-binding compounds inhibit TNF-induced necrosis in HT-29 cells. Results shown are averages of triplicates ±SEM.
(F) Clustering of RIP1 and RIP3 in necrotic MEFs shown by immunogold EM. Scale bars, 200 nm (RIP1) and 100 nm (RIP3).
(G) Costaining of ThT with RIP3 puncta in necrotic HeLa cells as visualized by confocal microscopy.
See also Figure S6.
346 Cell 150, 339–350, July 20, 2012 ª2012 Elsevier Inc.
formation of neurofibrillary tangles in Alzheimer’s and Parkin-
son’s diseases, respectively, as well as in other neurodegenera-
tive tauopathies and synucleinopathies (Avila et al., 2010;
Oueslati et al., 2010). Our data are consistent with a feed-
forward, gain-of-function mechanism in which kinase activation
and RIP1/RIP3 necrosome formation are mutually reinforcing. In
long-range interactions in the unstructured flanking sequences
of RIP1 and RIP3 and possibly by RIP1 ubiquitination. Indeed,
expression of RIP1 and RIP3 RHIM fragments, but not full-length
RIP1 and RIP3, induced spontaneous clustering in cells without
stimulation (Figure S7B). In line with the observation of autoinhi-
bition, it was reported that large proteins have a low propensity
to form amyloid fibrils, although they possess a great tendency
to form b-sheet-rich aggregates that are weak binders of ThT
and CR (Ramshini et al., 2011). In contrast, the RIP1/RIP3
complex binds ThT and CR robustly, even though the folded
KDs and the DD in these proteins are primarily a-helical. Kinase
activation and the resultant hyperphosphorylation may be the
key events that reduce this autoinhibition, perhaps as a result
of charge repulsion to expose the RHIM core, leading to
enhanced complex formation. In turn, complex formation further
potentiates kinase activation through autophosphorylation and
cross-phosphorylation, propagating the pronecrotic signal.
Is it possible that the amyloid structures per se also have
toxicity to cells and contribute to cell death? It is well known
that amyloids are toxic, likely as a consequence of their mass
and induction of inflammation or disruption of membrane integ-
rity (Eisenberg and Jucker, 2012). However, given that specific
RIP3 kinase substrates have been identified and are crucial for
induction of programmed necrosis (Sun et al., 2012; Zhao
et al., 2012), kinase activation seems to be the key consequence
of the formation of the RIP1/RIP3 amyloid scaffold. Drawing
a parallel with the structural model of the designed amyloid of
ribonuclease A (Sambashivan et al., 2005), the KDs of RIP1
and RIP3 would extend from the central amyloid spine to the
periphery and find space to retain their globular structures and
Previous structural studies have revealed several arrange-
ments of cross-b amyloid spines in the formation of dry steric
zippers, including those from both parallel and antiparallel
b sheets (Eisenberg and Jucker, 2012; Sawaya et al., 2007).
The fiber diffraction pattern of RIP1/3-RHIM (Figure 2F) showed
that each b sheet in the fibrils consists of parallel b strands rather
than antiparallel b strands, as evidenced by the strong 4.7 A˚
spacing along the fibril axis (meridional direction). Structures of
related self-complementing steric zippers have also led to
insights regarding complementary molecular surfaces in RIP1
Figure 7. The RIP1/RIP3 Interaction Is Crucial for Kinase Activation, Clustering, and Cell Death
of RIP3 at the VQVG RHIM sequence. Results shown are averages of triplicates ±SEM.
(B) Effects of RIP3 mutations on puncta formation in HeLa cells transfected with the indicated RIP3-YFP plasmids as examined by confocal microscopy.
(C) Effects of RIP3 mutations on kinase activity of the anti-RIP3 immunoprecipitates in RIP3?/?fibroblasts stably expressing the indicated RIP3-GFP mutants
using MBP as the substrate. Bottom panel shows the RIP3 western blot of the same membrane.
