Monomer/Dimer Transition of the Caspase-Recruitment Domain of Human Nod1†,‡
Thiagarajan Srimathi,§,|Sheila L. Robbins,§,|Rachel L. Dubas,§Mizuho Hasegawa,⊥Naohiro Inohara,⊥,4and
Young Chul Park*,§
Basic Science, Fox Chase Cancer Center, Philadelphia, PennsylVania 19111, Department of Pathology, UniVersity of Michigan
Medical School, Ann Arbor, Michigan 48109, and Department of Biochemistry 2nd, Interdisciplinary Graduate School of
Medicine and Engineering, UniVersity of Yamanashi, 1110 Shimokato, Chuou, Yamanashi 409-3898, Japan
ReceiVed August 16, 2007; ReVised Manuscript ReceiVed NoVember 20, 2007
ABSTRACT: Nod1 is an essential cytoplasmic sensor for bacterial peptidoglycans in the innate immune
system. The caspase-recruitment domain of Nod1 (Nod1_CARD) is indispensable for recruiting a
downstream kinase, receptor-interacting protein 2 (RIP2), that activates nuclear factor-κB (NF-κB). The
crystal structure of human Nod1_CARD at 1.9 Å resolution reveals a novel homodimeric conformation.
Our structural and biochemical analysis shows that the homodimerization of Nod1_CARD is achieved by
swapping the H6 helices at the carboxy termini and stabilized by forming an interchain disulfide bond
between the Cys39 residues of the two monomers in solution and in the crystal. In addition, we present
experimental evidence for a pH-sensitive conformational change of Nod1_CARD. Our results suggest
that the pH-sensitive monomer/dimer transition is a unique molecular property of Nod1_CARD.
The Nod1(nucleotide-binding and oligomerization domain)
proteins, in particular Nod1 and Nod2, are essential cyto-
plasmic sensors for bacterial peptidoglycan derivatives. Nod1
detects meso-diaminopimelic acid in Gram-negative and
certain Gram-positive bacterial cell walls, while Nod2 acts
as a general sensor of bacteria by detecting muramyl
dipeptide (1-3) present in all Gram-positive and Gram-
negative bacterial cell walls. It is known that Nod2 is a
susceptibility gene for Crohn’s disease (4, 5) and that the
polymorphisms in Nod1 are associated with inflammatory
bowel disease (6) and asthma (7). Moreover, Nod1 is
involved in host defense against Helicobacter pylori infection
of the gastric mucosa, a chronic infection that can lead to
peptic ulcers and gastric cancer (8).
Nod1 and Nod2 have a common tripartite domain
structuresa carboxy-terminal leucine-rich repeat domain
(LRR), a centrally located nucleotide-binding and oligomer-
ization domain (NOD), and an amino-terminal caspase-
recruitment domain (CARD) (9). Nod1 has one CARD,
whereas Nod2 has two CARDs. The LRR is involved in
ligand recognition (10, 11), and the NOD facilitates self-
oligomerization (12). The CARDs of Nod1 and Nod2 are
indispensable for the recruitment of downstream effectors
(13-15), such as receptor-interacting protein 2 (RIP2, also
known as RICK). RIP2 is a CARD-containing serine/
threonine kinase that physically associates with Nod1 and
Nod2 through CARD-CARD interactions and activates the
transcription factor NF-κB. It has also been shown that the
oligomerization of Nod1 induces the proximity of RIP2 and
that the enforced oligomerization of RIP2 activates NF-κB
The CARD is a protein interaction module found in
remarkably diverse proteins, which are mainly involved in
the activation of caspases and NF-κB (16). In general, the
CARD participates in multiprotein complex formations where
caspases or protein kinases are brought together and activated
through the so-called induced proximity mechanism (17).
To investigate the interactions mediated by the CARD, we
purified human Nod1_CARD (residues Met1 to Glu106). The
purified Nod1_CARD appeared as a mixture of monomers
and dimers. To elucidate the structural aspects of the
oligomerization mechanism of Nod1, we solved the crystal
structure of homodimeric Nod1_CARD. During the prepara-
tion of this manuscript, two NMR structures of monomeric
Nod1_CARD (PDB 2B1W and 2DBD; PDB ) Protein Data
Bank; 18) and one crystal structure of a homodimeric
Nod1_CARD (PDB 2NSN; 19) were reported. However, the
reason for the structural heterogeneity of Nod1_CARD and
its functional significance are currently not understood.
