Signal Transduction:
Designer Nodal/BMP2 Chimeras Mimic
Nodal Signaling, Promote Chondrogenesis,
and Reveal a BMP2-like Structure
J. Biol. Chem. 2014, 289:1788-1797.
doi: 10.1074/jbc.M113.529180 originally published online December 5, 2013
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This article cites 27 references, 7 of which can be accessed free at
http://www.jbc.org/content/289/3/1788.full.html#ref-list-1
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Luis Esquivies, Alissa Blackler, Macarena
Peran, Concepcion Rodriguez-Esteban, Juan
Carlos Izpisua Belmonte, Evan Booker, Peter
C. Gray, Chihoon Ahn, Witek Kwiatkowski
and Senyon Choe
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 3, pp. 1788 –1797, January 17, 2014
© 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
Designer Nodal/BMP2 Chimeras Mimic Nodal Signaling,
Promote Chondrogenesis, and Reveal a BMP2-like Structure
Received for publication, October 22, 2013, and in revised form, November 20, 2013 Published, JBC Papers in Press, December 5, 2013, DOI 10.1074/jbc.M113.529180
Luis Esquivies‡1, Alissa Blackler§, Macarena Peran¶储2, Concepcion Rodriguez-Esteban¶,
Juan Carlos Izpisua Belmonte¶, Evan Booker§, Peter C. Gray§3, Chihoon Ahn**‡‡4, Witek Kwiatkowski‡,
and Senyon Choe‡ ‡‡5
From the ‡Structural Biology Laboratory, the §Clayton Foundation Laboratories for Peptide Biology, and the ¶Gene Expression
Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, the 储Department of Health Sciences, University of
Jaen, 23071 Jaen, Spain, the **Department of Bio-Analytical Science, University of Science and Technology, Daejeon, 350-333,
Korea, and the ‡‡Joint Center for Biosciences at Lee Gil Ya Cancer and Diabetes Research Institute, Gachon University of Medicine
and Science, Incheon, 406-840, Korea
Nodal, a member of the TGF-␤ superfamily, plays an important role in vertebrate and invertebrate early development. The
biochemical study of Nodal and its signaling pathway has been a
challenge, mainly because of difficulties in producing the protein in sufficient quantities. We have developed a library of stable, chemically refoldable Nodal/BMP2 chimeric ligands (NB2
library). Three chimeras, named NB250, NB260, and NB264,
show Nodal-like signaling properties including dependence on
the co-receptor Cripto and activation of the Smad2 pathway.
NB250, like Nodal, alters heart looping during the establishment of embryonic left-right asymmetry, and both NB250 and
NB260, as well as Nodal, induce chondrogenic differentiation of
human adipose-derived stem cells. This Nodal-induced differentiation is shown to be more efficient than BPM2-induced differentiation. Interestingly, the crystal structure of NB250 shows
a backbone scaffold similar to that of BMP2. Our results show
that these chimeric ligands may have therapeutic implications
in cartilage injuries.
The TGF-␤ superfamily of proteins plays crucial roles during
development and homeostasis of both vertebrate and invertebrate organisms. Nodal is a TGF-␤ superfamily member that
The atomic coordinates and structure factors (code 4N1D) have been deposited
in the Protein Data Bank (http://wwpdb.org/).
1
Supported by the Jesse and Caryl Phillips Foundation Graduate Student
Endowment.
2
Supported first by the Spanish Ministry of Education (National Human
Resources Mobility National Plan I-D⫹i) and continued with a grant from
Jaen University.
3
Supported by the Clayton Medical Research Foundation, Inc. and Cancer
Center Core Grant P30 CA014195-38.
4
Supported by World Class University Program Grant R32-10215 and the
Incheon Free Economy Zone of Korea (joint Center for Biosciences).
5
To whom correspondence should be addressed: Structural Biology Laboratory, Salk Inst. for Biological Studies, 10010 N. Torrey Pines Rd., La Jolla, CA
92037. Tel.: 858-453-4100, Ext. 1652; E-mail: [email protected].
1788 JOURNAL OF BIOLOGICAL CHEMISTRY
controls mesoderm formation (1), establishes left-right asymmetry in vertebrates (2), and maintains pluripotency of human
embryonic stem cells (3), among other roles. Activins, which
comprise another TGF-␤ subfamily, also regulate wide ranging
biological processes including erythropoiesis (4), follicle-stimulating hormone secretion (5), and differentiation of macrophages (6).
Remarkably, despite the fact that they have very different
biological roles, Activins and Nodal signal via the same type II
(ActRII/IIB) and type I (ALK4) serine/threonine kinase receptors and activation of cytoplasmic Smad2/3 proteins. Unlike
Activin, however, Nodal signaling requires a co-receptor from
the epidermal growth factor-Cripto/FRL1/Cryptic protein
family such as Cripto. Cripto interacts with Nodal through its
EGF-like domain and with ALK4 through its CFC domain (7).
Cripto is not required for Activin signaling but rather binds to
the Activin-receptor complex to reduce Activin signaling
capacity (8). It is intriguing and remains unclear from a structural and mechanistic standpoint how Cripto is required for
Nodal signaling but inhibits Activin signaling via the same signaling receptors.
