Method of using a crystal of the N-terminal domain of a signal transducer and activator of transcription

Abstract

The present invention provides a crystal containing the N-terminal domain of a STAT protein that is of sufficient quality to perform X-ray crystallographic studies. Methods of preparing the crystals are include in the invention. The present invention further discloses the three-dimensional structure of the crystal. The present invention also provides methods of using the structural information in drug discovery and drug development.

Description

GOVERNMENTAL SUPPORT

FIELD OF THE INVENTION

[0002] The present invention relates generally to structural studies of STAT proteins, modified STAT proteins and more particularly the N-terminal domain of STAT proteins. Included in the present invention is a crystal of the N-terminal domain of a STAT protein and corresponding structural information obtained by X-ray crystallography. The present invention also relates to methods of using the crystal and related structural information in drug screening assays.

BACKGROUND OF THE INVENTION

[0003] Transcription factors play a major role in cellular function by inducing the transcription of specific mRNAs. Transcription factors, in turn, are controlled by distinct signaling molecules. The STATs (Signal Transducer and Activator of Transcription) constitute a family of transcription factors necessary to activate distinct sets of target genes in response to cytokines and growth factors [Darnell et al. WO 95/08629, (1995)]. The STAT proteins are activated in the cytoplasm by phosphorylation on a single tyrosine residue [Darnell et al., Science 264:1415 (1994)]. The responsible kinases are either ligand-activated transmembrane receptors with intrinsic tyrosine kinase activity, such as EGF- or PDGF-receptors, or cytokine receptors that lack intrinsic kinase activity but have associated JAK kinases, such as those for interferons and interleukins [Ihle, Nature 377:591-594 (1995)]. One distinctive characteristic of the STAT proteins are their apparent lack of requirement for changes in second messenger, e.g., cAMP or Ca++, concentrations. Presently, there are seven known mammalian STAT family members. The recent discovery of a Drosophila STAT protein, suggests that these proteins have played an important role in signal transduction since the early stages of our evolution [Darnell, PNAS, 94:11767-11769 (1997)].

[0004] Each STAT protein contains a SRC homology domain (SH2 domain). When activated, the STAT proteins are phosphorylated, and form homo- or heterodimeric structures in which the phosphotyrosine of one partner binds to the SH2 domain of the other. The reciprocal SH2-phosphotyrosine interactions between two STAT proteins result in the formation of an active dimer that translocates to the nucleus and activates specific gene expression [Darnell et al., Science 264:1415 (1994)] by binding to a canonical recognition site for the STAT dimer. This canonical recognition site encompasses 9-10 base pairs (TTCN3-4GAA) of DNA [Horvath et al., Genes & Devel. 9:984 (1995); Seidel et al., Proc. Natl. Acad. Sci. USA 92:3041 (1995); Ihle, Cell 84:331 (1996); Mikita et al., Mol. Cell. Biol. 16:5811 (1996)]. Analysis of the binding of activated STATs to DNA targets has revealed that the STAT binding sites can extend over two or more adjacent canonical sites [Xu et al., Science 273:750 (1996); Meier and Groner, Mol. Cell. Biol. 14:128 (1994); Symes et al., Molecular Endocrinology 8;1750 (1994); Dajee et al., Molecular Endocrinology 10:171 (1996); John et al., EMBO J. 15:5627 (1996)].

[0005] STAT proteins serve in the capacity as a direct messengers between the cytokine or growth factor receptor present on the cell surface, and the cell nucleus. However, since each cytokine and growth factor produce a specific cellular effect by activating a distinct set of genes, the means in which such a limited number of STAT proteins mediate this result remains a mystery. Indeed, at least twenty-five different ligand-receptor complexes signal the nucleus through the seven known mammalian STAT proteins [Yan et al., Cell 84:421-430 (1996)].

[0006] There is increasing evidence that mammalian transcription factors activate transcription and achieve biological specificity by interactions with other transcription factors, trans-activators or the general transcription machinery [McKnight, Genes & Development 10:367 (1996); Roeder, Trends in Biochemical Sciences 21:327 (1996)]. Although the molecular basis for these phenomena is poorly understood, direct protein:protein interactions among multiple promoter bound proteins appear to mediate this synergistic activation [Tijan and T. Maniatis, Cell 77:5 (1994)].