(D) Effects of RIP1 mutations on TNF-induced necrosis in RIP1?/?fibroblasts transfected with the indicated RIP1 fused to GFP. Results shown are averages of
(E) Effects of RIP1 mutations on kinase activity of the anti-GFP immunoprecipitates in 293T cells transfected with the indicated RIP1-GFP constructs using MBP
as the substrate. The same membrane was probed for RIP1 on western blot in the lower panel.
See also Figure S7.
Cell 150, 339–350, July 20, 2012 ª2012 Elsevier Inc. 347
and RIP3 (Sawaya et al., 2007). The core RHIM sequences of
RIP1 and RIP3 and IQIG and VQVG, respectively, suggest that
the hydrophobic Ile and Val residues pack to form the double
b sheet in the steric zipper. This type of packing is remarkably
similar to the structure of the VQIVYK sequence from tau (PDB
2ON9) (Sawaya et al., 2007), a microtubule-associated protein
that plays an important role in stabilizing axonal microtubules.
Particularly, the first 3 residues of the RHIM core sequences
and the tau sequence are basically identical. We built two
alternative models of the RHIM steric zipper based on the tau
structure (Figure S7C). In both models, heterosteric zippers
(Eisenberg and Jucker, 2012) for the b-amyloid spine are
proposed. Notably, the hydrophobic packing between Ile and
Val residues is reminiscent of tau. It is likely that packing in
RIP1 or RIP3 homo-oligomeric fibrils (with Ile-Ile or Val-Val
contacts) is less optimal than the Ile-Val contacts in the hetero-
oligomeric complex. This preference may potentiate the unusual
assembly of the RIP1/RIP3 heterodimeric complex and explain
the apparent 1:1 stoichiometry between RIP1 and RIP3.
A number of additional proteins, including the cytoplasmic
DNA sensor DAI, the Toll-like receptor signaling adaptor TRIF,
and the murine cytomegalovirus protein M45, have been shown
to contain RHIMs (Figure S7D). Both DAI and TRIF recruit RIP1
and/or RIP3 through their RHIMs to activate NF-kB or to induce
cell death (Kaiser and Offermann, 2005; Meylan et al., 2004;
Takaoka et al., 2007). Indeed, we show that DAI and RIP1 also
form filamentous structures (Figure S7E). M45 binds to RIP1
and functions as a viral inhibitor in DNA sensing, Toll-like
receptor signaling, and the TNF receptor pathway (Mack et al.,
2008; Rebsamen et al., 2009; Upton et al., 2010). In all of these
signaling processes, the high-order oligomeric scaffold of
amyloids may be the key feature that brings signaling proteins
into proximity to allow their activation. Similarly, yet distinctly,
the oligomeric scaffold of the DD superfamily mediates caspase
activation and apoptosis (Wang et al., 2010), as well as MyD88-
dependent Toll-like receptor signaling (Lin et al., 2010). In addi-
tion, this DD superfamily scaffold exhibits functional characteris-
tics akin to amyloids and prions in their abilities to seed and to
propagate (Hou et al., 2011). For both the amyloid scaffold and
the DD superfamily scaffold, the slow seeding phase and the
fast growing phase in the assembly of these complexes may
dictate a highly cooperative process and a digital threshold
response mechanism. Assembly of highly oligomeric signalo-
somes may be an emerging principle in signal transduction.
Cloning, Protein Expression, Purification, and Mutagenesis
The RIP1 (residues 496–583)/RIP3 (residues 388–518) complex and its trunca-
tion complexes werepolycistronicallysubcloned intothepET28a(Novagen) or
pDB-His-MBP vector (Berkeley Structural Genomics Center). The RIP1 (resi-
dues 496–583) and RIP3 (residues 388–518) constructs were subcloned into
the pET28a or pSMT3 vectorwithaHis-Sumotag. All proteins wereexpressed
in E. coli BL21 (DE3) RIPL cells (Novagen) and were purified by Ni-affinity and
gel filtration chromatography.
