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INTRODUCTION |
Src homology 2 (SH2)1
domains are highly conserved regions common to a series of cytoplasmic
signaling proteins such as the Src family of tyrosine kinases,
phospholipase C-
, the p85 subunit of phosphatidylinositol 3-kinase,
and the STAT family of transcription factors (1, 2). These noncatalytic
domains target the proteins to specific phosphotyrosyl peptide
sequences within their binding partners, thereby regulating a wide
range of intracellular signaling events. The specificity of this
interaction is determined by the amino acid sequence surrounding the
phosphotyrosine on the one hand and by the SH2 domain on the other
(3).
Activation of transcription factors of the STAT family has been shown
to require the transient association of the STATs with cytokine
receptors (4, 5). STAT factors interact through their SH2 domains with
specific phosphotyrosine motifs within the cytoplasmic parts of the
activated receptors. In the case of the activation of STAT1 and STAT3
by interleukin-6, four such tyrosine motifs within the interleukin-6
signal-transducing receptor subunit gp130 have been identified (6, 7).
Two of these motifs (Y767RHQ and Y814FKQ) give
rise to specific STAT3 activation, whereas two others (Y905LPQ and Y915MPQ) are able to recruit both
STAT1 and STAT3 (6). Subsequent to receptor binding, the STAT factors
are phosphorylated on a single tyrosine residue by receptor-associated
tyrosine kinases of the Janus kinase family (8-10). This activation of
the STAT factors leads to homo- or heterodimerization and translocation to the nucleus, where they bind to enhancers of interleukin-6-inducible genes resulting in the activation of transcription of, e.g.
acute phase protein genes (11-13). The dimerization of STAT factors
has also been shown to be mediated by the SH2 domains (9). This has
been confirmed recently by x-ray structures of the STAT1 and STAT3
dimers bound to DNA (14, 15). In this complex the two SH2 domains form
a tunnel that is passed by the two phosphotyrosine-containing tail segments.
Previous experiments have shown that the SH2 domain is also the sole
determinant of specific STAT factor activation via gp130 and the
interferon-
receptor (16, 17). The mechanism for the binding of STAT
monomers to the phosphotyrosine-containing recruitment sites of the
cytoplasmic region of signal-transducing receptor subunits still needs
to be elucidated.
Here we describe the expression, refolding, and structural
characterization of the STAT3-SH2 domain as well as its specific binding to phosphotyrosine peptides. Furthermore, we demonstrate that
this interaction requires a monomeric domain.
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EXPERIMENTAL PROCEDURES |
Peptide Synthesis--
Biotinylated peptides were synthesized as
described earlier (18).
Plasmid Construction--
Constructions were carried out using
standard procedures (19). For construction of pB-STAT3-BS, the cDNA
encoding murine STAT3 (kindly supplied by J. Darnell, Jr., Rockefeller
University, New York) was provided with BglII and
SalI restriction sites at the 5'- and 3'-ends, respectively,
and cloned into a pBluescript vector (Stratagene, Heidelberg, Germany).
The sequence encoding the STAT3-SH2 domain (amino acid residues
582-702) was amplified by polymerase chain reaction, and
BamHI and AvrII restriction sites were introduced
by the 5'- and 3'-primers, respectively. The
BamHI/AvrII DNA fragment was ligated with a
modified pRSet5c vector carrying an adaptor consisting of an
amino-terminal MRGS(H)6-tag and a BamHI and an
AvrII restriction site. The resulting vector pRSetS3SH2
coding for the amino-terminally His-tagged STAT3-SH2 domain was
verified by DNA sequence analysis.