Here, we present biochemical evidence that homodimeric
Nod1_CARD in solution is formed through helix swapping
and is stabilized by a subsequent interchain disulfide bond
formation, as observed in our crystal structure. In addition,
our results indicate that pH-sensitive conformational changes
of Nod1_CARD may cause the structural differences between
†This work was supported, in part, by the Crohn’s and Colitis
Foundation of America. Y.C.P. is a Principal Investigator of the Cancer
‡The atomic coordinates and structure factors (PDB 2NZ7) have
been deposited in the Protein Data Bank, Research Collaboratory for
Structural Bioinformatics, Rutgers University, New Brunswick, NJ.
* To whom correspondence should be addressed. Phone: (215) 728-
5652. Fax: (215) 728-3574. E-mail: Young.Park@fccc.edu.
§Fox Chase Cancer Center.
|These authors contributed equally to this work.
⊥University of Michigan Medical School.
4University of Yamanashi.
1Abbreviations: ?ME, ?-mercaptoethanol; DTT, dithiothreitol; iE-
DAP, D-glutamyl-meso-diaminopimelic acid; IPTG, isopropyl thioga-
lactoside; MES, 2-(N-morpholino)ethanesulfonic acid; NF-κB, nuclear
factor-κB; Nod1, nucleotide-binding and oligomerization domain protein
1; Nod1_CARD, caspase-recruitment domain of Nod1; PDB, Protein
Data Bank; RIP2, receptor-interacting protein 2; RMSD, root mean
square deviation; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide
Biochemistry 2008, 47, 1319-1325
10.1021/bi7016602 CCC: $40.75© 2008 American Chemical Society
Published on Web 01/11/2008
the NMR structures (PDB 2B1W and 2DBD) and the crystal
structures (PDB 2NSN and the structure presented here).
Protein Expression and Purification. The DNA fragments
of human Nod1, encoding residues Met1 to Glu106
(Nod1_CARD) and residues Met1 to Asp95 (short_CARD),
were inserted into pET24d vector (Novagen) and TOPO100D
vector (Invitrogen), respectively, following instructions from
the manufacturer. Mutant Nod1_CARDs were generated with
the QuikChange II kit (Stratagene), using the plasmid
containing Nod1_CARD as a template. The sequences of
cloned DNAs were confirmed using DNA sequencers (Ap-
plied Biosystems) in the DNA Sequencing Facility at Fox
Chase Cancer Center. The oligonucleotide primers used for
cloning were made by the DNA Synthesis Facility at Fox
Chase Cancer Center. Nod1_CARD and its mutants were
expressed and purified as described previously (20). Briefly,
protein expression was induced in Escherichia coli Rosetta-
pLysS cells (Novagen) in LB medium supplemented with
0.5% (w/v) glucose by adding 1 mM IPTG at 37 °C for 3 h.
Harvested cells were resuspended in a lysis buffer consisting
of 50 mM sodium phosphate (pH 7.0), 1.5 M NaCl, 1.5 M
urea, 10% (v/v) glycerol, and 3 mM ?ME. Cleared cell
extract was incubated with nickel resin (Qiagen) at room
temperature for 1 h, and then the resin was washed with a
wash buffer consisting of 50 mM sodium phosphate (pH 6.0),
300 mM NaCl, 10% (v/v) glycerol, 20 mM imidazole, and
3 mM ?ME. The protein was eluted from the resin with an
elution buffer consisting of 50 mM sodium phosphate (pH
6.0), 300 mM NaCl, 10% (v/v) glycerol, 300 mM imidazole,
and 3 mM ?ME. The fractions containing Nod1_CARD were
combined and further purified through a Superdex 75 size-
exclusion column (Amersham Bioscience) in a buffer
consisting of 10 mM sodium phosphate (pH 6.0), 130 mM
NaCl, and 1 mM DTT. The fractions containing dimeric
Nod1_CARD were concentrated using Amicon Ultra 100K
centrifugal filter devices (Millipore). The bacterially ex-
pressed Nod1_CARD showed two, very closely located
bands on SDS-PAGE gels due to proteolysis during
expression (see Supporting Information, Figure S1). Before
use, the conformational integrity of the purified proteins was
confirmed by circular dichroism using an Aviv 62ABS
spectrometer in the Spectroscopy Supporting Facility at Fox
Chase Cancer Center to ensure the purified Nod1_CARD
consisted of six helices as expected.