The full-length RIP1(1–671)/RIP3(1–518) complex was subcloned into
pFastBacDual vector (Invitrogen) with an N-terminal His-tag on RIP1 and an
N-terminal MBP-tag on RIP3. The WT and mutant RIP1 and RIP3 (RIP1-FL,
RIP3-FL, RIP1-FL-I539D, and RIP3-FL-V460P) were subcloned into pFast-
BacHTA vector (Invitrogen) with an N-terminal His-tag. The proteins were
expressed in Hi5 cells and were purified with either Ni-NTA or amylose resin
followed by gel filtration chromatography.
Electron Microscopy and Image Processing
RIP1/RIP3-RHIM, RIP1-RHIM, and RIP3-RHIM were negatively stained with
uranyl formate and imaged with a 1K 3 1K charge-coupled device (CCD)
camera (Gatan) on a CM10 electron microscope (FEI). For subsequent
image processing, images were collected on imaging plates with a Tecnai
T12 electron microscope. The particles were subjected to ten cycles of
multireference alignment, each followed by K-means classification into
For full-length RIP1, RIP3, RIP1/RIP3 complex, and endogenous RIP1/3
complexes, the samples were stained using uranyl acetate and imaged with
a Veleeta 2K 3 2K cooled CCD camera (Olympus-Soft Imaging Solutions,
Munster, Germany) on a JEM-1400 (JEOL, Ltd., Tokyo, Japan) electron
X-Ray Diffraction from Fibrils
The RIP1/RIP3-RHIM complex was dried and partially aligned by using
aSpeedVac System(Savant) andmountedinacryoloop.Thediffractionimage
was collected by using a Rigaku RU-H3R X-ray generator on a Rigaku R-Axis
IV imaging plate detector.
Congo Red Binding
CR absorption spectra were recorded from 430–600 nm in 96-well plates by
using a SpectraMax M2 plate reader (Molecular Devices).
Thioflavin T Fluorescence
Fluorescence measurements were performed in 96-well plates on a Spectra-
Max M2 plate reader (Molecular Devices) with an excitation wavelength of
Circular Dichroism Spectroscopy
CD spectra of RIP1, RIP3, and the RIP1/RIP3 complex were measured on an
Aviv Model 410 spectropolarimeter at a cell length of 0.1 cm at 20?C with five
scans per measurement. Buffer control was subtracted from each sample
Fourier Transform Infrared Spectroscopy
Infraredspectra wererecorded byusingaBrukerTensorspectrometer(Bruker
Optik, Germany) at room temperature and with a resolution of 4 cm?1. The
spectra represent the sum of 40 scans after reference subtraction.
Renaturation and Seeding Experiments Using ThT Fluorescence
complex was denatured in 150 mM NaOH. The denatured proteins were
diluted 100-fold into a native buffer and 50 mM ThT. The final concentration
for RIP1 or the RIP1/RIP3 complex was 5mM. Seeding experiments were per-
formed as described above with the exception that RIP1, RIP3, or RIP1/RIP3
complex seeds were added to the reaction to a final concentration of 0.5 mM.
and emission wavelengths of 430 nm and 485 nm, respectively.
Solid-State NMR Experiments
2D DARR spectra were recorded on a Bruker Advance III 400 spectrometer.
The 2D13C-13C DARR and DREAM spectra used for the resonance assign-
ment, aswellasthe 1D13C spectra, wererecorded on anAdvanceII900spec-
trometer. Data were collected at ?10?C if not mentioned otherwise. All triple
resonance spectra used for the assignment were recorded on an Advance I
750 spectrometer. All spectra were processed by using Topspin 3.1 and
analyzed using the program CARA.