Expression, Purification, and Refolding of the Recombinant
MRGS(H)6-tagged STAT3-SH2 Domain--
Escherichia
colistrain BL21(DE3)pLysS transformed with the pRSetS3SH2 plasmid
was grown at 37 °C in LB medium containing chloramphenicol (50 µg/ml) and ampicillin (100 µg/ml) to an A595
of 0.6-0.7. Cells were induced with 0.4 mM
isopropyl-
-D-thiogalactopyranoside for 3 h at
37 °C and subsequently harvested by centifugation. The bacterial
pellet was resuspended in lysis buffer (26 mM Tris-HCl, pH
7.5, 10 mM EDTA, 1 mM phenylmethylsulfonyl
fluoride, 1 mM dithiothreitol), cells were lysed by
repeated freezing and thawing, and the inclusion bodies were harvested
by centrifugation and purified by five cycles of sonication (constant
pulse for 2 min at 0 °C), each followed by centrifugation at 3,700 rpm for 30 min. The inclusion bodies were solubilized in buffer S (50 mM Tris-HCl, pH 8.0, 6 M GdnHCl, 1 mM EDTA, 100 mM dithiothreitol). After
incubation at 42 °C for 30 min, insoluble particles were removed by
filtration through a 0.45-µm sterile filter, and the buffer pH was
adjusted to 2. The denatured STAT3-SH2 domain was purified by reverse
phase HPLC using a Polygosil 60-5 C18 column (CS-Chromatographie
Service GmbH, Langerwehe, Germany) equilibrated in buffer A (0.1%
trifluoroacetic acid). Elution of the STAT3-SH2 domain occurred at
50.3% buffer B (80% acetonitrile, 0.1% trifluoroacetic acid). The
purified protein was isolated by lyophilization and solubilized in
buffer S. Refolding of the purified SH2 domain was achieved by dialysis at 4 °C against different buffers (buffer C: 20 mM
Na2HPO4/KH2PO4, pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 1 mM
dithiothreitol; buffer D: 20 mM
Na2HPO4/KH2PO4, pH 5.5, 150 mM NaCl, 0.5 mM EDTA; buffer E: 20 mM CH3COONa/CH3COOH, pH 4.5, 150 mM NaCl, 0.5 mM EDTA). For purity
determination, the proteins were resolved on a 15% polyacrylamide gel
by SDS-PAGE and visualized using SyproTM Orange protein
stain reagent (Molecular Probes, Eugene, OR). The quantitative analysis
was performed on a Storm 840 fluorescence scanner (Molecular Dynamics,
Krefeld, Germany) using Image QuantTM software.
Immunoblot Analysis--
The harvested cell fragments containing
the inclusion bodies were resolved by SDS-PAGE and transferred to an
Immobilon polyvinylidene difluoride membrane (Millipore, Eschborn,
Germany) using a semidry electroblotting apparatus. STAT3-SH2 detection
was performed using a monoclonal MRGSHis antibody (QIAGEN,
Hilden, Germany). A polyclonal goat anti-mouse horseradish
peroxidase-conjugated secondary antibody (DAKO, Hamburg, Germany) was
used to visualize the immunoreactive bands by Western blot techniques.
Peptide Binding Assay--
Peptide binding of the isolated pure
STAT3-SH2 domain was performed by the means of an ELISA. A 96-well
ELISA Maxisorb plate (NUNC, Roskilde, Denmark) was coated with 2.5 µg/ml streptavidin (100 µl/well; 16 h at room temperature).
Unoccupied binding sites were blocked with 2% bovine serum albumin in
phosphate-buffered saline (10 mM
Na2HPO4/KH2PO4, pH 7.4, 200 mM NaCl, 2.5 mM KCl) (200 µl/well; 2 h at room temperature). After four washings with phosphate-buffered
saline and 0.02%Tween (200 µl/well), the biotinylated peptides were
immobilized by incubating the streptavidin surface with 100 µl/well
of a 500 ng/ml peptide solution in phosphate-buffered saline for 1 h at room temperature. The wells were washed four times with 200 µl/well phosphate-buffered saline and 0.02% Tween, and the surface
was equilibrated with buffer C or D. STAT3-SH2 solutions were incubated
for 1 h at room temperature, and unbound protein was removed by
washing four times with the appropriate phosphate buffer. The
unoccupied phosphotyrosine residues were detected by incubation with
PY20 phosphotyrosine antibody (Transduction Laboratories, Lexington,
KY; 1:2,000, 100 µl/well) for 45 min at room temperature. Bound PY20
was visualized using a polyclonal goat anti-mouse horseradish
peroxidase-conjugated antibody (DAKO; 1:2,000, 100 µl/well, 45 min at
room temperature). Staining reagent was 0.1 mg/ml
3,3',5,5'-tetramethylbenzidine in 0.1 M acetate buffer,
pH 5.5, containing 0.003 vol % H2O2. The
reaction was stopped with 2 M sulfuric acid. Inhibition of
the PY20/phosphopeptide interaction by STAT3-SH2 was determined by
calculating the decrease in absorbance with increasing amounts of
STAT3-SH2 relative to a SH2-free sample.