Bone morphogenetic proteins (BMPs),6 the major subfamily
of the TGF-␤ superfamily, control development of the skeletal
system, as well as cartilage (9) and tendon formation (10). Even
though BMPs can utilize the same type II receptor as Activin
and Nodal, their signaling is different, being propagated
through BMP-specific type I receptors and activation of Smad
1/5/8 proteins. Nonetheless, studies have shown the existence
of cross-talk between the Activin and BMP signaling pathways
during chondrogenesis (11, 12).
The comparative biochemical and biophysical studies of
Nodal versus Activin signaling have been hampered by the
6
The abbreviations used are: BMP, bone morphogenetic protein; RASCH, random assembly of segmental chimera and heteromers; hASC, human adipose-derived stem cells; Col, collagen.
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Background: Nodal is a TGF-␤ superfamily member that plays important roles in development and diseases but is difficult
to produce for biochemical studies.
Results: Nodal promotes chondrogenesis, and Nodal/bone morphogenetic protein 2 chimeras function indistinguishably from
Nodal but have BMP2-like structure.
Conclusion: Nodal/BMP2 chimeras can substitute for Nodal in functional and structural studies.
Significance: Nodal/BMP2 chimeras have therapeutic potential.
Nodal/BMP2 Chimeras
inability to produce these proteins in quantities suitable for
structural and functional studies. A chemical refolding protocol
that has been successful for many ligands (13) has failed for
both Nodal and Activin. A strategy known as random assembly
of segmental chimera and heteromers (RASCH) has been
devised to combine sections of Activin and BMP2 to produce
Activin/BMP2 chimeras that we termed the AB2 library (14).
From the AB2 library, we found a chimera, AB208, which
achieved the refolding efficiency of BMP2 while exhibiting the
signaling properties of Activin (14). Additionally, the RASCH
strategy has yielded other chimeras such as AB204 and AB215
with enhanced SMAD 1/5/8 signaling capabilities compared
with wild type BMP2. Thus, this strategy enabled us not only to
produce properly refolded chimeras with the properties of their
parental ligands (e.g., AB208), but also to produce chimeras that
exhibit novel signaling characteristics.
In this study, we apply the RASCH strategy to combine Nodal
and BMP2 and create Nodal/BMP2 chimeras (NB2 library). We
identify three chimeras (NB250, NB260, and NB264) with the
refolding capabilities of BMP2 and the Cripto-dependent signaling properties of Nodal. Additionally, we find that NB250,
like Nodal, alters heart looping during the establishment of
embryonic left-right asymmetry. Interestingly, we observe that
NB250 and NB260 strongly induce chondrogenic differentiation of human adipose-derived stem cells (hASCs) in a manner
similar to Nodal and more efficiently than BMP2 or Activin A.
Finally, we solve the crystal structure of NB250 and demonstrate that its fundamental scaffold remains identical to that of
BMP2 except for small local conformational changes in the
receptor-binding regions. This suggests that Nodal itself likely
adopts a BMP2-like fold. Together, our results show that these
Nodal-like chimeric ligands will be useful for additional structural studies and may have therapeutic implications for the
treatment of cartilage injuries and other disorders.
EXPERIMENTAL PROCEDURES
NB2 Chimera Expression and Purification—To create the
NB2 chimera library, the mature domains of human Nodal and
human BMP2 were divided into six segments. Twelve Nodal/
JANUARY 17, 2014 • VOLUME 289 • NUMBER 3
BMP2 primers corresponding to each segment (Integrated
DNA Technologies, Coralville, IA) were combined by an overlapping PCR strategy to produce a full-length PCR fragment of
each chimera. To create overlapping regions for PCR, we substituted residues Ala77 and Leu95 in Nodal to valine. The constructs were cloned into pET21A vector (EMD Biosciences,
Darmstadt, Germany) and propagated in Nova Blue cells (EMD
Biosciences). Final expression constructs were confirmed by
DNA sequencing.
All NB2 chimeras were expressed in Escherichia coli as inclusion bodies and isolated, purified, and refolded using a modified
protocol (13). The refolded ligands were initially purified using
Hi-trap heparin sulfate (GE Healthcare) and a sodium chloride
gradient. Fractions containing the refolded ligand were submitted to a second round of purification using a C4 reverse phase
column (GraceVydac). The protein was eluted using an acetonitrile gradient. Nodal was purchased from R&D Systems.
NB250 Crystallization—Lyophilized NB250 was resuspended
in water at 10 mg/ml, and crystallization trials with several commercial screening kits, Crystal Screen, Crystal Screen 2 (Hampton Research), Wizard Screens I and II (Emerald BioStructures), PEG/ION, and Nextal Classics suite (Qiagen) were
conducted using the Mosquito crystallization robot (TTP
Labtech, Cambridge, MA). The trials yielded several crystallization hits, which were subsequently optimized. The final crystal was obtained using the hanging-drop vapor diffusion
method, in which 1 ␮l of 10 mg/ml protein was mixed with 1 ␮l
of a reservoir solution of 0.2 M NaCl, 0.1 M sodium acetate, pH
4.6, 30% 2-methyl-2,4-pentanediol. Crystals appeared after 3
weeks at 15 °C and were frozen in liquid N2 using a cryoprotectant comprised of the original reservoir solution supplemented with 15% (v/v) glycerol. Diffraction data were collected
at the Stanford Synchrotron Radiation Laboratory using Beamline 11-1 at the resolution of 1.9 Å. The space group of NB250
was found to be R32:H with the following cell dimensions: a,
b ⫽ 95.57; c ⫽ 97.49; ␣, ␤ ⫽ 90; ␥ ⫽ 120. Data were processed
using XDS software (15), and the structure was solved using
molecular replacement with BMP2 as a model. Model building
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FIGURE 1. NB2 library chimera design strategy. BMP2 and Nodal sequence alignment is shown. Segments are color-coded and were defined by taking into
account conservation of structural motifs.
