From the Protein Engineering Group and the
¶ Macromolecular NMR Section, ABL Basic Research Program,
NCI-Frederick Cancer Research and Development Center, Frederick,
Maryland 21702-1201
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ABSTRACT |
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The multifunctional signal transducer and
activator of transcription (STAT) proteins relay signals from the cell
membrane to the nucleus in response to cytokines and growth factors.
STAT4 becomes activated when cells are treated with interleukin-12, a
key cytokine regulator of cell-mediated immunity. Upon activation, dimers of STAT4 bind cooperatively to tandem interferon- activation sequences (GAS elements) near the interferon-
gene and stimulate its
transcription. The amino-terminal domain of STAT4 (STAT4(1-124)) is required for cooperative binding interactions between STAT4 dimers and activation of interferon-
transcription in response to
interleukin-12. We have overproduced this domain of human STAT4 (hSTAT4(1-124)) in Escherichia coli and purified it to
homogeneity for structural studies. The circular dichroism spectrum of
hSTAT4(1-124) indicates that it has a well ordered conformation in
solution. The translational diffusion constant of hSTAT4(1-124) was
determined by nuclear magnetic resonance methods and found to be
consistent with that of a dimer. The rotational correlation time
(
c) of hSTAT4(1-124) was estimated from 15N
relaxation to be 16 ns; this value is consistent with a 29-kDa dimeric
protein. These results, together with the number of signals observed in
the two-dimensional 1H-15N heteronuclear single
quantum coherence spectrum of uniformly 15N-labeled
protein, indicate that hSTAT4(1-124) forms a stable, symmetric
homodimer in solution. Cooperativity in native STAT4 probably results
from a similar or identical interaction between the amino-terminal
domains of adjacent dimers bound to DNA.
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INTRODUCTION |
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Cytokines mediate communication between cells in the immune system
by binding to specific receptors and stimulating the transcription of
distinct sets of genes. The signals are relayed from the cell surface
to the nucleus via the
JAK-STAT1 pathway (1-4).
JAK-STAT signaling commences when cytokine binding induces receptor
dimerization, which brings cytoplasmic, receptor-associated JAKs into
apposition and enables them to self-activate by reciprocal transphosphorylation. The activated kinases phosphorylate a distal tyrosine on the cytoplasmic tail of the receptor, which can then be
recognized by the SH2 domain of a specific STAT protein. Upon association with the receptor, a tyrosine residue near the carboxyl terminus of the STAT protein is phosphorylated by the JAK kinase. Now
activated, the STAT protein can form homo- or heterodimers in which the
phosphotyrosine of one partner binds to the SH2 domain of the other.
The STAT dimers then migrate to the nucleus, where they participate in
transcriptional activation by binding to specific DNA sequences, termed
interferon- activation sequence (GAS) elements.
Thus far, only seven different STATs and four JAK family members have
been identified (2), which raises the question of how transcriptional
specificity is achieved in cytokine signal transduction: that is, how
can a relatively small number of JAKs and STATs elicit distinct
responses to a much larger number of cytokines and growth factors,
particularly when all but one of the STATs appear to bind
preferentially to the same DNA sequence (2). Recently, Xu et
al. (5) characterized authentic binding sites for STATs 1, 4, 5, and 6 within the first intron of the interferon- (IFN-
) gene by
DNase I footprinting. Remarkably, these experiments revealed that
rather than binding to the same sites, as might have been expected,
STATs 1, 4, and 5 each bound to a distinct pattern of adjacent sites,
none of which bears a close resemblance to the high-affinity, consensus
sequence identified by the random selection method. Instead, these
binding sites consist of tandem arrays of imperfect GAS elements that
are separated by 10 base pairs or about one turn of the helix in B-form
DNA. The binding of STAT4 to these tandem sites is cooperative in
nature; simultaneous occupancy of multiple sites is required to achieve a stable association with the DNA. The amino-terminal 124 residues of
STAT4 are essential for cooperative binding to these adjacent low-affinity sites, but not for its ability to be phosphorylated, to
dimerize, or to bind to, a single high-affinity site (5). Xu et
al. (5) proposed that this cooperativity results from a direct
interaction between the amino-terminal domains of STAT dimers bound to
adjacent sites on DNA. A similar result has been obtained with STAT1
(6), and it seems likely that the other STATs also use their
amino-terminal domains for cooperative binding (2).