Crystallization and Data Collection. Nod1_CARD was
crystallized as described previously (20). Briefly, crystal-
lization drops consisting of 2.0 µL of purified Nod1_CARD
(13.0 mg/ mL) in 10 mM sodium phosphate (pH 6.0), 130
mM NaCl, and 1 mM DTT and 2.0 µL of crystallization
solution consisting of 20% (w/v) PEG6000, 67 mM MES
(pH 6.0), 100 mM diammonium hydrogen citrate, 90 mM
NaI, and 1 mM DTT were equilibrated against 0.9 mL of
crystallization solution using the sitting-drop vapor-diffusion
method. Small crystals appeared within two weeks. To
produce diffraction-quality crystals, small crushed crystals
were seeded into new drops containing the same crystalliza-
tion solution and an equal volume of protein. Crystals
appeared within two weeks and reached their maximum
dimensions within a month. To obtain heavy atom derivative
crystals, native crystals were soaked in cryo solution A
consisting of 20% (w/v) PEG6000, 67 mM MES (pH 6.0),
100 mM diammonium hydrogen citrate, and 20% (v/v)
glycerol for 30 min and then transferred into a heavy atom
solution, consisting of 10 mM KAu(CN)2, KAuBr4, or K2-
PtBr4in cryo solution A. After 1 h of incubation in the heavy
atom solution, the crystals were transferred into cryo solution
A for 10 min and then into cryo solution B (cryo solution A
with 25% (v/v) glycerol) for 3 min before being flash-cooled
in liquid nitrogen. All the experiments, including crystal-
lization and preparation of heavy atom derivative crystals,
were performed at 4 °C. The diffraction data sets were
collected on beamline F-2 at MacCHESS, Ithaca, NY, using
X-rays at a wavelength of 0.977 Å. The native and heavy
atom derivative data sets were processed using HKL-2000
Structure Determination and Analysis. The experimental
phase information was obtained by the multiple isomorphous
replacement method using the Phenix package (22). The
partial model generated from Phenix was completed using
Coot (23) and refined using Refmac5 (24) from the CCP4
suite (25). The statistics for data collection and refinement
are summarized in Supporting Information Table S1. The
root mean square deviations (RMSDs) for the three-
dimensional structure comparisons were calculated using the
program Superpose, and the various solvent-accessible
surface areas were calculated using Areaimol from the CCP4
suite (25). The area of interchain interface and the hydrogen
bonds were obtained using PISA (26). Protein models shown
in the figures were prepared using Pymol (DeLano Scientific
Size-Exclusion Chromatography Experiments. The con-
formations of Nod1_CARD and its mutants were tested using
200 µL of proteins that were purified through Ni-NTA
affinity chromatography and extensively dialyzed at 4 °C in
buffers consisting of 10 mM sodium phosphate (pH 6.0) and
130 mM NaCl with either 1 mM DTT or no DTT.
Short_CARD has a higher molecular mass than Nod1_CARD,
due to about 3 kDa of extra amino acid residues from
TOPO100D vector. The conformations of Nod1_CARD at
various pHs were tested using 200 µL of the dimeric protein
(about 5 mg/mL) that was purified through size-exclusion
chromatography at pH 6.0 with a Superdex 75 column
(Amersham Pharmacia). The column was pre-equilibrated
with the buffers consisting of 130 mM NaCl, 1 mM DTT,
and 10 mM sodium phosphate (pH 6.0), 10 mM Tris-HCl
(pH 7.0 or 8.0), or 10 mM sodium acetate (pH 5.0).
Fluorescence Spectroscopy. All fluorescence measure-
ments were performed at 25 °C using a Quantum Master
2000 fluorometer (PTI) in the Spectroscopic Facility at Fox
Chase Cancer Center. Homodimeric Nod1_CARD was
purified through size-exclusion chromatography using a
Superdex 75 column (Amersham Pharmacia) in a buffer
consisting of 10 mM sodium acetate (pH 5.0), 130 mM NaCl,
and 1 mM DTT. The purified proteins were concentrated to
4 mg/mL and then diluted to 400 µg/mL using buffers
consisting of 150 mM NaCl, 1 mM DTT, and 30 mM sodium
phosphate (pH 6.0), 30 mM Tris-HCl (pH 7.0 or 8.0), or
30 mM sodium acetate (pH 5.0). The diluted protein samples
were dialyzed against the buffers used for the dilution at 4
°C for 3 h. Emission fluorescence of Nod1_CARD, from
300 to 400 nm, was recorded using excitation wavelengths
at 280 and 295 nm. To measure the fluorescence of the
1320 Biochemistry, Vol. 47, No. 5, 2008
Srimathi et al.