Nodal/BMP2 Chimeras
1790 JOURNAL OF BIOLOGICAL CHEMISTRY
TABLE 1
List of NB2 chimeras
Chimeras not successfully refolded are denoted with a minus sign. Dimer yield was
divided into three categories based on refolding efficiency (RE): lower than 50 ␮g
(⫹), 50 –100 ␮g (⫹⫹), and higher than 100 ␮g (⫹⫹⫹). Bolded rows indicate successful refolding.
Name
Sequence
RE
NB201 (Nodal)
NB203
NB205
NB206
NB207
NB208
NB209
NB210
NB211
NB212
NB213
NB214
NB215
NB216
NB217
NB218
NB219
NB220
NB221
NB222
NB223
NB224
NB225
NB226
NB227
NB228
NB229
NB230
NB231
NB232
NB233
NB234
NB202 (BMP2)
NB204
NB235
NB236
NB237
NB238
NB239
NB240
NB241
NB242
NB243
NB244
NB245
NB246
NB247
NB248
NB249
NB250
NB251
NB252
NB253
NB254
NB255
NB256
NB257
NB258
NB259
NB260
NB261
NB262
NB263
NB264
NNNNNN
NBNBNB
NNNNNB
NNNNBB
NNNBBB
NNBBBB
NBBBBB
NBNBBB
NBBNBN
NBNNBB
NNNNBN
NNNBNN
NNBNNN
NBNNNN
NBBNNN
NBNBNN
NBNNBN
NBNNNB
NNBBNN
NNBNBN
NNBNNB
NNNBBN
NNNBNB
NBBBNN
NBNBBN
NNBBBN
NBBNNB
NNBBNB
NNBNBB
NBBBBN
NBBBNB
NBBNBB
BBBBBB
BNBNBN
BBBBBN
BBBBNB
BBBNBB
BBNBBB
BNBBBB
BNNBBB
BNBNBB
BNBBNB
BNBBBN
BBNNBB
BBNBNB
BBNBBN
BBBNNB
BBBNBN
BBBBNN
BNNNBB
BNNBNB
BNBNNB
BBNNNB
BNNBBN
BBNNBN
BNBBNN
BBNBNN
BBBNNN
BNNNNB
BNNNBN
BNNBNN
BNBNNN
BBNNNN
BNNNNN
⫺
⫺
⫺
⫺
⫺
⫺
ⴙⴙ
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
ⴙ
⫺
⫺
⫺
⫹⫹⫹
ⴙ
ⴙⴙ
ⴙ
ⴙⴙⴙ
ⴙ
ⴙ
ⴙ
⫺
⫺
ⴙⴙ
ⴙⴙ
ⴙⴙⴙ
ⴙⴙ
ⴙ
ⴙⴙ
⫺
ⴙ
⫺
⫺
ⴙ
ⴙ
ⴙⴙⴙ
⫺
⫺
⫺
⫺
ⴙ
⫺
⫺
ⴙ
ⴙ
5⬘-AGGATGGCTTCCACCAGTGC-3⬘ and 5⬘-TGCGTAAAAGACCTCACCCTCC-3⬘ for Aggrecan (ACN); 5⬘-GAGACAGCATGACGCCGAG-3⬘ and 5⬘-GCGGATGCTCTCAATCTGGT-3⬘ for type II Collagen (COL2A1); and 5⬘-ACTCCGAGACGTGGACATC-3⬘ and 5⬘-TGTAGGTGACCTGGCCGTG-3⬘ for Sox9 (SOX9). The levels of expression of the
respective genes were normalized to corresponding GAPDH
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was done in COOT version 0.6.2 (16). Refinement of the structure was completed using PHENIX (17) to R and Rfree of 0.175
and 0.203, respectively. The final data processing and refinement statistics are listed in Table 2. The root mean square deviations in bond lengths and bond angles are 0.006 Å and 1.14°,
respectively. All structure figures were generated using PyMOL
(version 1.3; Schrödinger) and Molscript version 2.1.2 (19).
Luciferase, Phospho-SMAD2 Assays, and ASCs Cell Culture—
Luciferase and phospho-Smad2 assays were performed as
described previously (8). Human mesenchymal stem cells
were derived from the adipose tissue of the subcutaneous
abdomen of a 37-year-old Caucasian female (lot number
9061601.12; PromoCell, Heidelberg, Germany). Cells were
cultured in growing medium (high glucose DMEM (Invitrogen) with 10% fetal bovine serum and 1% penicillin/streptomycin (Invitrogen)).
Induction of Monolayer Expanded Cells toward Chondrocytes—
Human ASCs were induced to chondrogenic phenotype as
described previously (20). Briefly, cells were seeded at 30 – 40%
of confluence in 12-well plates. Control cells were grown in
incomplete chondrogenic medium (DMEM high glucose
(Invitrogen) supplemented with 10% fetal bovine serum and 1%
ITS ⫹ Premix (Collaborative Biomedical-Becton Dickinson,
Bedford, MA), plus 1% penicillin-streptomycin (Invitrogen)).
