© 2001 Nature Publishing Group http://structbio.nature.com
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© 2001 Nature Publishing Group http://structbio.nature.com
The L3 loop and C-terminal
phosphorylation jointly
define Smad protein
trimerization
Benoy M. Chacko1, Bin Qin1, John J. Correia2,
Suvana S. Lam1, Mark P. de Caestecker3 and Kai Lin1
1Department of Biochemistry and Molecular Pharmacology, University of
Massachusetts Medical School, 55 Lake Ave. North, Worcester, Massachusetts
01655, USA. 2Department of Biochemistry, University of Mississippi Medical
Center, 2500 North State Street, Jackson, Mississippi 39216, USA.
3Department of Nephrology, Vanderbilt University Medical Center,
Nashville, Tennessee 37232, USA.
Smad proteins mediate the transforming growth factor β
responses. C-terminal phosphorylation of R-Smads leads to
the recruitment of Smad4 and the formation of active signaling complexes. We investigated the mechanism of phos-
phorylation-induced Smad complex formation with an activating pseudo-phosphorylated Smad3. Pseudo-phosphorylated Smad3 has a greater propensity to homotrimerize, and
recruits Smad4 to form a heterotrimer containing two
Smad3 and one Smad4. The trimeric interaction is mediated
through conserved interfaces to which tumorigenic mutations map. Furthermore, a conserved Arg residue within the
L3 loop, located near the C-terminal phosphorylation sites
of the neighboring subunit, is essential for trimerization.
We propose that the phosphorylated C-terminal residues
interact with the L3 loop of the neighboring subunit to stabilize the trimer interaction.
Members of the transforming growth factor β (TGF-β)
superfamily of ligands play important roles in diverse physiological and pathological processes1,2. Characterization of the
TGF-β signaling mechanism led to the discovery of a family of
signal transducers and transcription regulators named Smad
proteins3,4. Two classes of Smad protein are responsible for signal activation. The receptor-mediated Smad proteins
(R-Smads), which include Smad1, Smad2, Smad3, Smad5 and
Smad8, function in ligand specific pathways, and are
phosphorylated at the C-terminus upon transmembrane
a
b
c
d
Fig. 1 Oligomerization states of S4AF, S3LC and S3LC(3E). a, The S3LC and S4AF constructs and a domain comparison between Smad3 and Smad4. The
potential sites of Ser phosphorylation in Smad3 are marked with black dots11–13. b, Size exclusion chromatography elution profiles of S3LC and S4AF
at four different protein concentrations. Protein concentrations before loading on the column were 6, 17, 50 and 150 µM. The y-axis (mAU) plots
absorption at 280 nm. The calculated subunit molecular weights for S4AF and S3LC are 30,884 and 31,565, respectively. c, Smad3(3E) superactivates
Smad3/4 dependent transcriptional responses. NMuMg cells were transfected with SBE-lux along with full-length wild type (WT) Smad3 or the
Smad3(3E) mutant with or without full-length WT Smad4. The basal activity observed in the absence of Smad4 was due to the NMuMg cells expressing endogenous Smad2/3 and Smad4, as confirmed by western blot (de Caestecker, M.P., unpublished data). Similar levels of WT and mutant Smad3
protein expression were seen in parallel experiments in COS-1 cells (data not shown). d, Size exclusion chromatography elution profiles of S3LC and
S3LC(3E). The concentration of the samples at loading was 50 µM. The y-axis (mAU) plots absorption at 280 nm. The SDS-PAGE of the eluted fractions
stained with Commassie blue is shown.
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c
receptor kinase activation. The common Smad protein, Smad4,
serves as a signaling partner by forming hetero-oligomers with
phosphorylated R-Smads. The active heteromeric Smad complexes then enter the nucleus to modulate transcription3,4.