[0007] In the case of the STATs, a small N-terminal domain has been shown to mediate a number of important protein:protein interactions that influence transcriptional outcome [Leung et al., Science 273:750 (1996); Vinkemeier et al., EMBO J. 15: 5616 (1996)]. This domain allows cooperative interactions between STAT dimers bound to adjacent target sites on DNA, leading to a drastically prolonged half-life of the protein-DNA complex [Vinkemeier et al., EMBO J 15: 5616 (1996)]. Functional assays exploring the induction of the hepatic Spi 2.1 gene revealed the necessity for cooperative STAT binding to two adjacent recognition sites for a full growth hormone response [Bergad et al., J. Biol. Chem. 270, 24903 (1995)]. In addition, it was observed that these cooperative contacts affect the binding site selection of different STATs on a natural promoter that contains multiple potential STAT recognition sites [Xu et al., Science 273:750 (1996)]. Each of the oligomerized STAT-1, -4, and -5 dimers were shown to bind to a different combination of canonical sites [Xu et al., Science 273:750 (1996)]. Deletion of the N-terminal ˜100 residues of STAT-1 and STAT-4 abolishes cooperative binding to DNA [Xu et al., Science 273:750 (1996); Vinkemeier et al., EMBO J 15: 5616 (1996)]. The truncated protein fully retains binding to a single target site as a dimer, suggesting that the N-terminal domain is dispensable for dimer formation and DNA binding [Xu et al., Science 273:750 (1996); Vinkemeier et al., EMBO J. 15: 5616 (1996)], but is necessary for interaction between STAT dimers and binding site discrimination [Xu et al., Science 273:750 (1996)]. Also, the N-domain of STAT-1 is required for interaction between STAT-1 and the transcriptional co-activator protein CBP, a large (˜2500 amino acids) polypeptide with transacetylase activity [Zhang et al., Proc. Natl Acad. Sci. USA 93:15092 (1996)]. Additionally, the amino-terminal region of STAT-2 is involved in binding to the intracellular region of the interferon-a receptor [Leung et al., Mol. Cell. Biol. 15:1312 (1995)].

[0008] Therefore, there is a need to obtain agonists and antagonists that can modulate the effect of STAT proteins during specific gene activation. In particular, there is a need to obtain drugs that will directly interact with the important N-terminal domain of STAT proteins. Unfortunately, identification of such drugs have heretofore relied on serendipity and/or systematic screening of large numbers of natural and synthetic compounds. A far superior method of drug-screening relies on structure based drug design. In this case, the three dimensional structure of a protein or protein fragment is determined and potential agonists and/or potential antagonists are designed with the aid of computer modeling [Bugg et al., Scientific American, December:92-98 (1993); West et al., TIPS, 16:67-74 (1995)]. However, heretofore the three-dimensional structure of a STAT protein or fragment thereof has remained unknown, essentially because no such protein crystals had been produced of sufficient quality to allow the required X-ray crystallographic data to be obtained.

[0009] Therefore, there is presently a need for obtaining an N-terminal STAT domain fragment that can be crystallized to form a crystal with sufficient quality to allow such crystallographic data to be obtained. Further, there is a need for such crystals. Furthermore there is a need for the determination of the three-dimensional structure of such crystals. Finally, there is a need for procedures for related structural based drug design based on such crystallographic data.

[0010] The citation of any reference herein should not be construed as an admission that such reference is available as “Prior Art” to the instant application.

SUMMARY OF THE INVENTION

[0011] The present invention provides a crystal containing the N-terminal domain of a STAT protein which effectively diffracts X-rays and thereby allows the determination of the atomic coordinates of the N-terminal domain to a resolution of greater than 5.0 Angstroms. In a preferred embodiment of this type the crystal effectively diffracts X-rays for the determination of the atomic coordinates of the N-terminus to a resolution of greater than 3.0 Angstroms. In a more preferred embodiment of this type the crystal effectively diffracts X-rays for the determination of the atomic coordinates of the N-terminus to a resolution of greater than 2.0 Angstroms.