Size Exclusion Chromatography--
Size exclusion chromatography
was performed on a Bio-SilectTM SEC 125-5 column
(Bio-Rad). The column was equilibrated with the refolding buffer C (pH
7.5) or D (pH 5.5), respectively, loaded with 500 µl of STAT3-SH2
(70-80 µg/ml), and run at a constant flow rate of 0.7 ml/min. The
collected 0.7-ml fractions were resolved on a 15% polyacrylamide gel
by SDS-PAGE and visualized by silver staining.
Circular Dichroism Spectroscopy--
CD measurements were
carried out on an AVIV (Lakewood, NJ) 62DS CD spectrometer, equipped
with a temperature control unit, and a Jasco J-600 spectropolarimeter,
both calibrated with a 0.1% aqueous solution of
D-10-camphorsulfonic acid according to Chen and Yang (20).
The spectral band width was 1.5 nm. The time constant ranged between 1 and 4 s and the cell path length between 0.1 and 10 mm.
Fluorescence Spectroscopy--
Steady-state fluorescence spectra
were recorded on a Spex Fluorolog 211 photon-counting
spectrofluorometer (Spex Industries, NY) with a band width of 2.7 nm
(excitation monochromator) and 2.2 nm (emission monochromator). The
excitation wavelength was 295 nm. The spectra are corrected for changes
in lamp intensity and for spectral sensitivity of the
emission-monochromator/photomultiplier system. All fluorescence
measurements were carried out at 20 °C.
Fluorescence lifetimes and anisotropy decay were measured in the single
photon-counting mode with an Edinburgh Instruments Ltd. (U. K.)
spectrometer, model 199. The full width at half maximum of the lamp
pulse from the hydrogen flashlamp was 1.4 ns. The excitation wavelength
was 295 nm and the band width 8 nm. The emitted light was passed
through a combination of a UV-transmitting black glass and a cutoff
glass filter to create a band pass (WG320, DUG11, Schott, Mainz,
Germany). At least 80,000 counts were accumulated in the peak channel
of the total fluorescence intensity, I(t). The
lamp pulse was recorded with a suspension of Ludox (NEN Life Science
Products) at 345 nm. Data handling and the iterative nonlinear least
squares fit of the decays were accomplished with a program supplied by
Edinburgh Instruments Ltd. Intensity decays
(I(t)) were fit to the multiexponential model
using I(t) =
biexp(
t/
i), where bi values are the amplitudes associated
with the decay time
i. The fractional
intensity Bj = bj
j/
bi
i permits
calculation of the mean lifetime <
> =
Bi
i.
Fluorescence anisotropy decays were analyzed by an exponential fit.
where r0 = r + r
.
The parameters of r(t) are as follows.
r is the anisotropy;
, the rotational correlation time;
r0 and r
are the
limiting anisotropies, r(t)(t
0) = r0 and
r(t)(t
) = r
. The quality of the fits was gathered from
plots of weighted residuals and from the statistical parameter
2 (21).
Protein Concentrations--
Protein concentrations were
calculated from absorption spectra in the range of 240-320 nm using
the method of Waxman et al. (22).
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RESULTS |
Expression and Purification of Recombinant STAT3-SH2--
To
obtain sufficient amounts of protein, the amino-terminally His-tagged
STAT3-SH2 domain was expressed in E. coli. The recombinant protein was found entirely in inclusion bodies (Fig.
1A). Repeated sonication and
centrifugation yielded inclusion bodies containing about 90% STAT3-SH2
protein. 1 liter of medium contained 40-50 mg of inclusion body
proteins. After solubilization of the inclusion bodies in GdnHCl the
proteins were separated on a reverse phase HPLC column, and
STAT3-SH2-containing peak fractions were lyophilized. The STAT3-SH2
protein proved to be at least 99% pure (Fig. 1B). This
procedure yielded 10-15 mg of pure STAT3-SH2/liter of culture.

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Fig. 1.
Expression and purification of
STAT3-SH2. A, SDS-PAGE analysis of bacterially
expressed STAT3-SH2. Protein bands are visualized by Coomassie staining
(lanes 1-5) or by immunoblot analysis (lane 6).