In addition, 50 mg/ml of l-ascorbic acid 2-phosphate (SigmaAldrich) was freshly added at each media exchange. To direct
chondrogenic differentiation, cells were cultured in chondrogenic medium (incomplete chondrogenic medium containing
10 ng/ml of the Nodal ligand). The medium was changed every
other day. The cells were cultured in chondrogenic media for 2,
4, and 6 weeks, depending on the experiment. Complete chondrogenic medium with 10 ng/ml of BMP2, known to be capable
of chondrogenesis, was used as positive control. To compare
the potential of Nodal with the Nodal-like chimeras, recombinant human Nodal (R&D Systems, Minneapolis, MN) or
Nodal-like ligand was added to the incomplete chondrogenic
medium at 10 ng/ml.
Chondrogenic Differentiation in Cell Pellet Culture—Cells
were grown on 12-well plates in chondrogenic medium. Upon
reaching confluence, the cell monolayer detached itself from
the plate and took the form of a crumpled ball. Control cells
grown in incomplete chondrogenic medium did not detach
spontaneously from the plastic, and therefore, the monolayer
was manually separated using a sterile tip. The pellets were
transferred to 15-ml conical tubes and incubated with loosened
caps at 37 °C and 5% CO2. The medium was exchanged every
other day for the duration of the experiment, and the tubes were
gently agitated to avoid the adherence of the pellet to the tube
walls.
RNA Isolation and Real Time PCR Analysis—Total cellular
RNA was isolated using TRIzol reagent (Invitrogen) according
to the manufacturer’s recommendations. 2 ␮g of DNase 1
(Invitrogen)-treated total RNA was used for cDNA synthesis
using the SuperScript II reverse transcriptase kit for RT-PCR
(Invitrogen). Real time PCR was performed using the SYBRGreen PCR Master mix (Applied Biosystems). Sequences of
primers were 5⬘-GGACTCATGACCACAGTCCATGCC-3⬘
and 5⬘-TCAGGGATGACCTTGCCCACAG-3⬘ for GAPDH;
Nodal/BMP2 Chimeras
values and are shown as fold change relative to the value of the
control sample. All the samples were done in triplicate.
Cell Monolayer and Cell Pellet Processing—For histological
and immunocytochemistry analysis, culture pellets were fixed
with 4% paraformaldehyde for 20 min at room temperature and
embedded in 2.3 M sucrose for 1 h. Cell pellets were embedded
in tissue freezing medium, Blue (Electron Microscopy Sciences)
and frozen on dry ice. Sections of 2– 6 ␮m in thickness were cut
with a microtome and placed in the center of a coated slide.
Sections were washed with PBS in a humid chamber, until
excess sucrose was washed away.
Toluidine Blue Staining—Briefly, 0.1 g of toluidine blue
(Sigma) was dissolved in 100 ml of distilled H2O. Pellet sections
were stained in toluidine blue solution for 1–5 min at room
temperature and rinsed with distilled H2O until excess stain
was washed away.
In Vivo Assays—Chick embryos were explanted and grown in
vitro as described (21). RCAS-Nodal retroviral stocks were produced as described (21) and used to infect, by air pressure, the
right side of Hensen’s node in developing chick embryos. Beads
were soaked in Nodal-like NB250 compound and similarly
applied locally on the right side of Hensen’s node. Embryos
were processed for whole mount Pitx2 in situ hybridization as
JANUARY 17, 2014 • VOLUME 289 • NUMBER 3
described previously (21) or fixed in paraformaldehyde for
heart looping visualization.
Fluorescence Microscopy—Briefly, after fixation, pellet and
sections were blocked and permeabilized for 1 h at 37 °C with
5% BSA, 5% appropriate serum, 1⫻ PBS with 0.1% Triton
X-100. Subsequently, cells and sections were incubated with
the indicated primary antibody overnight at 4 °C. Pellet sections
were then washed thrice with 1⫻ PBS and incubated for 2 h at
37 °C with the respective secondary antibody. Pellet sections
were then washed thrice with 1⫻ PBS; DAPI (0.5 ␮g/ml in PBS)
was added to the last wash. These sections were mounted with
aqueous mounting medium before analysis.
The primary antibodies used were anti-type I Collagen antibody (rabbit polyclonal antibody (ab292); Abcam, Cambridge,
MA), anti-type II Collagen antibody (mouse monoclonal antibody (ab3092); Abcam), and anti-Sox9 antibody (rabbit polyclonal antibody (AB5535); Chemicon). Alexa Fluor 568 or 488
(Neomarkers) were used as secondary antibodies. The localizations of the proteins were observed with a Leica TCS SP2 AOBS
confocal or Nikon E-800 microscope.
Surface Plasmon Resonance (BIAcore) Affinity Studies—The
affinity of NB250 to ActRII-ECD was measured using a Biacore
3000 (GE Healthcare). Using primary amine coupling, the
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FIGURE 2. NB2 chimeras have signaling properties indistinguishable from those of Nodal. A and B, Nodal, NB250, NB260, and NB264 are Cripto-dependent
and induce Smad2 signaling as measured by Smad2-dependent luciferase expression (A) and Western blotting using a phospho-Smad2 antibody (B). C, Nodal
and NB250 exhibit a similar dose dependence of Smad2 activation. 293T cells transfected with Cripto or control vector were treated with varying concentrations of R&D Nodal or NB250, and the resulting luciferase activity was measured. D, NB250 signaling resembles Nodal but not BMP2. 293T cells were transfected
with Smad2-responsive (A3-luc) or Smad1/5/8-responsive (BRE-luc) luciferase plasmids and either Cripto or empty vector. Wells were treated in triplicate with
Nodal, NB250, or BMP2, and the resulting luciferase induction was measured. IB, immunoblot.