R-Smads and Smad4 share a common domain configuration
(Fig. 1a). The C-terminal MH2 domain is responsible for
oligomerization and transcriptional regulation, whereas the
N-terminal MH1 domain has DNA binding activity5–7. The
linker domain contains a Pro-rich acidic sequence, which in
conjunction with the MH2 domain contributes to the transcriptional regulatory activity8–10. A distinct feature of
R-Smads is the presence of the conserved C-terminal sequence,
SSXS, which is the substrate for receptor kinase-mediated
phosphorylation11–14.
The mechanism of phosphorylation-induced heterooligomer formation between Smad4 and R-Smads is unclear.
One hypothesis, based on the crystal structure of Smad4, suggests that the hetero-oligomer is hexameric, resulting from a
Smad4 trimer interacting with an R-Smad trimer6. Another
hypothesis, based on biochemical analysis of a cellular extract,
suggests that the hetero-oligomer is trimeric, consisting of a
mixture of Smad4 and R-Smad subunits15. Furthermore, the
role of the C-terminal phosphorylation of R-Smad in oligomerization is unknown. In this study, we investigated the molecular
form and mechanism of the phosphorylation-induced interaction between Smad3 and Smad4. Our results suggest a novel
mechanism that explains the activation of the Smad proteins by
the TGF-β superfamily of ligands.
Phosphorylation promotes Smad3 trimerization
To elucidate the molecular basis of the Smad3–Smad4 interaction, we studied their oligomerization states in solution using
purified proteins. Reports have indicated that the MH2 domain
of Smad proteins mediates both homomeric and heteromeric
interactions16. Since full-length Smad3 and Smad4 tended to
form irreversible aggregates, truncated proteins were used
nature structural biology • volume 8 number 3 • march 2001
b
Fig. 2 Ratio of Smad subunits within the heteromeric complex and the
basis of subunit association. a, S4AF interacts directly with S3LC(3E) to
form a heterotrimeric complex. Size exclusion chromatography of
S3LC(3E) and S4AF mixtures at different S4AF concentrations. The eluted
fractions were analyzed by SDS-PAGE and the gels were stained with
Coomassie blue. The ‘total’ relative mole ratio refers to the ratio of S4AF
to S3LC(3E) before loading on the size exclusion column. The ‘complex’
relative mole ratio refers to the ratio of S4AF to S3LC(3E) in fraction 15
(elution peak of the complex), as determined by analysis of Coomassiestained bands with the Fluor-S MultiImager and MultiAnalyst software
(Bio-Rad). The standard deviations were obtained through multiple measurements of background at different regions of the gel.
b, Crystal structure of S4AF showing the homotrimer in the asymmetric
unit and the sulfate binding sites9. The three subunits are colored green,
blue and red. The side chains involved in the trimer interface contacts are
shown in black. Corresponding residues in Smad3 are shown in parentheses. Asp 351 does not directly contact the neighboring subunit, but
forms part of a hydrogen bonding network with Arg 361 and Asp 537,
which directly link the subunits. The L3 loops are colored yellow. The
locations of the four distinct sulfate binding sites within the S4AF asymmetric trimer are boxed. Site 1 is located at the base of the elongated
TOWER structure, towards the outer edge of the trimer. Site 2 is located
at the base of the TOWER towards the trimer interface. Site 3 is located
in the L3 loop, a conserved structural element proposed to be important
for receptor kinase recognition14,21,25. These three sulfates are present in
the corresponding positions in all three subunits. Between subunit B and
C, an additional sulfate ion was located adjacent to site 2, forming a
unique tandem sulfate binding site, site bc. c, Mutation of trimer interface residues of S4AF abolishes heteromeric interaction between
S3LC(3E) and S4AF. S3LC(3E) and the trimer interface mutants of S4AF
were mixed at a 1:1 mole ratio and loaded on the size exclusion column.
The eluted fractions were analyzed by SDS-PAGE and the gels were
stained with Coomassie blue.
(Fig. 1a). An S3LC construct included the linker and the MH2
domain of Smad3 (residues 145–424). An S4AF construct
included the MH2 domain and part of the linker region of
Smad4 (residues 273–552), which had been previously defined
as the minimal transcriptionally active fragment8,9.