Lanes 1 and 2 show the total proteins of E. coli BL21(DE3)pLysS transformed with pRSetS3SH2 before (lane
1) and after (lane 2) induction with
isopropyl- -D-thiogalactopyranoside. Lane 3 shows the supernatant of the bacterial lysate after removal of
insoluble cell fragments by centrifugation. Repeated sonication of the
harvested cell fragments (lane 4) yielded purified inclusion
bodies (lane 5). Proteins of the inclusion bodies were
resolved by SDS-PAGE, transferred to an Immobilon membrane, and
detected with a monoclonal MRGSHis antibody (lane
6). The apparent molecular mass is in good agreement with the
calculated mass of 15 kDa. B, reverse phase HPLC-purified
STAT3-SH2 domain was refolded by dialysis. For purity determination,
the proteins were resolved by SDS-PAGE. Panel 1 shows silver
staining of the refolded STAT3-SH2 domain. For quantification, the
polyacrylamide gel was stained with SyproTM Orange reagent
and analyzed using a Storm 840 scanner. The quantitative analysis
(panel 2) proved the protein to be more than 99% pure
(peak a) with the sole detectable impurity being a
disulfide bonded STAT3-SH2 dimer (peak b).
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Refolding and CD Spectroscopy--
The purified protein was
dissolved in 6 M GdnHCl, 1 mM EDTA, and 100 mM dithiothreitol and dialyzed for refolding against buffers of pH 7.5, 5.5, and 4.5, respectively. Subsequently the protein
samples were characterized by CD spectroscopy. Fig.
2 shows the far UV and near UV CD spectra
of the STAT3-SH2 domain at pH 7.5 (solid line) and pH 4.5 (dashed line). Although the far UV CD spectra are remarkably
different at the two pH values, they look similar to spectra of other
SH2 domains (23, 24). Even more pronounced differences were detected
between the near UV CD spectra at the two different pH values (Fig.
2B). For instance, at pH 4.5 a distinct band appeared
at 292 nm which can be assigned to a tryptophan residue. This effect
can be attributed to a local change rather than to a change of the
overall fold of the protein. The reversibility of this structural
change was determined by changing the pH of the solution from pH 4.5 to
7.5 and vice versa (data not shown).

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Fig. 2.
CD spectra of the refolded SH2 domain.
The figure shows the far UV (A) and near UV (B)
CD spectra of the refolded STAT3-SH2 domain at pH 7.5 (solid
line) and pH 4.5 (dashed line).
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To determine the thermal stability of the folded proteins at the
different pH values we recorded a series of CD spectra with increasing
temperature. At pH 7.5 a melting curve with a midpoint around
43 °C was obtained (Fig. 3). At pH
4.5, however, the protein precipitated with increasing temperature
(data not shown).

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Fig. 3.
Thermal stability of the refolded STAT3-SH2
domain at pH 7.5. Thermal stability was determined by CD
spectroscopy. The graph shows the ellipticity MRW (MRW,
mean residue weight) at 215 nm as a function of temperature.
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Unfolding by GdnHCl--
Because the thermal stability of the
protein could only be estimated at pH 7.5, we monitored the
GdnHCl-induced unfolding of the protein by fluorescence spectroscopy at
pH 4.5, 5.5, and 7.5. With increasing GdnHCl concentration the
fluorescence intensity decreased (not shown), and the maximum of the
tryptophan emission shifted from 333 to 353 nm at pH 5.5. This behavior
is characteristic of a transition of a protein from a folded to an
unfolded state. Whereas the midpoint of the denaturation curve at pH
7.5 (Fig. 4, solid line) was
found at about 1.7 M GdnHCl, this point is shifted to 2.1 M GdnHCl at acidic pH values of 5.5 (dotted
line) and 4.5 (dashed line).

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Fig. 4.
Unfolding of the recombinant STAT3-SH2
domain. The figure shows unfolding of the STAT3-SH2 domain by
increasing concentrations of GdnHCl at pH 4.5 ( , dashed
line), pH 5.5 ( , dotted line) and pH 7.5 ( ,
solid line). The wavelength shift of maximum tryptophan
emission max was monitored as a function of GdnHCl
concentration. Because max also changes with pH, the
data were normalized. The max values observed at GdnHCl
concentrations of 0 and 4 M were taken as representative of
the 100% native and 0% native, i.e. fully denatured state,
respectively.