Nodal/BMP2 Chimeras
receptor ECD was immobilized on a CM5 chip independently
using flow cell 2. No protein was immobilized on flow cell 1 as a
negative control. For kinetic analysis, all tests were performed
in duplicate using a minimum of five concentrations, plus a zero
concentration. Binding data were analyzed with BIAevaluation
software ver. 4.1 (GE Healthcare) and fit using a global 1:1 Langmuir binding with mass transfer model.
RESULTS
NB2 Chimera Production—NB2 chimeras were designed
using the RASCH strategy (14). We exploited the sequence similarity between BMP2 and Nodal to enable proper chemical
refolding under in vitro conditions to develop NB2 chimeras.
Nodal and BMP2 were each divided into six segments based on
the alignment of their sequences, thereby minimizing disruption of secondary structural motifs (Fig. 1). These segments
were recombined using the overlapping PCR method to produce 26 ⫽ 64 constructs and were given an identification number, NB2XX, where N (Nodal) and B2 (BMP2) are followed by
the serial numbers ranging from 1 to 64 for XX. The collection
of these 64 chimeras is referred to as the NB2 library. NB2
chimeras can also be identified using a code comprised of a
six-letter sequence of N and B that indicates their segmental
1792 JOURNAL OF BIOLOGICAL CHEMISTRY
makeup. For instance, NB250 can be referred to as BNNNBB,
because segments 1, 5, and 6 are derived from BMP2, and segments 2, 3, and 4 are derived from Nodal.
In short, these 64 NB2 library chimeras were purified from
E. coli inclusion bodies, denatured, chemically refolded, and
purified using affinity chromatography and reverse phase chromatography. Their production efficiency is reported in Table 1.
Interestingly, chimeras beginning with segment 1 derived from
Nodal did not produce a stable dimer during refolding, with the
exception of two ligands that refolded correctly with noticeable
efficiency. On the other hand, 21 of 32 chimeras with BMP2
segment 1 can be refolded and purified. As a result, we
obtained a total of 23 properly refolded NB2 ligands of 64
possible. Based on these results, it appears that BMP2 segment 1 significantly improves proper chemical refolding of
the NB2 chimera sequences.
NB250, NB260, and NB264 Signal Like Nodal in a Cripto-dependent Manner—After screening several chimeras, we found
that NB250 (BNNNBB), NB260 (BNNNBN), and NB264
(BNNNNN) have Cripto-dependent activation of a Smad2-responsive luciferase reporter (Fig. 2A). These three chimeras
were also similar to Nodal in their ability to induce Criptodependent Smad2 phosphorylation (Fig. 2B). Additionally,
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FIGURE 3. NB250 mimics Nodal effects on Pitx2 expression and heart looping in developing chick embryos. A, Pitx2 transcripts are detected along the
entire left side of the lateral plate mesoderm (arrow). B, ectopic expression of Nodal on the right side of chick embryos inducing Pitx2 transcripts on the right
side of the lateral plate mesoderm (red arrow). C, a bead soaked with NB250 induces ectopic Pitx2 transcripts along the right lateral plate mesoderm (red arrow).
D, wild type chick embryos develop with a rightward heart looping (arrow). E and F, both Nodal (E) and NB250 (F) reverse heart looping in a leftward orientation
(arrows).
Nodal/BMP2 Chimeras
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FIGURE 4. Nodal, NB250, and NB260 induce chondrogenic differentiation
of human adipose-derived stem cells. The results shown are from real time
PCR analysis of selected chondrogenic markers (Collagen II, Sox9, and Aggrecan) after the cells were cultured for 14 days in complete chondrogenic
medium containing different ligands. Gene expression is shown as fold
change compared with cells cultured in incomplete chondrogenic medium
(CTL). The data are shown as relative averages ⫾ S.D. of two independent
experiments analyzed in triplicate (n ⫽ 6).
denser extracellular matrix. On the other hand, the control cells
did not display tissue-like organization and rather present the
appearance of aggregated cells with no clear organization.
Additionally, the degree of maturation after chondrogenic differentiation was evaluated by immunohistochemical techniques using cells derived from a pellet culture system. Immunostaining of Col II and Col I in cell pellet sections showed a
substantial number of Col II-positive cells, and a dense filamentous network of Col II connecting the ligand-treated cells (Fig.
5C). Interestingly, we did not detect co-localization of Collagen
I and II, as Col I-positive cells were located surrounding Col
II-positive cells, indicating the resemblance of a stratified structure similar to that found in cartilage tissue (Fig. 5E). Expression of the chondrogenic transcription factor Sox9 was highly
enriched in ligand-treated cells when compared with control
cells (Fig. 5F). The detailed picture of Sox 9 nuclear localization
is created by a Z-stack of the NB260-treated sections and shown
in the upper left corner of the far right panel of Fig. 5F. Altogether, these data demonstrate that NB250 and NB260 can
cause hASCs to acquire a mature chondrocyte-like phenotype
in a more efficient manner than BMP2.