The oligomerization behavior of S3LC and S4AF was initially
analyzed using size exclusion chromatography. S3LC elutes as
an apparent monomer when loaded at a low concentration, but
self-associates into higher molecular weight species approaching a trimer when its concentration is increased (Fig. 1b). In
contrast, S4AF behaves as an apparent monomer even at the
highest concentration (150 µM). The association model was
confirmed by sedimentation. S4AF is best described as a noninteracting monomer while S3LC is best described as existing
in a monomer–trimer equilibrium with an association constant
K3 of 1.2–3.1 × 109 M-2. The details of the sedimentation analysis will be reported elsewhere33.
To investigate the structural basis of phospho-activation, the
C-terminal Ser phosphorylation sites of S3LC were mutated to
Glu residues to mimic the structural and electrostatic proper249
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c
d
b
Fig. 3 The sulfate binding site within the L3 loop is critical for Smad heteromeric interaction. a, The sulfate binding sites shown in detail. The
location of the sulfate ions in the context of the trimeric S4AF structure is
shown in Fig. 2b. b, Mutation of the sulfate binding site at the L3 loop
specifically reduces heteromeric interaction between S3LC(3E) and S4AF.
S3LC(3E) and the sulfate binding site mutants of S4AF were mixed at a
1:1 mole ratio and loaded on the size exclusion column. The eluted fractions were analyzed by SDS-PAGE and the gels were stained with
Coomassie blue. c, Stereo view of the Fo - Fc omit map at the L3 loop of
the S4AF(R515S) mutant. The mutant and wild type coordinates are
shown in black and gray, respectively. d, The Smad4 Arg 515 mutant only
weakly activates Smad3/4 dependent transcriptional responses. Smad4
null MDA-MB468 cells were transfected with SBE-Lux along with
the indicated FLAG-Smad3(3E) and Smad4-Myc mutant constructs.
e, Homotrimerization of S3LC(3E) is abolished by mutation of residues at
the trimer interface or the conserved Arg residue within the L3 loop.
Proteins (50 µM) were loaded onto the size-exclusion column and the
eluted fractions were analyzed by SDS-PAGE and the gels were stained
by Coomassie-Blue.
ties of phosphorylation. Two mutants were generated, one with
the last two Ser residues substituted (S3LC(2E)), and the other
with all three Ser residues substituted (S3LC(3E)). Both
mutants behaved similarly in cell signaling assays and in in vitro
biochemical analyses. The S3LC(3E) mutant was chosen for all
subsequent studies because S3LC(2E) was more prone to timedependent aggregation, although later we found that addition
of the reducing agent tris(2-carboxyethyl)phosphine
hydrochloride (TCEP) prevented aggregation of both mutants.
The S3LC(3E) mutant mimicked the effect of physiological
phosphorylation, as judged by the ability of this pseudo-phosphorylated Smad3 to superactivate a Smad3/Smad4 dependent
signaling response (Fig. 1c). The effect was similar to the
reported ligand-dependent transcriptional activation associat250
e
ed with overexpression of wild type Smad317–20. When analyzed
by size exclusion chromatography, the S3LC(3E) mutant eluted
as a larger molecular weight species than the wild type
(Fig. 1d). From the sedimentation analysis we concluded that
the model of subunit association of S3LC(3E) is a
monomer–trimer transition with a 17–35-fold increase in the
overall association constant relative to the wild type33.
Stoichiometry of the heteromeric Smad complex
Proposals concerning the oligomeric state of the Smad4–
R-Smads interaction have been based on the crystal structures
of Smad4 and biochemical analysis of Smad proteins in cell
extracts6,9,15. However, the interpretation of crystal packing in
the former and the heterogeneity of the cellular components in
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Fig. 4 Proposed model of phosphorylation induced Smad protein activation. Left, phosphorylation induced heterotrimer between Smad4 and
R-Smad subunits. Right, phosphorylation induced homotrimer of
R-Smad. The Smad4 subunit is shown in pink. The R-Smad subunits are
shown in blue. The conserved L3 loops are shown in black. The phosphorylated C-terminal tails of the R-Smad subunits are represented by red
arrows.
the latter give rise to conflicting results. To overcome these
problems, we tested whether Smad3 and Smad4 interact directly in the purified state.