STAT4 is activated in response to interleukin-12 (IL-12), which plays a
primary role in the development of T helper 1 (TH1) cells
and in the induction of organ-specific autoimmune diseases (7, 8).
Cooperative binding of STAT4 dimers to adjacent low-affinity GAS sites
is required for transcriptional activation of IFN- (5), which is
thought to mediate many of the effects of IL-12 (9). Therefore, a small
molecule that can bind to the amino-terminal domain of STAT4 and
prevent its self-association might be an effective immunosuppressant.
To facilitate the discovery of such a therapeutic agent, we have
undertaken an effort to determine the three-dimensional structure of
this domain in solution and elucidate the molecular details of its
self-association. To this end, we have overproduced the amino-terminal
domain of human STAT4 (hSTAT4(1-124)) in Escherichia coli
and purified it to homogeneity. Furthermore, we have characterized this
fragment of STAT4 by circular dichroism (CD) and nuclear magnetic
resonance (NMR) techniques and report that it has a well ordered
conformation in solution that is amenable to structure
determination.
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MATERIALS AND METHODS |
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Plasmid Expression Vector-- The segment of a human STAT4 cDNA clone that corresponds to amino acid residues 1-124 was amplified by the polymerase chain reaction (PCR), using primers PE-11 (5'-TATTATCATGAGTCAGTGGAATCAAGTCCAACAG-3') and PE-12 (5'-ATTATAAGCTTGGATCCTTA CTGGACAGGCATGTTGGCTGCAGCCAAaATaCgaCgCTCTTCCCT-3'). The restriction sites within these primers that were used to cleave the PCR fragment prior to ligation (BspHI and BamHI) are underlined. Mismatches that were introduced within the PE-12 primer to change rarely used arginine and isoleucine codons to more common ones are indicated in lowercase type (see text for discussion). PCR amplification was performed with Pfu polymerase under the conditions recommended by the supplier (Stratagene). The PCR regimen entailed 25 cycles in a Perkin-Elmer Thermal Cycler 2400, with each cycle consisting of 30 s at 94, 55, and 72 °C, respectively. The PCR fragment was cleaved with BspHI and BamHI and then ligated with the NcoI/BamHI vector backbone of pET3d (10) to construct pDW474. The nucleotide sequence of the STAT4 DNA in pDW474 was confirmed experimentally.
Protein Expression--
Cells from single, drug-resistant
colonies of E. coli BL21/DE3 containing pDW474 and pDC952
were grown to saturation in LB broth (11) supplemented with 100 µg/ml
ampicillin and 30 µg/ml chloramphenicol at 37 °C. The saturated
cultures were diluted 100-fold in the same medium and grown in
shake-flasks to mid-log phase (A600 = 0.5-0.7), at which
time isopropyl-1-thio--D-galactopyranoside was added to
a final concentration of 1 mM. After 3 h, the cells were recovered by centrifugation. 15N-Labeled
hSTAT4(1-124) was expressed in M9 minimal medium (11) containing 1.5 g/L of 15NH4Cl and 2.6 g/L of
15N-Celtone (Martek Biosciences Corp.).