The Structure of NB250 Closely Resembles That of BMP2—
We determined the crystal structure of NB250 at 1.9 Å resolution by the molecular replacement method using the known
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NB250, the most divergent from Nodal of these three chimeras,
shows a dose dependence similar to that of Nodal. Our results
indicate that Cripto-dependent activation of Smad2 by NB250
closely follows that of Nodal in the concentration range of
0.01–30 nM (Fig. 2C). Moreover, like Nodal, NB250 does not
activate a BMP-specific luciferase reporter that is activated by
BMP2 (Fig. 2D).
NB250 Alters Heart Looping in a Manner Very Similar to
That of Nodal—We compared the activity of NB250 and Nodal
in vivo and found that both ligands induce ectopic expression of
Pitx2 and alter heart looping during the establishment of vertebrate embryonic left-right asymmetry in a very similar manner. During normal development, Pitx2 transcripts are detected
asymmetrically along the entire left side of the lateral plate mesoderm in developing chick embryos (Fig. 3A). We discovered
that NB250 induces ectopic expression of Pitx2 and alters heart
looping during the establishment of vertebrate embryonic leftright asymmetry. This activity is very similar to that of Nodal.
Ectopic expression of Nodal on the right side of chick embryos
leads to induction of Pitx2 transcripts on the right side of the
lateral plate mesoderm (Fig. 3B, red arrow). Similarly, application of a bead soaked in NB250 induces ectopic Pitx2 transcripts along the right lateral plate mesoderm (Fig. 3C, red
arrow). The wild type chick embryo develops with a rightward
heart looping (Fig. 3D). However, Nodal and the NB250 chimera are each able to reverse heart looping in a similar leftward
orientation (Fig. 3, E and F), indicating that the in vivo action by
NB250 mimics that of Nodal.
NB250 and NB260 Induce Chondrogenic Differentiation of
Human Adipose-derived Stem Cells—BMP2 (22) and Activin
(23) have each been observed to play a role in promoting chondrogenesis, whereas a role for Nodal in this process has not yet
been established. However, because Nodal shares the same signaling pathway with Activin, we inferred that it might also
induce chondrogenesis. We treated monolayers of expanded
hASCs with 10 ng/ml of Nodal, NB250, NB260, or BMP2 for 2
weeks and compared the ability of each of these ligands to
induce expression of chondrogenic genes using real time PCR.
The results show that each of these ligands induced the expression of Col II, Sox9, and Aggrecan in a very similar manner
(Fig. 4).
To further evaluate the chondrogenic potential of the chimeras, we used a cell pellet culture model to mimic the cellular
condensation process occurring in normal limb development
(24). Representative images of two independent experiments,
using duplicates for each, are shown in Fig. 5. After 6 weeks of
culture, pellets supplemented with BMP2, NB250, or NB260
achieved a cartilage-like appearance with a white shiny look
resembling cartilage tissue and showed a marked increase in
size compared with cell pellets grown in incomplete chondrogenic medium (control) (Fig. 5A).
We explored the formation of cartilage-like tissue by staining
sections of the pellets with toluidine blue. Active matrix production and cartilage-like tissue formation was confirmed in
BMP2, NB250, and NB260 supplemented pellets (Fig. 5B). Purple-dark blue metachromasia is even more apparent in NB2
chimera-treated pellet than in BMP2-treated pellet, suggesting
higher deposition of glycosaminoglycans and formation of
Nodal/BMP2 Chimeras
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FIGURE 5. NB250 and NB260 induce the formation of cartilage-like tissue. A, hASCs cultured in a pellet system treated with Nodal-like ligands (NB250 and
NB260) or BMP2 or grown in incomplete chondrogenic medium as negative control (CTL). Scale bar, 1 mm. B, toluidine blue stain of sections of hASC pellets
grown in incomplete chondrogenic medium or supplemented with BMP2, NB250, or NB260. Scale bar, 350 ␮m. C–E, immunochemistry assays of monolayer
cells treated with BMP2, NB50, or NB260 for 4 weeks. Type II collagen is stained green, type I collagen is stained red, and DAPI is stained blue. Scale bar, 65 ␮m.
F, expression of chondrogenic transcription factor Sox9 in culture pellet sections treated with BMP2, NB250, or NB260. Sox9 is shown in green, and cell nuclei
are in blue. Ortho view of Z-stack, a representative cross-sectional image of x-y (box a), y-z (box b), and x-z (box c) coordinates is shown in the far right panel. Scale
bar, 4 ␮m. The data are representative of two independent experiments performed in duplicate.
BMP2 structure. Processing and refinement statistics are presented in Table 2. Similar to BMP2, the NB250 monomer has
seven cysteine residues. These residues allow the monomer to
form three intradisulfide bonds (Cys14/Cys80, Cys43/Cys112,
and Cys47/Cys114) and one interdisulfide bond, which is formed
between the Cys79 of each monomer. These disulfide bonds,
along with ␣ helix ␣1 formed by the region between residues
Asn59 and Tyr70, comprise the core of the protein. The overall
architecture of the dimer is dictated by these disulfide bonds
1794 JOURNAL OF BIOLOGICAL CHEMISTRY
(cysteine knot motif) coupled with four anti-parallel ␤-strands
extending outward from the core, giving it the appearance of
the spread wing shape of a butterfly, similar to that of BMP2
(Fig. 6A). The pre-helix loop, helix ␣1, and ␤ sheet ␤2 consist of
segments derived from Nodal and are depicted in yellow in Fig.