S4AF interacts directly with S3LC(3E) as judged by their coelution on a size exclusion column (Fig. 2a). When mixed at a
1:1 mole ratio, a significant portion of S4AF co-eluted in the
same fractions as the trimeric S3LC(3E). The remaining
uncomplexed S4AF eluted in a monomeric form. The position
of the elution peak of the S4AF–S3LC(3E) complex coincides
with that of trimeric S3LC(3E), suggesting that the heteromeric complex is also a trimer. In addition, the height and position
of the elution peak of the complex remained unchanged when
either a four-fold excess or three-fold less of S4AF was used,
suggesting that the extent of the complex formation is limited
by the amount of S3LC(3E) present (Fig. 2a; chromatograms
not shown). Interestingly, regardless of the amount of S4AF
used, there is twice as much S3LC(3E) as S4AF in the complex
fractions, as measured by fluorometric counting of the
Commassie blue stained gels. These results suggest that the
complex is a heterotrimer with a preferred stoichiometry of
two subunits of S3LC(3E) and one subunit of S4AF. The
heterotrimeric assembly was also identified by sedimentation33.
The subunit packing arrangement in the crystal structure of
the Smad4 MH2 domain and S4AF suggests that trimerization
has an important role6,9. Although both proteins are monomeric in solution, the subunit packing interfaces include highly
conserved residues to which the majority of the tumor-derived
missense mutations of S4AF map (Fig. 2b). The MH2 domains
of Smad4 and R-Smads are highly homologous (50% identity),
except that Smad4 has an insertion of ~35 residues, which
forms a tower-like structural extension from the core (previously referred to as the TOWER)9. One of the three subunits of
S4AF has a disordered TOWER that mimics an R-Smad subunit
(Fig. 2b, blue subunit). The homotrimeric interaction observed
in the crystal structure may represent mimicry of the heterotrimer detected in the current study. To test this hypothesis,
selected residues of S4AF at the interfaces in the trimer were
mutated to their tumorigenic counterparts and the mutants
were tested for their ability to interact with S3LC(3E); the
S4AF(D351H) and S4AF(D537E) trimer interface mutants lost
their ability to interact with S3LC(3E) (Fig. 2c). This result
demonstrates that the heteromeric interaction is mediated
directly through Asp 351 and Asp 537 of Smad4.
Mechanism of the phosphorylation trigger
We have identified four distinct sulfate binding sites in the
crystal structure of S4AF9 (Figs 2b, 3a). Since the chemistry and
the mode of binding of a sulfate ion are similar to those of a
phosphate ion, we postulated that the sulfate binding sites
might correspond to the ‘receptor’ sites for the phosphorylated
C-terminal residues of an R-Smad through direct electrostatic
nature structural biology • volume 8 number 3 • march 2001
interaction. To test whether these sulfate binding sites may play
such a role in heteromeric interaction, selected basic residues
involved in coordinating the sulfate ions in S4AF were mutated
to Ser and examined for their ability to mediate the heteromeric interaction with S3LC(3E).
Mutation of Arg 416 in site 1 as well as mutation of Arg 496
or Arg 502 in site 2/site bc (see Figs 2b, 3a for nomenclature)
had no effect on the heteromeric interaction between S4AF and
S3LC(3E) (Fig. 3b). In contrast, mutation of Arg 515 of site 3
within the L3 loop significantly reduced the interaction. To verify that the loss of interaction did not result from conformational disturbance, the crystal structure of the S4AF(R515S)
mutant was determined (Table 1). The root mean square
(r.m.s.) deviation for Cα atoms between wild type S4AF and
S4AF(R515S) over the entire structure was 0.4 Å, suggesting
that the mutation does not introduce gross structural changes.