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Fluorescence Spectroscopy--
Steady-state fluorescence,
fluorescence lifetime, and anisotropy decay measurements were performed
at pH 7.5, 5.5, and 4.5. The steady-state fluorescence data of the SH2
domain upon excitation at 295 nm are compiled in Table
I. With a pH decrease from 7.5 to 4.5, the maximum of the emission band shifted from 336 to 330. This shift
was accompanied by a decrease of the full width at half maximum from 56 to 51 nm. The shift of the emission maximum and the decrease of the
full width at half maximum are indicative of an increasingly
hydrophobic environment of the sole tryptophan present in the SH2
domain.
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Table I
Fluorescence-steady state data of the STAT3-SH2 domain at different pH
values
max is the wavelength of the maximum tryptophan
fluorescence. FWHM is the full width at half maximum
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The results of the fluorescence lifetime and anisotropy decay
measurements are compiled in Tables II and III. The decays of the
tryptophan fluorescence can be fitted by a sum of three exponentials with fractional intensities Bi and corresponding
lifetimes
i and lead to calculated mean lifetimes
(<
>) of 4.7, 4.1, and 3.7 ns for the pH values of 7.5, 5.5, and
4.5, respectively (Table II). Such a
behavior is in good agreement with the blue shift of the emission
maximum in Table I (25).
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Table II
Fluorescence intensity decay data of the STAT3-SH2 domain at different
pH values
Bi is the fractional amplitude associated with the
decay time i;   is the mean lifetime (see
"Experimental Procedures").
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The fluorescence anisotropy decays of the SH2 domain at different pH
values were fitted with one exponential and led to rotational correlation times of
= 12.4, 6.4, and 6.1 ns at pH values of 7.5, 5.5, and 4.5, respectively (Table III).
Rotational correlation times
can be used to calculate the molecular
mass from the equation Mr = f ×
(f = 2.6 kDa/ns) for spherical particles on the
basis of the Stokes-Einstein relationship (26). The expected rotational correlation time
for a monomeric SH2 domain is therefore about 5.7 ns assuming a spherical shape. The measured rotational correlation times at acidic pH values are in good agreement with the overall tumbling rate expected for a monomer. The twice as high value found at
neutral pH indicates the existence of a dimeric SH2 domain. The
reversibility of the monomer/dimer transition was determined by
changing the pH from 4.5 to 7.5 and vice versa by dialysis (data not
shown).
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Table III
Fluorescence anisotropy decay data of the STAT3-SH2 domain at different
pH values
ri are the anisotropies, the rotational
correlation time (see "Experimental Procedures").
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Size Exclusion Chromatography--
Additional evidence for the
dimerization was provided by size exclusion chromatography experiments
(Fig. 5) using a calibrated column. Fig.
5A shows the elution profile of the SH2 domain (monomer, dotted line; dimer, solid line). Both peaks
contain STAT3-SH2 as shown by SDS-PAGE (see inset in Fig.
5A). The apparent molecular masses of both the monomer (13 kDa) and the dimer (32 kDa) are in good agreement with the expected
values (Fig. 5B) of 15 and 30 kDa, respectively.

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Fig. 5.
Size exclusion chromatography.
A, elution profile of the STAT3-SH2 domain under monomeric
(pH 5.5, dotted line) and dimeric (pH 7.5, solid
line) conditions. The elution profile of the marker proteins
(a, bovine thyroglobulin, 670 kDa; b, bovine
-globulin, 158 kDa; c, chicken ovalbumin, 44 kDa;
d, horse myoglobin, 17 kDa; e, vitamin
B12, 1.35 kDa) is drawn as a dashed line. and denote the STAT-SH2 domain eluted at pH 7.5 and 5.5, respectively. The inset displays a SDS-PAGE analysis of
fractions 3-13 showing STAT3-SH2. Protein bands were visualized by
silver staining. The marker proteins b, c, and
d were used for linear regression. B, plot of the
log(Mr) as a function of the retention times of
the marker proteins (a-d). Elution times of the STAT3-SH2
domain at pH 7.5 ( ) and 5.5 ( ) correspond to molecular masses of
32 and 13 kDa, respectively.