homodimeric Nod1_CARD with a disulfide bond at various
pHs, the proteins were prepared as described above, but
without DTT. The secondary structure contents were moni-
tored by circular dichroism using an Aviv 62ABS spectrom-
eter in the Spectroscopy Supporting Facility at Fox Chase
SDS-PAGE Analysis. The existence of the interchain
disulfide bond was examined after mixing with 2× SDS
sample buffer (BioRad), supplemented either with 715 mM
?ME for a reducing condition or without ?ME for a
nonreducing condition. The protein species were separated
on a NuPAGE SDS-PAGE gel (Invitrogen) after a 1 min
incubation in a boiling water bath.
OVerall Structure of Nod1_CARD. The crystal structure
of homodimeric Nod1_CARD was determined at 1.9 Å
resolution using the multiple isomorphous replacement
method (see Supporting Information Table S1). The electron
density map unambiguously shows two chains in one
asymmetric unit; one extends from Ser14 to Glu106 and the
other from Glu8 to Glu106. The structure of Nod1_CARD
reveals that the H6 helix (residues Ala96 to Glu106) is
swapped with the adjacent monomer to form a homodimeric
conformation (Figure 1). In the structure, the H6 helix of
one monomer has swung out and bound itself between the
H1 and H5 helices of the other monomer. The hinge loop
(Asp95 to Asp99) between the H5 and H6 helices of one
monomer and the H1-H2 loop (His33 to Thr37) that
connects the H1 and H2 helices of the other monomer interact
through hydrogen bonds and salt bridges (Supporting Infor-
mation Table S2). The electron density map also reveals an
interchain disulfide bond between the Cys39 of one monomer
and the Cys39 of the other monomer (Figure 2). The
existence of an interchain disulfide bond in the Nod1_CARD
crystals used for X-ray diffraction data collections was
confirmed with SDS-PAGE analysis in a nonreducing
condition (Supporting Information Figure S1). The residues
from Pro18 to Asp95 (H1 to H5 helix) adopt a compact
globular fold well-packed around a central hydrophobic core.
Four helices, from H2 to H5, form an antiparallel four-helix
bundle, and the H1 helix is bent over to cover the
hydrophobic core. In addition, the hydrophobic residues of
the swapped H6 helix (Leu100, Trp103, and Leu104)
participate in covering the remaining exposed surface of the
hydrophobic core. The formation of the Nod1_CARD
homodimer buries a total of 3145 Å2solvent-accessible
surface area, 1582 Å2from one monomer and 1563 Å2from
the other monomer. About 87% of this total interface area
is provided by the swapping of the H6 helices.
Comparison with Other CARD Structures. To investigate
the conformational changes caused by homodimerization, our
homodimeric crystal structure of Nod1-CARD was compared
to the two recently reported monomeric NMR structures
(PDB 2B1W and 2DBD) and the homodimeric crystal
structure (PDB 2NSN). The three-dimensional structure
alignment shows that our structure is very similar to PDB
2NSN with an RMSD of 0.63 Å. The main difference
between these two crystal structures is that our structure has
an interchain disulfide bond. Our structure also shows a
reasonable RMSD of 1.3 Å from PDB 2DBD (Figure 3).
The three-dimensional structure alignment shows that the H6
helix of monomeric Nod1_CARD (PDB 2DBD) is exactly
replaced by the swapped H6 helix in our homodimeric
structure. The difference between these two structures is that
FIGURE 1: Overall structure of homodimeric Nod1_CARD. On the
top is the cartoon representation of homodimeric Nod1_CARD. At
the bottom the Nod1_CARD is rotated by 90° about the horizontal
axis. One monomer (residues Glu8 to Glu106) is colored cyan, and
the other monomer (residue Ser14 to Glu106) is colored magenta.
The disulfide bond is shown in stick representation.