Superposition of the L3 loop region from wild type S4AF and
the mutant revealed that the conformation of the L3 loop is
unaltered (Fig. 3c); the r.m.s. deviation between the L3 loop (all
atoms within residues 505–520, superimposed on the Cα trace
of the entire structure) of wild type S4AF and S4AF(R507S)
was 0.4 Å.
To establish the functional significance of these observations,
we determined the ability of these Smad4 mutants to activate
Smad3/Smad4 dependent transcription in Smad4 null MDA
MB 468 cells8. The Smad3(3E) mutant activated the SBE-lux
reporter only when cotransfected with Smad4 (Fig. 3d). In contrast, while mutations of Smad4 residues Arg 416, 496 and 502
partially diminished the ability of Smad4 to activate this transcriptional response, the Arg 515 mutant could only weakly
activate this response in the presence of Smad3(3E), despite
similar levels of protein expression (data not shown). These
data are consistent with the in vitro data, indicating that
Arg 515 is a critical determinant of Smad3–Smad4 heterotrimerization in vivo. The partially diminished ability of the
other Arg mutants may involve signaling defects other than
Smad3–Smad4 heteromeric interaction. Further studies will be
required to dissect the structural determinants that govern
other Smad interactions in the signaling pathway.
These results demonstrate that the conserved Arg 515 within
the L3 loop of Smad4 plays a specific and essential role in heterotrimerization. The L3 loop is not involved in intermolecular
contacts in the complex but is located on a solvent accessible
surface of the trimer (Fig. 2b). Consequently, its role in facilitating intersubunit interactions is likely to occur through additional contacts. The L3 loop is located close to the C-terminus of
the neighboring subunit in the trimer. Structural analysis of the
MH2 domain from Smad4 and Smad2 reveals that the 10 C-terminal residues of R-Smads form flexible extensions from the
core6,9,21. It is possible that phosphorylation of the C-terminal
serine residues of R-Smads induces them to bind at the L3 loop
of the neighboring subunit through electrostatic interactions.
The 10 C-terminal residues of R-Smad would presumably form
an extended structure to reach the neighboring L3 loop, as the
distance is ∼28 Å (Fig. 4, left). This model is consistent with the
data presented here, demonstrating that both the negative
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electrostatic potential at the C-terminal phosphorylation sites
of Smad3, as well as Arg 515 within the L3 loop of Smad4, are
essential for heteromeric interaction. A prediction based on this
model is that phosphorylation induced homotrimerization of
the R-Smads also occurs through the same mechanism (Fig. 4,
right) since both the L3 loop Arg residue and the trimer interface residues are conserved in the R-Smads. Indeed, mutation of
either the conserved L3 loop Arg 385 or the trimer interface
residues Asp 257, Val 276, or Asp 407 (see Fig. 2b) of S3LC(3E)
prevents homotrimerization (Fig. 3e). These results demonstrate that phosphorylation-mediated homotrimerization and
heterotrimerization proceed via a common mechanism.
Mechanistic implications
An interesting feature of the proposed heteromeric complex is
the asymmetry of the trimer. Since the C-terminal tail of
Smad4 is not phosphorylated, the L3 loop ‘receptor’ site of the
neighboring R-Smad subunit is presumably unoccupied. With
one less intersubunit bridge, the heterotrimeric interaction
would be expected to be weaker than the homotrimeric interaction, in contrast to the result of the sedimentation analysis and
the signaling model. Several mechanisms could account for the
tighter interaction of the heterotrimer. The interface between
Smad4 and Smad3 subunits may involve better contacts than
those between the R-Smad subunits due to specific variations
within the interface. In addition, the L3 loop of Smad4 may be
a better ‘receptor’ than the L3 loop of R-Smads for interaction
with the phosphorylated C-terminal extension. Furthermore,
the unique TOWER structure of Smad4 may undergo a conformational change to facilitate interaction with the neighboring
R-Smad subunit. The details of the interactions await the crystal structure determination of the heteromeric complex.