Protein Purification-- E. coli cells containing hSTAT4(1-124) were resuspended in 0.01 culture volumes of lysis buffer (50 mM Tris (pH 8.0), 150 mM NaCl, 1 mM EDTA) and disrupted by three successive passes through a French press at 4 °C. The soluble extract was prepared by centrifuging the disrupted cell suspension at 14,000 × g for 15 min at 4 °C. To remove nucleic acids and some acidic proteins from the soluble extract, polyethyleneimine (PEI) was added to a final concentration of 0.15%. After the mixture was kept on ice for 5 min, the precipitate was pelleted by centrifugation as above. Residual PEI was removed by a two-step ammonium sulfate precipitation at concentrations of 20 and 60%. The pellet from the 60% ammonium sulfate precipitation was resuspended in 50 mM Tris (pH 8.0), 1 mM EDTA, 5 mM dithiothreitol (DTT) (10 ml/liter of culture volume) and dialyzed extensively against the same at 4 °C. The dialyzed material was clarified by centrifugation at 14,000 × g for 10 min and then passed over an anion exchange column (Q-Sepharose, 1.6 cm × 10 cm, Amersham Pharmacia Biotech) at 4 °C. The bound protein was eluted from the column with a linear gradient of 0-1 M NaCl in 50 mM Tris (pH 8.0) and 5 mM DTT; hSTAT4(1-124) eluted at approximately 250 mM NaCl. Fractions containing hSTAT4(1-124) were pooled and dialyzed against 25 mM sodium phosphate, 12 mM sodium formate, 12 mM sodium acetate (pH 5.25), 5 mM DTT at 4 °C. This material was then passed over a cation exchange column (SP-Sepharose, 1.6 × 10 cm, Amersham Pharmacia Biotech), and the bound protein was eluted from the column with a linear gradient of 0-1 M NaCl in 25 mM sodium phosphate, 12 mM sodium formate, 12 mM sodium acetate (pH 5.25), 5 mM DTT. Again, hSTAT4(1-124) eluted at approximately 250 mM NaCl. The fractions containing hSTAT4(1-124) were pooled, concentrated to at least 2 mg/ml, and dialyzed against 20 mM sodium acetate (pH 5.25), 25 mM NaCl, 5 mM DTT at 4 °C. The final polishing step was performed on a preparative-grade gel filtration column (Superdex 75, 1.6 × 60 cm, Amersham Pharmacia Biotech) equilibrated with the same buffer.
CD Spectroscopy-- The CD spectrum was recorded on a JASCO 720 spectropolarimeter. The protein sample was 23 µM in 20 mM sodium acetate-d3 (pH 5.3), 50 mM NaCl, 5 mM DTT. Spectra were recorded from 195 to 350 nm using a 0.1-mm path length demountable cell (Uvonics) at 22 °C. Ellipticity was calculated per residue.
NMR Spectroscopy-- All NMR spectra were acquired on a Varian Unity-plus 600 spectrometer equipped with a Z-spec triple resonance, pulsed-field gradient probe (Nalorac Corp., Martinez, CA) at 25 °C. Self-diffusion measurements were made using the water-sLED experiment as described (12, 13), with a diffusion time of 134 ms, a recycle delay of 10 s, and 128 transients for each of the 25 values of the pulsed-field gradient ranging from 4 to 42 G/cm. The 1H-15N HSQC spectrum was acquired using coherence selection (14) and flipback pulses (15). 15N T1 and T2 relaxation times were measured using sensitivity enhanced experiments (16). The protein concentration for all NMR experiments was 0.8 mM in 20 mM sodium acetate-d3 (pH 5.3), 50 mM NaCl, 5 mM DTT, 90% H2O/10% D2O.
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RESULTS |
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Overproduction of hSTAT4(1-124) in E. coli-- Xu et al. (5) produced amino acids 1-124 of human STAT4 as a hexahistidine-tagged polypeptide in E. coli and demonstrated that this fragment could competitively inhibit the cooperative binding of STAT4 homodimers to adjacent low-affinity GAS sites. This result indicates that the isolated domain is capable of folding into an "active" conformation in the E. coli cytoplasm. We elected to express the same fragment of human STAT4 in an untagged (native) form to alleviate any concern that the polyhistidine tag might reduce the solubility or otherwise alter the behavior of the protein in solution. Accordingly, residues 1-124 were amplified from a cDNA clone by PCR and inserted into the bacteriophage T7 promoter vector pET3d (10), as described under "Materials and Methods."