For ThT staining, HeLa cells were plated on coverslips and transfected with
RIP3-mCherry. After 15–18 hr, cells were treated with zVAD-fmk, LBW242,
and TNF for 3 hr. 20 mM ThT was added to cells 1 hr prior to fixation and
imaging with a Leica SP5 confocal microscope. For microscopy of WT and
348 Cell 150, 339–350, July 20, 2012 ª2012 Elsevier Inc.
mutant RIP1-CFP, RIP1-GFP, or RIP3-YFP, transfected NIH 3T3 fibroblasts or
HeLa cells were induced to undergo necrosis and visualized by confocal
Cell Death Assays
For inhibition of programmed necrosis, CR, ThT, or HBX were used at the
indicated concentrations on HT-29 cells treated with TNF, LBW242, and
zVAD-fmk. Cell death was determined by lactate dehydrogenase (LDH)
release or propidium iodide (PI) exclusion using flow cytometry. For induction
of apoptosis, HT-29 cells were pretreated with the indicated concentrations of
inhibitors prior to stimulation with IFN-g and anti-Fas antibody.
For effects of RIP3 mutations, HeLa cells were transfected with WT or
mutant RIP3 fused to YFP. Cells were treated with zVAD-fmk, LBW242, and
TNF, and cell death was determined in the YFP-positive population with PI
by flow cytometry. For effects of RIP1 mutations, RIP1?/?fibroblasts were
transfected with WT or mutant RIP1 fused to GFP. Cells were similarly treated,
and cell death was determined in the GFP-positive population by PI flow
ThT Binding and Immunogold EM of the Endogenous RIP1/RIP3
RIP1 immunoprecipitates were purified from necrotic HT-29 cells, and ThT
fluorescence was measured from 450–550 nm by using a Spex Fluorolog-3
spectrofluorometer with excitation at 430 nm. For immunogold EM analyses,
necrotic MEFs were fixed in 4% paraformaldehyde. RIP1 (BD PharMingen)
and RIP3 (ProSci) antibodies were applied at 1:50 dilutions. Secondary immu-
nogold conjugated antibodies (15 nm for RIP1 and 6 nm for RIP3) were used at
Stability Assessment of the Endogenous RIP1/RIP3 Complex
HT-29 cells were lysed in lysis buffer supplemented with 4 M urea, 8 M urea, or
150 mM NaOH. Thirty minutes after lysis, cell lysates were diluted 10-fold with
regular lysis buffer and immunoprecipitated with anti-RIP3 antibody. For
TNFR1 complexes, WT MEFs were treated with TNF for 2 min. Cell lysis was
performed as for the RIP1/RIP3 complexes.
In Vitro Kinase Assays
293T cells were transfected with indicated RIP1 or RIP3 mutants fused to GFP
or YFP, respectively. The fusion proteins were immunoprecipitated 24 hr later
with anti-GFP antibody (Roche). For RIP3 stable fibroblasts, RIP3 complexes
were immunoprecipitated after necrosis induction. The resulting immune
complexes were used for in vitro kinase assay using myelin basic protein
(MBP, Stressgen) as substrate.
Supplemental Information includes Extended Experimental Procedures and
seven figures and can be found with this article online at http://dx.doi.org/
WethankL.Cohen-Gould forhelp withEM imagingattheWeillCornellMicros-
copy Facility, S.M. Damo for initial work on this project, Q. Li for insect cell
expression, V. Kumar for ThT measurement of endogenous complexes, and
D. Porter (Novartis) for the generous gift of LBW242. This work was supported
by grants to F.K.C. (AI083497 and AI088502) and H.W. (AI045937). F.K.C. is
a member of the UMass DERC (DK32520). J.N. is an Irvington Institute post-
doctoral fellow of the Cancer Research Institute, and D.M. is supported by
NIH training grant T32 AI07349. T.W. is an investigator in the Howard Hughes
Received: April 6, 2012
Revised: May 7, 2012
Accepted: June 8, 2012
Published: July 19, 2012
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