FIGURE 2: 2Fo- Fcelectron density map for the interchain disulfide
bond. The electron density map (gray) shows the disulfide bond
that connects Cys39 residues between the two monomers. Residues
around Cys39 (Asn36 to Asn43 of each monomer) are shown as
sticks colored by atom type (carbon, cyan; each monomer, magenta;
nitrogen, blue; oxygen, red; sulfur, yellow).
FIGURE 3: Three-dimensional structure alignment of monomeric
and homodimeric Nod1_CARDs. The dimeric crystal structure and
monomeric NMR structure are superimposed using the program
Superpose in the CCP4 suite (25). Ribbon representation of
homodimeric Nod1_CARD with subunits colored blue and cyan.
The NMR structure of monomeric Nod1_CARD (PDB 2DBD) is
Monomer/Dimer Transition of the CARD of Nod1
Biochemistry, Vol. 47, No. 5, 2008 1321
in PDB 2DBD the hinge loop (Asp95 to Asp99) does not
interact with the H1-H2 loop (His33 to Thr37). The RMSDs
between our structure and PDB 2B1W and between PDB
2DBD and PDB 2B1W are 2.7 Å, which is rather large. In
addition, the structure alignment shows that the three-
dimensional positions of the amino acid residues in the region
between the H2 helix and the H6 helix of PDB 2B1W are
inconsistent with PDB 2DBD, PDB 2NSN, and our structure.
The overall structure of the homodimeric Nod1_CARD is
similar to other CARD structures of Iceberg (PDB 1DGN;
27) and Apaf-1 (PDB 2YGS and 1CY5; 28, 29), with
RMSDs of 1.87 and 1.57 Å, respectively.
Role of the H6 Helix in Homodimerization. The structural
analysis of Nod1_CARD suggests that the H6 helix plays a
key role in homodimerization. To test the role of the H6
helix in the homodimerization of Nod1_CARD, we generated
a shorter Nod1_CARD (short_CARD; residues Met1 to
Asp95), in which the H6 helix was removed but Cys39 was
short_CARD is monomeric (Figure 4A). The short_CARD
retains its monomeric conformation even at high protein
concentrations (up to 5 mg/mL) in the absence of a reducing
agent. The crystal structure of Nod1_CARD suggests that
the interactions of the hinge region, between the H5 and H6
helices, of one monomer and the loop region that connects
the H1 and H2 helices of the other monomer (Supporting
Information Table S2) play a role in homodimerization. In
addition, the NMR structure of monomeric Nod1_CARD
(PDB 2B1W) shows that both the hinge region and loop
region are generally exposed to the solvent and do not
form significant interactions with other amino acid residues.
To understand the importance of the interactions mediated
by these regions, we generated mutant Nod1_CARD proteins,
in which His33, Arg35, Asn36, Asp95, Tyr97, Asp99, and
Arg101 are replaced with Ala. Size-exclusion chromatog-
raphy of purified mutant Nod1_CARD proteins without a
and Nod1_CARD_D95A are mainly monomeric (Supporting
Information Figure S2).
The deletion of the H6 helix (short_CARD) and the
mutations, which interrupt the interaction between the hinge
and the loop regions, significantly reduce the homodimer-
ization of Nod1_CARD. These results indicate that the H6
helix swapping in Nod1_CARD is a prerequisite for ho-
modimerization and the formation of an interchain disulfide
bond. Additionally, it appears that the amino acid residues,
Arg35 in the loop region and Asp95 in the hinge region, are
involved in the H6 helix swapping of Nod1_CARD.
Role of the Interchain Disulfide Bond in Homodimeriza-
tion. The homodimeric Nod1_CARD has an interchain
disulfide bond in solution and in the crystal (Figure 2B and
Supporting Information Figure S1). To evaluate the role of
the disulfide bond in the homodimer formation, we generated
an Nod1_CARD mutant (Nod1_CARD_C39A), where Cys39
is replaced with Ala. The Nod1_CARD_C39A shows a
predominantly monomeric conformation on size-exclusion
chromatography under a nonreducing condition (Figure 4B).
The importance of the disulfide bond in homodimerization
was tested using purified homodimeric Nod1_CARD at
various concentrations of ?ME. This result clearly shows
that increasing the concentration of ?ME results in a
proportional increase of the monomeric population of
Nod1_CARD (Figure 5). Since the short_CARD remains as
a monomer, even in a nonreducing condition (Figure 4A), it
appears that the homodimerization process of Nod1_CARD
is initiated by H6 helix swapping and the conformation is
Nod1_CARD (about 5 mg/mL) in 1 mM DTT and short_CARD
(about 5 mg/mL) in a nonreducing condition. Purified Nod1_CARD
and short_CARD were passed through a Superdex 75 column.