We noted that this interval of the human STAT4 cDNA contains four arginine codons that are rarely used in E. coli (AGG and AGA), including two consecutive ones. Several studies have demonstrated that these arginine codons, which are frequently present in eukaryotic cDNAs, can significantly impair the yield of a recombinant protein in E. coli (17, 18) or even cause misincorporation of amino acids (19), particularly when they occur in tandem. Fortuitously, the pair of consecutive arginine codons in STAT4 was close enough to the carboxyl terminus of this domain that they could be mutated to alternative codons simply by incorporating the appropriate mismatches into the PCR primer that was used to amplify the coding sequence for ligation with the expression vector. Another way to circumvent the problems associated with rare arginine codons is to overproduce the cognate tRNA (the product of the argU gene) on a compatible plasmid vector (17, 19). Thus, as a precautionary measure, cells also contained the argU plasmid pDC952 (20). E. coli BL21/DE3 cells containing the hSTAT4(1-124) expression vector (pDW474) and pDC952 were grown to mid-log phase in LB broth at 37 °C and induced for several hours with isopropyl-1-thio-
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Purification of hSTAT4(1-124)-- We used a combination of bulk fractionation methods and column chromatography to purify the recombinant hSTAT4(1-124) to homogeneity from an E. coli cell extract. The purity was monitored by SDS-PAGE after each step (Fig. 2A). The cells were lysed with a French press and then the nucleic acids and some endogenous proteins were precipitated by the addition of PEI. Next, additional E. coli proteins were precipitated by adding ammonium sulfate to a final concentration of 20% (w/v), which is the highest concentration that did not precipitate any of the hSTAT4(1-124). After the insoluble debris was removed by centrifugation, the ammonium sulfate concentration was increased to 60% (w/v), at which point nearly all of the hSTAT4(1-124) was recovered in the pellet. Some contaminating proteins remained in the supernatant at this point, which was discarded. Following bulk fractionation with PEI and ammonium sulfate, the hSTAT4(1-124) was already approximately 50% pure, as gauged by densitometric scanning of SDS-polyacrylamide gels (Fig. 2A, lane 5).
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CD Study--
The secondary structure of hSTAT4(1-124) is
predicted (22) to be composed entirely of -helices and connecting
loops, with no
-structure (Fig.
3A). As shown in Fig.
3B, the far-UV CD spectrum of hSTAT4(1-124) corroborates
this prediction. The CD spectrum exhibits a strong peak of negative
ellipticity at 222 nm, which is characteristic of a high degree of
-helical character and further serves to demonstrate that
hSTAT4(1-124) has a well ordered structure in solution. Quantitative
analysis of the spectrum yields an estimate of ~75%
-helix (23),
which is in good agreement with the predicted secondary structure (Fig.
3A).
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NMR Analysis--
Because it was proposed that the amino-terminal
domain of STAT4 mediates cooperative DNA binding through
self-association, we sought to determine whether the isolated domain
exists as a dimer in solution. Initially, we used NMR pulsed-field
gradient self-diffusion measurements (12) as an experimental tool for this purpose. The data obtained from these experiments are summarized in Fig. 4. The self-diffusion
coefficient, DS, of 0.86 × 106 cm2 s
1 corresponds to a
dimer for hSTAT4(1-124), based on comparisons with two monomeric
proteins of similar size (lysozyme, 14.1 kDa, DS = 1.04 × 10
6 cm2 s
1;
NusB, 15.7 kDa, DS = 1.01 × 10
6 cm2 s
1) and a dimeric
protein, which is slightly larger than hSTAT4(1-124) (IL-10, 37.4 kDa
dimer, DS = 0.82 × 10
6
cm2 s
1) (12, 13).