The derived model reconciles several biological observations
and provides new insights into established paradigms of the
TGF-β dependent signaling mechanism. For example, a variety
of missense mutations both in and around the L3 loop of
R-Smads and Smad4 have been implicated in developmental
and tumorigenic processes6,22–24. Studies have suggested that
the principal effects of these mutations are likely to result from
abnormal interactions between the R-Smad and receptor
kinase25, and decreased R-Smad/Smad4 complex formation6.
We now show that mutation of a putative phosphoserine binding residue in the L3 loop of Smad4 dramatically reduces the
binding affinity for the phosphorylated R-Smad. This suggests
a new model in which tumorigenic mutations in the vicinity of
the L3 loop may interfere specifically with the formation of
R-Smad–Smad4 heterotrimers by decreasing the affinity of the
L3 loop for the phosphorylated R-Smad tail. In addition, the
different requirements for Smad2, Smad3 and Smad4 in specific signaling responses suggests that the relative amounts of
these proteins in the signaling complex serves to define signaling specificity26,27. While these complexes have been identified
in vivo, we now provide a structural model that supports the
idea that two different R-Smad molecules may coexist in heterotrimeric Smad complexes. Lastly, the phosphorylation
mechanism sheds light on how R-Smads dissociate from
SARA28. SARA is a membrane-anchored protein that recruits
Smad3/Smad2 to the TGF-β receptor kinase. Phosphorylation
of Smad3/Smad2 triggers dissociation from SARA, leading to
hetero-oligomerization with Smad4. A crystal structure
revealed that SARA makes van der Waal contacts with one side
of the Smad2 L3 loop, opposite the receptor site for the phosphorylated C-terminal sequence21. It is likely that through tin252
Table 1 Summary of structural data
Crystal parameters and crystallographic data
Space group
I4(1)22
Unit cell dimensions (Å)
a = b = 140.6, c = 193.0
Diffraction limits (Å)1
100–3.0 (3.11–3.0)
Total reflections
72,845
Unique reflections
19,552
Completeness (%)
97.9 (88.0)
I/σ
9.5 (2.3)
Rmerge (%)2
12.6 (48.5)
Refinement statistics
Number of protein atoms
R-factor (%)3
Rfree (%)4
R.m.s. deviations from ideal
Bond lengths (Å)
Bond angles (°)
B-factor r.m.s. deviations
Main chain (Å2)
Side chain (Å2)
5,434
20.01
26.27
0.007
1.3
2.1
2.8
Values in parentheses are for the highest resolution shell.
R-merge = Σ|Ihkl - <Ihkl>| / ΣIhkl
3R-factor = Σ ||F | - |F || / Σ |F | for all data.
hkl
o
c
hkl o
4R
free = Σhkl||Fo| - |Fc|| / Σhkl|Fo| for 10% of the data not used in refinement.
1
2
kering of the L3 loop conformation, binding of the phosphorylated C-terminal sequence at the L3 loop receptor site can
weaken Smad–SARA interactions. The L3 loop of Smad proteins could thus function as a switch controlling two distinct
steps of the signaling pathway.
In conclusion, this work defines the structure and
phosphorylation mechanism of the central signaling engine in
the TGF-β pathway. Phosphorylation mediated assembly of the
trimeric scaffold may be a critical switch for Smad nuclear
interactions involving the C-terminal oligomerization domain.
This work establishes a framework to further investigate how
the active heteromeric Smad complexes interact in the nucleus.
Methods
Construction of expression plasmids and mutagenesis. The
cDNA fragments of S3LC and S4AF were generated by PCR and were
subcloned into the pGEX-6P-1 and pGEX-4T-2 vectors, respectively.
Site-directed mutants were generated using the QuikChange kit
(Stratagene) and confirmed by sequencing.