[0012] In one embodiment the N-terminal domain of the crystal comprises the amino acid sequence of:

[0013] Arg Xaa HXaa Leu Xaa Xaa Trp HXaa Glu Xaa Gln Xaa Trp (SEQ ID NO: 1), where HXaa can be either Ile, Leu, Val, Phe, or Tyr and Xaa can be any amino acid. In another embodiment the crystal of the N-terminal domain of the STAT protein is contained in a STAT fragment that consists of 100 to 150 amino acids. In a preferred embodiment the STAT fragment comprises amino acids 4-112 of SEQ ID NO:2. In a more preferred embodiment the crystal contains an N-terminal domain of a STAT protein comprising amino acid residues 2-123 of SEQ ID NO:2 with 5 additional amino acid residues N-terminal to amino acid residue number 2, i.e., from the N-terminus GLY SER GLY GLY GLY, amino acid residue 2. In one embodiment of this type the crystal effectively diffracts X-rays to allow the determination of the atomic coordinates of the N-terminus to a resolution of 1.45 Angstroms.

[0014] The present invention provides a crystal of the N-terminal domain having a space group of P6522 and a unit cell of dimensions a=79.51 Å, b=79.51 Å, and c=84.68 Å. The present invention further provides a crystal of the N-terminal domain having secondary structural elements comprising eight helices (&agr;1-&agr;8) that are assembled into a hook-like structure that has an inner and outer surface. The first four helices (&agr;1-&agr;4) form a ring-shaped element having a proximal and a distal surface, whereas helices six (&agr;6) and seven (&agr;7) form an anti-parallel coiled-coil that also has a proximal and a distal surface. Helix five (&agr;5) connects the ring-shaped element to the anti-parallel coiled-coil, while helix eight (&agr;8) is wrapped around the distal surface of the ring-shaped element. The inner surface of the hook-like structure is formed by the intersection of the proximal surface of the ring-shaped element with the proximal surface of the antiparallel coiled-coil.

[0015] The present invention also provides a method of growing a crystal of an N-terminal domain of a STAT protein. The method of making the crystal comprises placing an aliquot of a solution containing a STAT N-terminal domain fragment of the present invention on a cover slip as a hanging drop above a well containing a reservoir buffer that comprises 0.2 M Na+CH3COO−, 0.1 M Tris/HCL pH 8.0, 17% PEG4000. All of the STAT N-terminal domain fragments of the present invention may be used in this manner to prepare such a crystal. In one specific embodiment the solution containing a STAT N-terminal domain fragment is prepared by combining a preparation of the STAT N-terminal domain fragment that contains 20 mg/ml of the STAT N-terminal domain fragment in 50 mM Hepes/HCl pH 8.0, 150 mM KCl, 2.5 mM CaCl2, and 5 mM DTT with an equal volume of the reservoir buffer. In a preferred embodiment the aliquot comprises approximately 1 &mgr;l of the solution. In another preferred embodiment the STAT N-terminal domain fragment comprises the amino acid sequence of:

[0016] Arg Xaa HXaa Leu Xaa Xaa Trp HXaa Glu Xaa Gln Xaa Trp (SEQ ID NO: 1), where HXaa can be either Ile, Leu, Val, Phe, or Tyr. In a more preferred embodiment the N-terminal domain is a STAT-4 N-terminal domain fragment. In a still more preferred embodiment the STAT-4 N-terminal domain fragment contains amino acid residues 2-123 of SEQ ID NO:2 with 5 additional amino acid residues N-terminal to amino acid residue number 2, i.e., from the N-terminus GLY SER GLY GLY GLY, amino acid residue 2.

[0017] The present invention also provides methods of screening drugs that either enhance or inhibit STAT-STAT dimeric interactions. Such methods include those that identify drugs that effect the interaction of N-terminal domains of STAT proteins that are bound to adjacent DNA binding sites. In one such embodiment, a drug library is screened by assaying the binding activity of a STAT protein to its DNA binding site. This assay is based on the ability of the N-terminal domain of STAT proteins to substantially enhance the binding affinity of two adjacent STAT dimers to a pair of closely aligned DNA binding sites, i.e., binding sites separated by approximately 10 to 15 base pairs. Such drug libraries include phage libraries as described below, chemical libraries compiled by the major drug manufacturers, mixed libraries, and the like. Any of such compounds contained in the drug libraries are suitable for testing as a prospective drug in the assays described below, including in a high throughput assay based on the methods described below.

[0018] An antagonist of the STAT N-terminal dimeric interaction antagonizes one or more aspects of the binding of the STAT dimers to adjacent weak binding sites for the STAT dimers on a promoter of a gene. Such antagonists could be useful as drugs in the treatment of a variety of disease states, including inflammation, allergy, asthma, and leukemias.

