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• W2085395791 abstract "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-γ activation sequences (GAS elements) near the interferon-γ 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-γ 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 (τc) 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. 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-γ activation sequences (GAS elements) near the interferon-γ 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-γ 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 (τc) 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. 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-STAT 1The abbreviations used are: JAK, Janus kinase; STAT, signal transducer and activator of transcription; IL-12, interleukin-12; IFN-γ, interferon-γ; GAS, interferon-γ 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. 1The abbreviations used are: JAK, Janus kinase; STAT, signal transducer and activator of transcription; IL-12, interleukin-12; IFN-γ, interferon-γ; GAS, interferon-γ 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. pathway (1Pellegrini S. Dusanterfourt I. Eur. J. Biochem. 1997; 248: 615-633Crossref PubMed Scopus (236) Google Scholar, 2Darnell J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3345) Google Scholar, 3Schindler C. Darnell J.E. Annu. Rev. Biochem. 1995; 64: 621-651Crossref PubMed Scopus (1640) Google Scholar, 4Darnell J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (4948) Google Scholar). 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-γ activation sequence (GAS) elements.Thus far, only seven different STATs and four JAK family members have been identified (2Darnell J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3345) Google Scholar), 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 (2Darnell J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3345) Google Scholar). Recently, Xu et al. (5Xu X. Sun Y.-L. Hoey T. Science. 1996; 273: 794-797Crossref PubMed Scopus (405) Google Scholar) characterized authentic binding sites for STATs 1, 4, 5, and 6 within the first intron of the interferon-γ (IFN-γ) 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 (5Xu X. Sun Y.-L. Hoey T. Science. 1996; 273: 794-797Crossref PubMed Scopus (405) Google Scholar). Xu et al. (5Xu X. Sun Y.-L. Hoey T. Science. 1996; 273: 794-797Crossref PubMed Scopus (405) Google Scholar) 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 (6Vinkemeier U. Cohen S.L. Moarefi I. Chait B.T. Kuriyan J. Darnell J.E. EMBO J. 1996; 15: 5616-5626Crossref PubMed Scopus (247) Google Scholar), and it seems likely that the other STATs also use their amino-terminal domains for cooperative binding (2Darnell J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3345) Google Scholar).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 (7Seder R.A. Kelsall B.L. Jankovic D. J. Immunol. 1996; 157: 2745-2748PubMed Google Scholar, 8Trembleau S. Germann T. Gately M.K. Adorini L. Immunol. Today. 1995; 16: 383-386Abstract Full Text PDF PubMed Scopus (267) Google Scholar). Cooperative binding of STAT4 dimers to adjacent low-affinity GAS sites is required for transcriptional activation of IFN-γ (5Xu X. Sun Y.-L. Hoey T. Science. 1996; 273: 794-797Crossref PubMed Scopus (405) Google Scholar), which is thought to mediate many of the effects of IL-12 (9Trinchieri G. Annu. Rev. Immunol. 1995; 13: 251-276Crossref PubMed Scopus (2217) Google Scholar). 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 coliand 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.DISCUSSIONAlthough 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-γ gene and for the stimulation of its transcription in response to IL-12 (5Xu X. Sun Y.-L. Hoey T. Science. 1996; 273: 794-797Crossref PubMed Scopus (405) Google Scholar). Like IFN-γ, 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 (2Darnell J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3345) Google Scholar, 5Xu X. Sun Y.-L. Hoey T. Science. 1996; 273: 794-797Crossref PubMed Scopus (405) Google Scholar, 6Vinkemeier U. Cohen S.L. Moarefi I. Chait B.T. Kuriyan J. Darnell J.E. EMBO J. 1996; 15: 5616-5626Crossref PubMed Scopus (247) Google Scholar).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 (22Vinkemeier U. Moarefi I. Darnell J.E. Kuriyan J. Science. 1998; 279: 1048-1052Crossref PubMed Scopus (212) Google Scholar). Rather, such an association is evident only when pairs of STAT dimers are bound cooperatively to adjacent sites on DNA (5Xu X. Sun Y.-L. Hoey T. Science. 1996; 273: 794-797Crossref PubMed Scopus (405) Google Scholar, 6Vinkemeier U. Cohen S.L. Moarefi I. Chait B.T. Kuriyan J. Darnell J.E. EMBO J. 1996; 15: 5616-5626Crossref PubMed Scopus (247) Google Scholar). 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 (22Vinkemeier U. Moarefi I. Darnell J.E. Kuriyan J. Science. 1998; 279: 1048-1052Crossref PubMed Scopus (212) Google Scholar). This suggests that it should be possible to develop compounds of this type with high specificity for a particular STAT. 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-STAT 1The abbreviations used are: JAK, Janus kinase; STAT, signal transducer and activator of transcription; IL-12, interleukin-12; IFN-γ, interferon-γ; GAS, interferon-γ 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. 1The abbreviations used are: JAK, Janus kinase; STAT, signal transducer and activator of transcription; IL-12, interleukin-12; IFN-γ, interferon-γ; GAS, interferon-γ 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. pathway (1Pellegrini S. Dusanterfourt I. Eur. J. Biochem. 