Short_CARD showed lower optical absorbance due to the lack of
Trp residues in the amino acid sequence. (B) Size-exclusion
chromatography profiles for Nod1_CARD in 1 mM DTT and
(about 15 mg/mL) and Nod1_CARD_C39A (about 15 mg/mL) were
passed through a Superdex 75 column.
(A) Size-exclusion chromatography profiles for
FIGURE 5: Monomer/dimer transition of Nod1_CARD in buffers
containing various concentrations of ?ME. Purified Nod1_CARD
(200 µL at 5 mg/mL) was passed through a Superdex 75
size-exclusion column after dialysis in buffers consisting of 10 mM
sodium phosphate (pH 6.0), 130 mM NaCl, and 0, 2, 4, or 6 mM
1322 Biochemistry, Vol. 47, No. 5, 2008
Srimathi et al.
then stabilized by disulfide bond formation between the two
Effect of pH on the Quaternary Structure of Nod1_CARD.
It is known that the energy barrier between the monomer
and the swapped dimer can be reduced by a change in
solution conditions (30). In our experiments, we observed
that Nod1_CARD exists mostly as dimers at pH 6.0 and
monomers at pH 7.0. In addition, Nod1_CARD is monomeric
at pH 7.0 in NMR structures (18), but the crystal structures
are homodimeric at pH 6.0 in this paper and at pH 4.7 in
PDB 2NSN (19). To test the effect of pH on conformation,
the purified homodimeric Nod1_CARD at pH 6.0 was passed
through a size-exclusion column at various pHs. The result
shows that homodimeric Nod1_CARD completely dissoci-
ates to monomers at pH 8.0, but remains as dimers at pH
5.0. Nod1_CARD prefers the homodimeric conformation at
pH 6.0, but the monomeric conformation at pH 7.0 (Figure
6). The presence of the interchain disulfide bond in the dimer
fractions, which were eluted from the size-exclusion column,
was confirmed by nonreducing SDS-PAGE analysis (data
Effects of pH on the Tertiary Structure of Nod1_CARD.
Our results show that H6 helix swapping is a prerequisite
for the quaternary structural change of Nod1_CARD, which
is pH-sensitive. This observation suggests that the tertiary
structure of Nod1_CARD might also be sensitive to pH
change. Nod1_CARD has one Trp residue, Trp103, located
on the H6 helix that is swapped during homodimerization.
We examined the pH-dependent tertiary structural change
around the H6 helix by monitoring the intrinsic Trp
fluorescence changes of Nod1_CARD at various pHs (Figure
7A). The intrinsic Trp fluorescence of Nod1_CARD is the
lowest at pH 5.0. At pH 6.0 and 7.0, the Trp fluorescence is
increased from the fluorescence at pH 5.0 by about 20% and
25%, respectively. At pH 8.0 the fluorescence increases by
about 50% from the fluorescence at pH 5.0. The maximum
emission wavelength is not significantly shifted. The sub-
sequent nonreducing SDS-PAGE analysis of the samples
confirms that the increasing Trp fluorescence was ac-
companied by a reduction of the disulfide bond, which
correlates with monomerization (Figure 7B). In contrast, the
pH change does not affect the secondary structural content
of Nod1_CARD since circular dichroism spectra recorded
at various pHs are indistinguishable (Supporting Information
Figure S3). To test whether pH can affect the tertiary
structure around the H6 helix in a homodimeric Nod1_CARD
with a disulfide bond, we monitored the intrinsic Trp
fluorescence changes of homodimeric Nod1_CARD at vari-
ous pHs without a reducing agent. However, the result shows
that the Trp fluorescence of homodimeric Nod1_CARD is
not affected by pH changes (Supporting Information Figure
S4). Our results indicate that the homodimerization of
Nod1_CARD in solution is sensitive to pH and accompanies
the tertiary structural changes, in particular around the H6
helix, as seen in our crystal structure. The tertiary structure
around the H6 helix becomes stable as Nod1_CARD proteins
adopt dimers stabilized by a disulfide bond. On the basis of
these observations, we propose that differences in pH may
explain the conformational heterogeneity of Nod1_CARD
observed in the NMR and crystal structures.