[0019] On the other hand, a drug that acts as an agonist stabilizes the N-terminal dimeric interaction between STAT dimers bound to adjacent weak binding sites for the STAT dimers on a promoter of a gene, thereby enhancing STAT function. Such agonists can be used as drugs that are useful in the treatment of anemias, neutropenias, thrombocytopenia, cancer, obesity, viral diseases and growth retardation, or other diseases characterized by an insufficient STAT activity.

[0020] Therefore the present invention provides a method of using the crystals of the present invention in drug screening assays. In one such embodiment the method comprises selecting a potential drug by performing rational drug design with the three-dimensional structure determined for the crystal. The selecting is preferably performed in conjunction with computer modeling. The potential drug is contacted with a dimeric STAT protein N-terminal domain and the binding of the potential drug with the N-terminal domain is detected. A drug is selected which binds to the N-terminal domain of the dimeric STAT protein.

[0021] In a preferred embodiment of this type, contacting the potential drug with a dimeric STAT protein N-terminal domain is performed with dimeric STAT protein fragments containing the STAT protein N-terminal domain. In a more preferred embodiment the STAT protein N-terminal domain has an amino acid sequence comprising SEQ ID NO: 1. In one such embodiment the dimeric STAT protein fragment is labeled. In another such embodiment the dimeric STAT protein fragment is bound to a solid support.

[0022] Another method of using a crystal of the present invention in a drug screening assay comprises selecting a potential drug by performing rational drug design with the three-dimensional structure determined for the crystal. The selecting is preferably performed in conjunction with computer modeling. The potential drug is contacted with two or more dimeric STAT proteins in the presence of a nucleic acid containing at least two adjacent weak binding sites for STAT protein dimers and the effect of the potential drug on the binding of the dimeric STAT proteins to each other and/or to the nucleic acid is detected. A potential drug is selected as a candidate drug when it either enhances or diminishes the binding of the dimeric STAT proteins to each other and/or the nucleic acid. In a preferred embodiment of this type the method further comprises growing a supplemental crystal containing a protein-drug complex formed between the dimeric N-terminal domain of the STAT protein and the candidate drug. A crystal is chosen that effectively diffracts X-rays allowing the determination of the atomic coordinates of the protein-ligand complex to a resolution of greater than 5.0 Angstroms, preferably to a resolution greater than 3.0 Angstroms and more preferably to a resolution greater than 2.0 Angstroms. The three-dimensional structure of the supplemental crystal is determined by molecular replacement analysis and a drug is selected by performing rational drug design with the three-dimensional structure determined for the supplemental crystal. The selecting is preferably performed in conjunction with computer modeling.

[0023] The present invention also provides a method for identifying a drug that modulates the ability of adjacent STAT protein dimers to interact and bind to adjacent DNA binding sites. One such embodiment comprises selecting a potential drug by performing rational drug design with the three-dimensional structure determined for a crystal of the present invention. The selecting is preferably performed in conjunction with computer modeling. The binding affinity of the STAT protein (or of a fragment thereof that comprises the N-terminal domain) for a nucleic acid comprising two adjacent weak STAT DNA binding sites in the presence and absence of the potential drug is determined (i.e., measured). The binding affinity of the STAT protein (or the fragment) for a nucleic acid comprising a single strong STAT binding site in the presence and absence of the potential drug is also determined. Next a comparison is made between the binding affinities of the STAT protein (or the fragment) is measured for the two adjacent weak STAT DNA binding sites in the presence and absence of the potential drug with that determined for the STAT protein (or the fragment) for the single strong STAT binding site in the presence and absence of the potential drug. A potential drug which causes an increase in the binding affinity measured for the two adjacent weak STAT DNA binding sites but not in the binding affinity measured for the single strong STAT binding site is identified as a drug that enhances the interaction between adjacent activated STAT dimers. On the other hand, a potential drug which causes a decrease in the binding affinity measured for the two adjacent weak STAT DNA binding sites but not in the binding affinity measured for the single strong STAT binding site is identified as a drug that inhibits the interaction between adjacent activated STAT dimers.