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Specific Interaction of the Recombinant STAT3-SH2 Domain with
Phosphopeptides--
To determine the functionality of the recombinant
STAT3-SH2 domain, we worked out an ELISA using biotinylated
phosphopeptides as bait. Based on previous investigations, we chose all
of the phosphopeptide motifs of the signal transducer gp130, a mutant of the pY767 motif containing a Q/E exchange at the pY+3
position (pYQ770E) as well as the phosphotyrosine motif
encompassing pY705 of STAT3 or a peptide containing the
amino acids of the gp130 motif pY767 in random order
(pYX) (Table IV).
As the STAT3-SH2 domain turned out to undergo a pH-dependent dimerization, we investigated the specificity
of the interaction with the various phosphopeptide motifs under neutral (dimer) and acidic (monomer) conditions. For the interaction of the
peptides with the monomeric SH2 domain, we performed the assay at pH
5.5 to maintain the stability of streptavidin.
Fig. 6 shows a schematic representation
of the ELISA used. After incubation of the biotinylated phosphopeptides
with the streptavidin-coated surface, the immobilized phosphotyrosine
residues were detected with the phosphotyrosine antibody PY20 (Fig.
6A). Incubation with increasing amounts of STAT3-SH2 led to
a decrease in absorbance because PY20 was unable to recognize the
phosphopeptides bound to the SH2 domain (Fig. 6B). The
relative decrease in absorbance with increasing amounts of STAT3-SH2
compared with an SH2-free sample was used to determine the inhibition
of the PY20/phosphopeptide interaction by STAT3-SH2 which indicates
SH2/peptide binding.

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Fig. 6.
Schematic representation of the peptide
binding assay. A, detection of the biotinylated
phosphopeptides immobilized on a streptavidin surface by the
phosphotyrosine antibody PY20. B, inhibition of the
PY20/peptide interaction by STAT3-SH2 bound to the phosphotyrosine
motifs.
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Fig. 7A shows the inhibition
of the PY20/phosphopeptide interaction at pH 5.5. The STAT3-SH2 domain
interacts specifically with the four-membrane distal phosphotyrosine
motifs of gp130 (pY767, pY814,
pY905, and pY915) whereas the peptides
pY683 and pYX as well as the
pY759 motif, known to bind to the SH2 domain of SHP-2, show
only weak binding. In addition, the isolated STAT3-SH2 domain binds to
the motif pY705 of STAT3. Interestingly, the binding of the
SH2 domain to pY767 is significantly impaired by a Q/E
exchange at the pY+3 position (pYQ770E). This emphasizes
the importance of the pY+3 position for specific recognition of
receptor motifs by STAT3-SH2.

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Fig. 7.
Inhibition of the PY20/phosphopeptide
interaction by STAT3-SH2. Binding of the STAT3-SH2 domain to the
phosphopeptides listed in Table IV was determined by monitoring the
inhibition of the PY20/peptide interaction with increasing
concentrations of STAT3-SH2 at pH 5.5 (A) and pH 7.5 (B).
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Fig. 7B shows the results of the same experiment under
neutral buffer conditions (pH 7.5) where the SH2 domain exists as a dimer. Whereas the monomeric SH2 domain is able to distinguish between
the different phosphopeptides, the dimeric molecule is not. The
phosphopeptides show only low affinity to the dimer.
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DISCUSSION |
The SH2 domains of the STAT factors are involved in receptor
recognition as well as in their dimerization. Dimerization is induced
by tyrosine phosphorylation and is a prerequisite for DNA binding. To
investigate the interaction of the SH2 domains with the respective
phosphotyrosine motifs on a molecular level we expressed the SH2 domain
of STAT3 in E. coli. After purification the protein was
refolded. CD spectra of the refolded protein correspond to those
observed with other SH2 domains (23, 24). Spectral differences were
observed at neutral and acidic pH. The changes seen in the far UV are a
reflection of limited rearrangements of the overall structure (Fig. 2).
The near UV spectrum, for instance, shows a distinct band at 292 nm at
pH 4.5 which is not detectable at pH 7.5, indicating a loss in
conformational mobility for the side chain of the sole tryptophan. This
result correlates well with the corresponding fluorescence spectra
where a blue shift of the emission maximum and a decrease of the full
width at half maximum are observed with decreasing pH, reflecting a
transition of the sole tryptophan from a hydrophilic to a more
hydrophobic environment.