Protein expression and purification. S3LC and S4AF were
expressed as glutathione-S-transferase (GST) fusion proteins in
Escherichia coli, extracted by standard procedures, and released by
PreScission Protease (Pharmacia Biotech) and thrombin (Enzyme
Research Lab), respectively. Eluted proteins were dialyzed in a DEAE
buffer containing 20 mM Tris (pH 7.4 for S3LC, pH 8.2 for S4AF),
10 mM NaCl, 0.1 mM EDTA, 1 mM dithiothreitol (DTT), and 1 mM
phenylmethanesulfonyl fluoride (PMSF). Proteins were purified
using DEAE-sepharose columns equilibrated with the respective
DEAE buffers and eluted by 10 mM to 310 mM NaCl gradients.
Size-exclusion chromatography. Size-exclusion chromatography
was performed using the Superdex 200 HR column on the Akta
Explore10 FPLC (Pharmacia Biotech). The column was equilibrated
at room temperature with 20 mM HEPES (pH 7.4), 0.1 mM EDTA,
100 mM NaCl, and 1 mM DTT. Before loading to the column, protein
samples were incubated in 1 mM TCEP, a reducing agent, for 60 min
at room temperature. Sample injection, elution and data analysis
were performed using UNICORN software (Pharmacia Biotech). The
nature structural biology • volume 8 number 3 • march 2001
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letters
flow rate was 0.7 ml min-1 and the fraction volume was 0.5 ml. The
column was calibrated with molecular weight standards blue dextran, ovalbumin (43k), albumin (67k), aldolase (158k), catalase
(232k), and ferritin (440k).
and the wild type GST fusion Smad3/Smad4 constructs, W. Kruijer for the SBELux construct, S. Davis for technical assistance. This research is funded by the
Sidney Kimmel Foundation Scholar Award and the center grant from the
Diabetes and Endocrinology Research Center.
Crystallization and structure determination of the
S4AF(R515S). The well solution contained 100 mM HEPES (pH 7.5),
250 mM lithium sulfate and 10% (v/v) PEG 4000. Crystals were transferred to a cryo-solvent consisting of 25% (v/v) glycerol and 75%
well solution, and were flash frozen in liquid nitrogen. Diffraction
data were collected at -170 °C using an R-Axis IV image plate system
mounted on a Rigaku rotating anode generator. The data were collected at a detector distance of 140 mm with 1° oscillation per frame
and were integrated and reduced using DENZO and Scalepack29.
The structure was refined using the CNS package30. Model building
was performed using CHAIN31. The wild type S4AF coordinates9
were used as an initial model for refinement and map calculation.
Correspondence should be addressed to K.L. email: [email protected]
Transcriptional response assays. MDA-MB 468 and NMuMg cells
were seeded to 50% confluence and transfected with SBE-Lux32,
pSV-β Gal and the indicated combinations of full-length FLAGSmad3 and Smad4-Myc mutants, using Fugene-6 (Roche), or
Lipofectamine (Gibco), respectively. Cells were lysed after 36 h, and
luciferase and β-galactosidase activity determined as described8.
Luciferase values were corrected for transfection efficiency with
β-galactosidase, expressed as the means (± S.E.) of three independent transfections. All experiments were repeated at least twice
with similar results. Protein expression levels were determined in
parallel experiments using COS-1 cells transfected with the same
proportions of FLAG-Smad3 and Smad4-Myc mutants used in the
transcriptional response assays, and the cell lysates immunoblotted
with anti-FLAG M2 (Kodak) or anti-Myc (9E10) antibodies.
Coordinates. The coordinates of S4AF(R515S) have been deposited
in the Protein Data Bank (accession code 1G88).
Acknowledgments
We thank A. Roberts (NIH), M. Marinus (UMass), and K. Knight (UMass) for
helpful suggestions of the manuscript, R. Derynck for the Smad4-Myc construct
nature structural biology • volume 8 number 3 • march 2001
Received 16 August, 2000; accepted 29 November, 2000.
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