6A, whereas ␤ sheets ␤1, ␤3, and ␤4 are BMP2 segments and are
depicted in purple. When the structures are superimposed
based on their shared sections (i.e., 1B, 5B, and 6B, which are
NB250 segments derived from BMP2), the C␣ root mean
VOLUME 289 • NUMBER 3 • JANUARY 17, 2014
Nodal/BMP2 Chimeras
TABLE 2
Data collection and refinement statistics for NB250
Statistics for the highest resolution shell are shown in parentheses.
Wavelength (Å)
0.98
31.55–1.912 (1.98–1.912)
R 3 2:H
95.57, 95.57, 97.494, 90, 90, 120
128,925
13,316 (1219)
9.7 (7.4)
99.12 (91.31)
41.36 (4.28)
31.08
0.033 (0.342)
0.1754 (0.1965)
0.2039 (0.1894)
Number of atoms
Macromolecules
Water
924
852
72
Root mean square deviation (bonds)
Root mean square deviation (angles)
Ramachandran favored (%)
Ramachandran outliers (%)
Clashscore
0.006
1.14
98
0
10.20
Average B-factor
Macromolecules
Solvent
43.20
42.60
49.80
square deviation is 0.540 Å. As expected, the regions of highest
structural discrepancy lie in the 2N3N4N region of NB250.
We superimposed NB250 onto BMP2 of the BMP2BMPRIa-ActRII ternary complex to identify residues responsible for potential differences between BMP2 and NB250 in type
II receptor and type I receptor binding. Previous studies have
identified Ala34, Pro35, Ser88, Met89, and Leu90 in BMP2 as
being implicated in the formation of the hydrophobic interface
required for ActRII binding via its hydrophobic residues Phe83,
Phe42, and Trp60 (25). NB250 shares these residues with BMP2
except for Ala34, which is substituted by Tyr34 in NB250. After
aligning the NB250 structure with BMP2 of the BMP2BMPRIa-ActRII ternary complex (Fig. 6B), it can be seen that
the aromatic side chain in tyrosine can form pi-stacking interactions with Phe83 of ActRII providing a basis for enhanced
NB250 affinity for its type II receptor.
To test this possibility directly, we used surface plasmon resonance (Biacore) to measure the affinity of NB250 to ActRIIECD immobilized on a CM5 chip. The kinetics of the binding
described with sensogram curves fitted with ␹2 of 2.4 produced
kon and koff values of 2.5 ⫻ 104 (1/M*s) and 3.8 ⫻ 10⫺4 (1/s),
respectively, and a KD ⫽ 15 nM. The analogous data measured
previously for BMP2 gave kon and koff values of 1.1 ⫻ 106 (1/M*s)
and 4.1 ⫻ 10⫺2 (1/s), respectively, and a KD of 37 nM (14).
When the structure of NB250 is aligned to the BMP2 of the
BMP2-BMPRIa-ActRII complex, four residues that have been
found to play a major role in the formation of the two BMP2
hydrophobic pockets required for type I receptor binding (25)
are different: Val26 is an Ile in BMP2, Phe49 is an Asn, and both
Ile62 and Val70 have been substituted for Tyr. Of these substitutions, the most disruptive to BMPRIa binding appears to be
F49N, because Phe49 inserts tightly to the hydrophobic pocket
formed by Ile62, Ile99, and Phe60 in BMPRIa (Fig. 6C), and the
change of a hydrophobic residue to a polar residue most likely
disrupts this binding.
JANUARY 17, 2014 • VOLUME 289 • NUMBER 3
Our previous studies of chemical refolding of Activin/BMP2
chimeras (14) revealed that the first BMP2 segment increases
the probability of proper chemical refolding and dimer formation. This study reasserts the finding, because it shows that the
first BMP2 segment also facilitates proper refolding of NB2
chimeras. It is noteworthy that the first segment of BMP2 has
four fewer basic residues than the first segment of Nodal, and
this difference may allow this BMP2 segment to promote chimera refolding.
Our functional studies in vitro show that the NB2 chimeras
NB250, NB260, and NB264 signal like Nodal in a Cripto-dependent manner. It appears that the chimeras may require segments 2, 3, and 4 of Nodal to achieve Cripto-dependent receptor activation and Smad2 phosphorylation. Therefore, we infer
that these segments, which include residues structurally crucial
to the putative type I receptor binding epitope, may play a pivotal role in proper Cripto binding and Cripto-dependent signaling. Indeed, previous modeling studies suggest that sections
2N, 3N, and 4N are relevant for Cripto dependence because
they encompass structural elements proposed to play a role in
Nodal binding to ALK4 in the presence of Cripto (26). Our
activity assays are consistent with this idea. We propose that to
retain Nodal functionality the minimal required Nodal segmental sequence is 2N3N4N, with NB250 (BNNNBB) being a
“minimalistic” chimera to carry out Nodal functionality.
Nodal and NB2 ligands display strikingly similar effects in
complex bioassays. We demonstrate that NB250 alters heart
looping during the establishment of vertebrate embryonic leftright asymmetry in a manner indistinguishable from that of
Nodal. Further, we show for the first time the chondrogenic
effect of Nodal and that it is mimicked by the NB2 ligands.