Four structures of Nod1_CARD, including the one pre-
sented here, recently became available in the PDB. Through
structural and biochemical experiments, we show that the
homodimerization of Nod1_CARD can occur through helix
swapping and an interchain disulfide bond formation in both
solution and crystals.
Our close investigation found that all of the amino acid
residues in PDB 2B1W starting from Asp66 moved exactly
three residues back. For example, the three-dimensional
position of Leu72 in the H4 helix in our structure is occupied
by the same Leu residues in PDB 2NSN and 2DBD, but
this position is occupied by Arg69 in PDB 2B1W. Misas-
signment of residues in the loop between Pro63 and Pro65
of the NMR structure may explain the structural differences
FIGURE 6: Size-exclusion chromatography profiles at various pHs.
The purified homodimeric Nod1_CARD (about 5 mg/mL) was
passed through a Superdex 75 column at pH 5.0, 6.0, 7.0, and 8.0.
FIGURE 7: pH-dependent conformational change of Nod1_CARD.
(A) The emission fluorescence spectra of Nod1_CARD (400 µg/
mL) at pH 5.0, 6.0, 7.0, and 8.0 were recorded using an excitation
wavelength of 295 nm. (B) The protein samples used for recording
fluorescence spectra were analyzed using SDS-PAGE in a reducing
condition and a nonreducing condition. The lane marked “STD” is
the molecular weight standard.
Monomer/Dimer Transition of the CARD of Nod1
Biochemistry, Vol. 47, No. 5, 2008 1323
between PDB 2B1W and the other Nod1_CARD structures.
The crystal structure of PDB 2NSN was solved by the
molecular replacement method using the NMR structure of
Iceberg (PDB 1DGN) as a model. However, Nod1_CARD
has a conformation and amino sequence quite different from
those of Iceberg, which is monomeric and whose sequence
identity to Nod1_CARD is only 25.5%. We determined the
crystal structure of Nod1_CARD using the multiple isomor-
phous replacement method to obtain experimental phase
information through an extensive search for heavy atom
derivative crystals, since the molecular replacement method
did not provide a satisfying phase solution. Our biochemical
results suggest that the purified Nod1_CARD that was used
for the structural determination of PDB 2NSN might exist
as mostly monomers or a mixture of monomers and dimers
in solution because of the high concentration of reducing
agent (5 mM DTT) and the slightly basic pH (8.0) used
during purification. Then, the protein might form a dimeric
conformation in the crystal because of the high protein
concentration and the acidic crystallization condition (pH
4.7). The available structures and the structure reported here
may represent sequential steps in Nod1_CARD dimerization.
The NMR structures represent the monomeric status of Nod1
at neutral pH. Under this condition, Nod1_CARD has a
relatively flexible tertiary structure around the H6 helix. PDB
2NSN may be similar to the intermediate state of the
are placed in close proximity by the oligomerization of
activated Nod1 and H6 helix swapping is induced by the
high local protein concentration. This dimeric intermediate
conformation of Nod1_CARD is not stable in solution,
however. As seen in the crystal structure presented here, the
conformation is then stabilized by forming a disulfide bond
between the two Cys39 residues that are brought together at
a proper angle by the H6 helix swapping.
Three factors that affect free energy differences between
the monomers and the domain-swapped oligomers were
suggested through analysis of about 40 structurally character-
ized cases of domain-swapped proteins (30). First, greater
entropy of the monomer makes it more favored thermody-
namically. Second, hinge loops may form new interactions
in the domain-swapped dimer, which favor dimerization.
Third, new interactions at the open interface make the
domain-swapped oligomer more favorable thermodynami-
cally. Our structural and biochemical analyses show that the
Nod1_CARD favors the monomeric conformation at neutral
and basic pHs as seen in the NMR structures (Figure 6).
The hinge loop of Nod1_CARD generates six hydrogen
bonds with the H1-H2 loop, when it forms a dimer
(Supporting Information Table S2). In addition, interchain
disulfide bond formation at the open interface stabilizes the
homodimeric conformation of Nod1_CARD (Figure 4B).
Therefore, the suggested model of domain swapping (31)
fits well to the dimerization mechanism of Nod1_CARD.
In addition, the pH-sensitive monomer/dimer transition of
Nod1_CARD indicates that acidic pH reduces the high-
energy barrier between the monomer and the swapped dimer
and promotes homodimerization in solution and crystals.