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DISCUSSION |
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Although it is not required for binding to single, high-affinity
GAS sites, the amino-terminal domain of STAT4 is essential for
cooperative binding to tandem arrays of low affinity sites within the
IFN- gene and for the stimulation of its transcription in response
to IL-12 (5). Like IFN-
, many genes probably will utilize tandem
arrays of low-affinity sites for activation. If so, then the
amino-terminal domains of the STATs may be attractive targets for
therapeutic agents that seek to attenuate cytokine signal
transduction.
The experiments reported here indicate that hSTAT4(1-124) has a well ordered conformation in solution that is amenable to structure determination by heteronuclear NMR spectroscopy. Furthermore, they reveal that hSTAT4(1-124) forms a stable, symmetric homodimer at high micromolar concentrations. The existence of a dimeric form of hSTAT4(1-124) is consistent with current models of transcriptional activation, which postulate that cooperative binding of STATs to adjacent low affinity GAS sites involves a physical interaction between their amino-terminal domains (2, 5, 6).
But does the hSTAT4(1-124) dimer that we observe in solution at high micromolar concentrations have any physiological relevance? It has not been possible to detect a similar interaction between the amino-terminal domains of native (i.e. full-length) STATs in solution (22). Rather, such an association is evident only when pairs of STAT dimers are bound cooperatively to adjacent sites on DNA (5, 6). It is not known whether the failure to detect this interaction in the absence of DNA is because of its inherently weak nature or whether the higher order structural organization of the STATs somehow precludes an intermolecular association between amino-terminal domains in solution. Because of its unusually low exctinction coefficient, it will be difficult to determine the equilibrium dissociation constant for the hSTAT4(1-124) dimer. Using the technique of dynamic light scattering, however, we have been able to show that hSTAT4(1-124) is unquestionably dimeric at 10 µM, the lowest concentration at which we can obtain reliable data with our instrument (data not shown). Thus, the actual dissociation constant is probably on the order of 1 µM or less. This value is several orders of magnitude lower than the protein concentration in the NMR experiments and not much greater than what is generally regarded as a physiologically meaningful concentration for an intracellular protein. Besides, in the presence of DNA, which forms a bridge between two pairs of STAT dimers, the local concentration of the amino-terminal domains is probably much greater than 10 µM. For these reasons, we do not believe that the hSTAT4(1-124) dimer is merely an artifact that arises at unnaturally high protein concentrations.
If, as we expect, the structure of the hSTAT4(1-124) dimer is physiologically relevant, then this information could be exploited to design compounds that will bind to the monomeric form of the amino-terminal domain and prevent its self-association. In this regard, it is interesting to note that almost all of the amino acid side chains that compose the dimer interface in the crystal structure of murine STAT4(1-124) are not conserved among the different STATs (22). This suggests that it should be possible to develop compounds of this type with high specificity for a particular STAT.
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FOOTNOTES |
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* This work was supported by the NCI, DHHS, under contract with ABL.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These authors contributed equally to this work.
To whom correspondence should be addressed. Tel.:
301-846-1842; Fax: 301-846-7148; E-mail: waughd{at}ncifcrf.gov.
1
The abbreviations used are: JAK, Janus kinase;
STAT, signal transducer and activator of transcription; IL-12,
interleukin-12; IFN-, interferon-
; GAS, interferon-
activation
sequence; HSQC, heteronuclear single quantum coherence; SH2,
Src-homology 2 domain; hSTAT4(1-124), a fragment of human STAT4
consisting of amino acid residues 1-124; PCR, polymerase chain
reaction; PEI, polyetheleneimine; DTT, dithiothreitol; PAGE,
polyacrylamide gel electrophoresis; BisTris, 2,2 bis(hydroxymethyl)-2,2',2"-nitrilotriethanol.
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REFERENCES |
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