Previous studies, as well as our own assays, have shown that
Activin/Nodal/TGF-␤ signaling plays a role in chondrogenesis
(23). The Activin/BMP2 chimeric ligand AB235 was shown to
direct adipose-derived stem cells to chondrogenic differentiation and exhibit a pattern of enhanced gene marker expression
similar to that observed for Nodal, BMP2, and the NB250 and
NB260 chimeras (27). NB250 and NB260 also induce chondrogenesis of human adipose-derived stem cells with an efficacy
that appears to exceed that of BMP2 and AB235. Histological
and immunohistochemical studies further demonstrate that
NB260 and NB250 induce the growth of cartilage tissue and a
dense filamentous network that resembles the stratified structure found in cartilaginous tissue.
Intriguingly, the overall structure of NB250 is very similar to
that of BMP2. This is surprising but may shed some light on its
functional properties. Combining this with the fact that NB250
appears to be indistinguishable from Nodal in its mode of signaling and effects on left-right heart looping in chick embryos
and chondrogenesis, it can be concluded that Nodal itself
adopts a BMP2-like fold. Thus, we predict that NB250 will likely
be a structural as well as a functional mimic of Nodal. Importantly, its rigidity should facilitate structural studies of relevant
complexes including the NB250-Cripto-ActRII-Alk4 complex.
The elucidation of this complex, together with the ActivinActRII-Alk4 complex, will be instrumental to understanding
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Resolution range (Å)
Space group
Unit cell
Total reflections
Unique reflections
Multiplicity
Completeness (%)
Mean I/␴I
Wilson B-factor
Rsym
R-factor
Rfree
DISCUSSION
Nodal/BMP2 Chimeras
how Cripto exerts opposing effects on Nodal and Activin
signaling (8).
Our studies show that NB250 is functionally indistinguishable from Nodal. However, its sequence is approximately onehalf Nodal and one-half BMP2. Interestingly, NB250 does not
signal through the canonical BMP pathway, i.e., via Smads 1, 5,
and 8. Nodal and BMP2 share the same type II receptor, ActRII,
and the type II receptor binding epitope of NB250 largely
resembles that of BMP2. Nodal receptor assembly is critically
regulated by Cripto, which binds to Nodal via its EGF-like
domain and to the type I receptor ALK4 via its CFC domain
(18). It is perhaps not surprising, therefore, that replacing the
1796 JOURNAL OF BIOLOGICAL CHEMISTRY
type II receptor binding epitope of Nodal with that of BMP2 in
NB250 does not disrupt Nodal function because the Cripto
binding site is preserved.
The affinity of NB250/ActRII is two times higher than that of
BMP2/ActRII, which may be explained by a single residue
change in the NB250 ActRII binding epitope that could slightly
enhance the affinity of this chimera for its type II receptor. We
determined that an Ala to Tyr change in the NB250 type II
binding epitope may establish a novel interaction with the
ActRII residues that bind to this pocket.
On the other hand, failure to observe BMP2-like signaling
through Smad1/5/8 suggests that NB250 has a substantially
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FIGURE 6. Structure of NB250 closely resembles BMP2. A, superposition of NB250 and BMP2. BMP2 is depicted with a thin cyan line. NB250 is depicted with
a thicker line with its BMP2 segments in blue, and its Nodal segments in yellow. The N and C termini are indicated. B, key residues predicted to be responsible for
the increased affinity to type II receptor in NB250. Top panel, front view of the BMP2 (cyan)-ActRII (red) complex. The key region of the BMP2/ActRII interface is
boxed. Bottom left panel, close-up of this region showing the original BMP2 Ala34 residue (cyan) and ActRII Phe83, Phe42, and Trp60 residues (red). Bottom right
panel, same region, with BMP2 replaced by NB250 (purple) showing the substitution of the noninteracting BMP2 Ala34 residue with the putative interacting
Tyr34 residue from NB250. C, key residues responsible for the decreased affinity to type I receptor in NB250. Top panel, front view of the BMP2 (cyan)-BMPRIa
(red) complex. The key region of the BMP2/BMPRIa interface is boxed. Bottom left panel, close-up of this region showing the original BMP2 Phe49 residue (cyan)
and BMPRIa Ile62, Ile99, and Phe60 residues (red). Bottom right panel, same region, with BMP2 replaced by NB250 (purple) showing the substitution of the BMP2
Phe49 residue with the Asn49 residue from NB250.
Nodal/BMP2 Chimeras
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decreased affinity for the canonical BMP2 type I receptor,
BMPRIa. This may be caused by disruptive residue changes that
are the product of the chimeric makeup of NB250, the most
significant being a Phe to Asn substitution in the NB250 type I
receptor binding pocket. Indeed, a change from a hydrophobic
to a polar residue in this region would greatly reduce, if not
abolish, the hydrophobic interactions required for BMPRIa to
bind to this ligand.
The NB2 chimeras employed in this work are easily produced
and will accelerate the development of cost-effective protocols
for structural studies and for biological purposes such as deriving chondrocytes in vitro. We have shown that NB250 is a suitable substitute for Nodal and that it is able to mimic Nodal’s
heart looping properties as well as Nodal’s newly found chondrogenic property. Furthermore, the chondrocytes obtained by
the library chimeras may have important implications for cell
replacement therapies in cartilage repair with valuable clinical
applications.