It appears that the conformation of the H6 helix is
intrinsically flexible because conformational heterogeneity
of the H6 helix was observed during NMR structure
determination (18) and because monomeric Nod1_CARD can
coexist with homodimeric Nod1_CARD in solution. The
intrinsic flexibility of the H6 helix of Nod1_CARD may play
a role in the formation of the active and stable oligomeric
structure of Nod1 in the context of high local concentration
of the CARD, which is produced through the self-oligomer-
izion of NOD when Nod1 is activated by ligand detection
(12). We tested the physiological significance of the ho-
modimerization of Nod1_CARD using transfected HEK293T
cells with plasmids for full-length Nod1 and full-length Nod1
mutants. The Nod1 mutants tested included the Cys39Ser,
His33Ala, Asn36Ala, Asp95Ala, and Arg101Glu mutations
that disturb the formation of the interchain disulfide bond
and the hydrogen bond network between the H1-H2 loop
(His33 to Thr37) and the hinge loop (Asp95 to Asp99).
Although the mutations of residues Cys39 and Asp95
generate mainly monomeric Nod1_CARD in Vitro (Figure
5), in ViVo the Nod1 mutants produced levels of NF-κB
activation similar to those of wild-type Nod1, upon treatment
with the Nod1-specific ligand γ-D-glutamyl-meso-diami-
nopimelic acid (iE-DAP) (unpublished data). This might be
due to the following reasons: (1) Upon detecting its ligand,
Nod1 is expected to form an apoptosome-like oligomeric
structure to recruit and activate RIP2 (31). The oligomer-
ization of Nod1, leading to a high local concentration of
CARD, is achieved by the NOD of Nod1, not by CARD.
(2) The key amino acid residues of Nod1_CARD, Glu53,
Asp54, and Glu56, for Nod1-RIP2 interaction (18) are
located on the opposite side of the H6 helices, remote from
the Cys39 residues that we have shown mediate homodimer-
ization. (3) At the high local concentration of CARD in the
apoptosome-like structure, Nod1_CARD can form the domain-
swapped intermediate dimer as seen in PDB 2NSN (19).
Since the physiological significance of Nod1_CARD dimer-
ization is not clear from our in ViVo tests, we attempted to
investigate the in Vitro interaction between Nod1_CARD and
RIP2. However, our recombinant RIP2 proteins were not
stable and quickly aggregated or precipitated. Although more
biological and biochemical studies are required to understand
the functional implications of the monomer/dimer transition
of Nod1_CARD in Nod1-induced NF-κB activation, we
suggest that the disulfide bond formation in homodimeriza-
tion might be a stabilization mechanism of activated Nod1,
similar to that seen in the structures of caspase-2 (32) and
prion proteins (33).
To date, this is the first report of a CARD that can undergo
the pH-sensitive monomer/dimer transitions through helix
swapping and interchain disulfide bond formation without
major changes to the secondary structure. We propose that
this is a unique molecular property of Nod1_CARD. Since
there is insufficient accumulated data for the other members
of the CARD protein family, direct comparison of this
molecular property of Nod1_CARD to those of other CARDs
is limited. The three-dimensional structure of the complex
between Nod1_CARD and RIP2_CARD, as well as of full-
length Nod1, is required to improve our understanding of
the Nod1-mediated interactions and their physiological role
in the innate immune response.
We thank the staff at Macromolecular Diffraction at
CHESS (MacCHESS) Facility beamline F-2 and at NSLS
beamline X12C for their assistance with data collection.
1324 Biochemistry, Vol. 47, No. 5, 2008
Srimathi et al.
SUPPORTING INFORMATION AVAILABLE Download full-text
Figures and tables showing SDS-PAGE analysis of
Nod1_CARD in nonreducing and reducing conditions, size-
exclusion chromatography for Nod1_CARD_R35A and
Nod1_CARD_D95A, far-ultraviolet circular dichroism spec-
tra for purified homodimeric Nod1_CARD at various pHs,
pH effect on the Trp fluorescence of dimeric Nod1_CARD,
statistics of diffraction data collection and structural refine-
ment of Nod1_CARD, and interactions between two mono-
mers of Nod1_CARD. This material is available free of
charge via the Internet at http://pubs.acs.org.
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Biochemistry, Vol. 47, No. 5, 2008 1325