The fluorescence anisotropy decay measurements enabled us to assign
these spectral differences to a pH-dependent dimerization of the recombinant SH2 domain which exists as a monomer under acidic
conditions and as a dimer at neutral pH. The less exposed tryptophan in
the monomeric state correlates with a higher stability of the monomer
compared with the dimer as revealed by GdnHCl-induced denaturation.
Thus, the small structural changes induced by dimerization are
accompanied by a destabilization of the molecule.
Taken together, the fluorescence and CD measurements show that
dimerization leads to a conformational change in the SH2 domain involving tryptophan 623. In the x-ray structure of the
(STAT1)2-DNA complex the corresponding tryptophan is
located at the surface of the molecule and is accessible to water. The
higher B factors of this amino acid residue in the crystal structure
are a further indication of its enhanced flexibility in the dimer. For
other SH2 domains such as those of Src or Lck kinase it has been shown that the BG loop is attached to the body of the molecule (27, 28),
whereas it is completely detached in the (STAT3)2-DNA and (STAT1)2-DNA complexes (14, 15). Because the BG loop is
also involved in the dimer interface we raise the idea that in the monomeric state this loop resembles the situation seen in other SH2
domains. This would bury the tryptophan (Trp623) within the
structure, a fact that we indeed observe for the monomeric SH2 domain.
The two conformational states might reflect different modes of
SH2/phosphotyrosine peptide interactions in STAT receptor binding and
STAT dimer formation.
Recently, heterodimeric complexes of STAT1 with STAT2 or STAT3
prior to cytokine stimulation have been described (29). The ability of
the STAT3-SH2 domain to form dimers may reflect such an interaction
between unphosphorylated STAT molecules. On the other hand, it cannot
be ruled out that the observed dimerization is a property of the
isolated domain and that its formation is prevented within the entire protein.
To study the interaction of the STAT3-SH2 domain with different
phosphotyrosine peptides we established an ELISA based on the
competition of a phosphotyrosine monoclonal antibody with the
recombinant protein in binding to phosphotyrosine peptide motifs.
Whereas the monomeric STAT3-SH2 domain was able to bind to specific
phosphotyrosine peptides no such interaction could be observed with the
dimeric protein.
We detected a specific interaction between the monomeric STAT3-SH2
domain and the four distal phosphotyrosine motifs present in the
cytoplasmic part of the signal transducer gp130. In contrast, the
phosphotyrosine peptides corresponding to the two membrane-proximal tyrosine residues did not show a specific interaction (Fig. 7). These
results are in good agreement with the previous observation that in
transiently transfected COS cells STAT3 is activated only through the
four distal tyrosine motifs of gp130 (6, 7). Interestingly, we found a
Q770E substitution in the Y767 motif of gp130 to lead to a
loss in STAT3-SH2 binding, corroborating the finding that a glutamine
residue at the Y+3 position is important for STAT3 activation (Fig. 7).
Thus, our STAT3-SH2/phosphopeptide interaction studies fully confirmed
the results obtained with native STAT3 in COS cells. Furthermore, the
phosphotyrosine peptide of STAT3 itself (pY705) showed
specific binding to the recombinant STAT3-SH2 domain. A comparison of
the affinities would require a common binding mechanism. As deduced
from the x-ray structure of the (STAT1)2-DNA and
(STAT3)2-DNA complexes the mode of SH2/peptide binding
therein shows fundamental differences to SH2/peptide interactions known so far (2). An expected higher affinity to the phosphotyrosine peptide
of STAT3 itself (pY705) compared with receptor motifs was
not observed. This is presumably because the complex interaction seen
in the x-ray structures cannot be reconstituted in the ELISA.
Thus far, structure/function studies of recombinant STAT-SH2 domains
were hampered by the fact that they did not show specific binding to
phosphotyrosine peptides. We found that at physiological pH the
recombinant STAT3-SH2 domain is forming dimers that do not bind to
phosphopeptides. The observation that under acidic conditions, the
STAT3-SH2 domain exists as a monomer that specifically binds to
phosphotyrosine motifs will enable us to elucidate how STAT factors
interact with their receptors.