1997; 248: 615-633Crossref PubMed Scopus (236) Google Scholar, 2Darnell J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3345) Google Scholar, 3Schindler C. Darnell J.E. Annu. Rev. Biochem. 1995; 64: 621-651Crossref PubMed Scopus (1640) Google Scholar, 4Darnell J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (4948) Google Scholar). 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-γ activation sequence (GAS) elements. Thus far, only seven different STATs and four JAK family members have been identified (2Darnell J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3345) Google Scholar), 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 (2Darnell J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3345) Google Scholar). Recently, Xu et al. (5Xu X. Sun Y.-L. Hoey T. Science. 1996; 273: 794-797Crossref PubMed Scopus (405) Google Scholar) characterized authentic binding sites for STATs 1, 4, 5, and 6 within the first intron of the interferon-γ (IFN-γ) 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 (5Xu X. Sun Y.-L. Hoey T. Science. 1996; 273: 794-797Crossref PubMed Scopus (405) Google Scholar). Xu et al. (5Xu X. Sun Y.-L. Hoey T. Science. 1996; 273: 794-797Crossref PubMed Scopus (405) Google Scholar) 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 (6Vinkemeier U. Cohen S.L. Moarefi I. Chait B.T. Kuriyan J. Darnell J.E. EMBO J. 1996; 15: 5616-5626Crossref PubMed Scopus (247) Google Scholar), and it seems likely that the other STATs also use their amino-terminal domains for cooperative binding (2Darnell J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3345) Google Scholar). 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 (7Seder R.A. Kelsall B.L. Jankovic D. J. Immunol. 1996; 157: 2745-2748PubMed Google Scholar, 8Trembleau S. Germann T. Gately M.K. Adorini L. Immunol. Today. 1995; 16: 383-386Abstract Full Text PDF PubMed Scopus (267) Google Scholar). Cooperative binding of STAT4 dimers to adjacent low-affinity GAS sites is required for transcriptional activation of IFN-γ (5Xu X. Sun Y.-L. Hoey T. Science. 1996; 273: 794-797Crossref PubMed Scopus (405) Google Scholar), which is thought to mediate many of the effects of IL-12 (9Trinchieri G. Annu. Rev. Immunol. 1995; 13: 251-276Crossref PubMed Scopus (2217) Google Scholar). 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 coliand 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. DISCUSSIONAlthough 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-γ gene and for the stimulation of its transcription in response to IL-12 (5Xu X. Sun Y.-L. Hoey T. Science. 1996; 273: 794-797Crossref PubMed Scopus (405) Google Scholar). Like IFN-γ, 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 (2Darnell J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3345) Google Scholar, 5Xu X. Sun Y.-L. Hoey T. Science. 1996; 273: 794-797Crossref PubMed Scopus (405) Google Scholar, 6Vinkemeier U. Cohen S.L. Moarefi I. Chait B.T. Kuriyan J. Darnell J.E. EMBO J. 1996; 15: 5616-5626Crossref PubMed Scopus (247) Google Scholar).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 (22Vinkemeier U. Moarefi I. Darnell J.E. Kuriyan J. Science. 1998; 279: 1048-1052Crossref PubMed Scopus (212) Google Scholar). Rather, such an association is evident only when pairs of STAT dimers are bound cooperatively to adjacent sites on DNA (5Xu X. Sun Y.-L. Hoey T. Science. 1996; 273: 794-797Crossref PubMed Scopus (405) Google Scholar, 6Vinkemeier U. Cohen S.L. Moarefi I. Chait B.T. Kuriyan J. Darnell J.E. EMBO J. 1996; 15: 5616-5626Crossref PubMed Scopus (247) Google Scholar). 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 (22Vinkemeier U. Moarefi I. Darnell J.E. Kuriyan J. Science. 1998; 279: 1048-1052Crossref PubMed Scopus (212) Google Scholar). This suggests that it should be possible to develop compounds of this type with high specificity for a particular STAT. 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-γ gene and for the stimulation of its transcription in response to IL-12 (5Xu X. Sun Y.-L. Hoey T. Science. 1996; 273: 794-797Crossref PubMed Scopus (405) Google Scholar). Like IFN-γ, 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 (2Darnell J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3345) Google Scholar, 5Xu X. Sun Y.-L. Hoey T. Science. 1996; 273: 794-797Crossref PubMed Scopus (405) Google Scholar, 6Vinkemeier U. Cohen S.L. Moarefi I. Chait B.T. Kuriyan J. Darnell J.E. EMBO J. 1996; 15: 5616-5626Crossref PubMed Scopus (247) Google Scholar). 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 (22Vinkemeier U. Moarefi I. Darnell J.E. Kuriyan J. Science. 1998; 279: 1048-1052Crossref PubMed Scopus (212) Google Scholar). Rather, such an association is evident only when pairs of STAT dimers are bound cooperatively to adjacent sites on DNA (5Xu X. Sun Y.-L. Hoey T. Science. 1996; 273: 794-797Crossref PubMed Scopus (405) Google Scholar, 6Vinkemeier U. Cohen S.L. Moarefi I. Chait B.T. Kuriyan J. Darnell J.E. EMBO J. 1996; 15: 5616-5626Crossref PubMed Scopus (247) Google Scholar). 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 (22Vinkemeier U. Moarefi I. Darnell J.E. Kuriyan J. Science. 1998; 279: 1048-1052Crossref PubMed Scopus (212) Google Scholar). This suggests that it should be possible to develop compounds of this type with high specificity for a particular STAT." @default.
• W2085395791 created "2016-06-24" @default.
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• W2085395791 date "1998-07-01" @default.
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• W2085395791 title "The Amino-terminal Domain of Human STAT4" @default.
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