[0024] The present invention further provides a method for identifying a drug that modulates the ability of adjacent STAT protein dimers to interact and bind to adjacent DNA binding sites which comprises measuring the binding affinity of the STAT protein comprising a functional N-terminal domain to a nucleic acid comprising two adjacent weak STAT DNA binding sites in the presence and absence of a potential drug. The binding affinity of a modified form of the STAT protein that lacks a functional N-terminal domain is also determined for the nucleic acid in the presence and absence of the potential drug. The binding affinity measured for the STAT protein in the presence and absence of the potential drug is then compared with the binding affinity measured for the modified STAT protein in the presence and absence of the potential drug. A potential drug which causes an increase in the binding affinity measured for the functional STAT protein but not in the binding affinity measured for the modified STAT protein is identified as a drug that enhances the interaction between adjacent activated STAT dimers, and a potential drug which causes a decrease in the binding affinity measured for the STAT protein but not in the binding affinity measured for the modified STAT protein is identified as a drug that inhibits the interaction between adjacent activated STAT dimers. Variations of these assays are performed in in situ assays as described below with reporter genes operably under the control of weak and/or strong STAT binding sites.

[0025] In one such embodiment the modified STAT protein is a STAT protein that lacks the &agr;4-tryptophan. In another such embodiment the modified Stat protein lacks the &agr;4-glutamic acid. In still another embodiment the modified STAT protein is the truncated STAT protein Stat1tc identified in by Vinkemeier et al. [EMBO J. 15:5616 (1996)].

[0026] The present invention further provides a method for identifying a drug that enhances or diminishes the ability of STAT protein dimers to induce the expression of a gene operably under the control of a promoter containing at least two adjacent weak binding sites for STAT protein dimers. One such embodiment comprises selecting a potential drug by performing rational drug design with the three-dimensional structure determined for a crystal of the present invention. The selecting is preferably performed in conjunction with computer modeling. The level of expression of a first reporter gene and a second reporter gene contained by a host cell in the presence and absence of the potential drug is determined. The first reporter gene is operably linked to a first promoter containing at least two adjacent weak binding sites for STAT protein dimers, and the second reporter gene is operably linked to a second promoter comprising at least one strong binding site for a STAT protein dimer. The binding of STAT protein dimers to the two adjacent weak binding sites induces the expression of the first reporter gene, and the binding of the STAT protein dimer to the strong binding site induces the expression of the second reporter gene. In addition the host cell either naturally contains STAT protein dimers or is modified and/or induced to contain them. The level of expression of the first reporter gene is then compared with that of the second reporter gene in the presence and absence of the potential drug. When the presence of the potential drug results in an increase in the level of expression of the first reporter gene but not that of the second reporter gene, the potential drug is identified as a drug that enhances the ability of STAT protein dimers to induce the expression of a gene operably under the control of a promoter containing at least two adjacent weak binding sites for STAT protein dimers. On the other hand, when the presence of a potential drug results in a decrease in the level of expression of the first reporter gene but not that of the second reporter gene, the potential drug is identified as a drug that inhibits the ability of STAT protein dimers to induce the expression of a gene operably under the control of a promoter containing at least two adjacent weak binding sites for STAT protein dimers.

[0027] In a preferred embodiment of this type, the method further comprises growing a supplemental crystal containing a protein-drug complex formed between the dimeric N-terminal domain and the drug. The crystal effectively diffracts X-rays allowing the determination of the atomic coordinates of the protein-ligand complex to a resolution of greater than 5.0 Angstroms, preferably to a resolution of greater than 3.0 Angstroms and more preferably to a resolution of greater than 2.0 Angstroms. The three-dimensional structure of the supplemental crystal is then determined with molecular replacement analysis. A drug is selected by performing rational drug design with the three-dimensional structure determined for the supplemental crystal. The selecting is preferably performed in conjunction with computer modeling.

[0028] In an alternative embodiment, the first reporter gene is contained by a first host cell, and the second reporter gene is contained by a second host cell. In this case, both the first host cell and second host cell contain STAT protein dimers. In one embodiment, the weak STAT binding sites are from sites present in the regulatory regions of the MIG gene. In another embodiment the weak STAT binding sites are from sites present in the regulatory regions of the c-fos gene. In still another embodiment the weak STAT binding sites are from sites present in the regulatory regions of the interferon-&ggr; gene. In a related embodiment the mutated cfos-promotor element, the M67 site [Wagner et al., EMBO J. 9:4477 (1990)] is used as the strong STAT binding site. In another embodiment of this type the strong STAT binding site is the S1 site [Horvath et al., Genes & Devel 9:984 (1995)]. In still another such embodiment strong STAT binding site is obtained from the IRF-1 gene promoter. In preferred embodiments, the host cell or host cells are mammalian cells.

[0029] These and other aspects of the present invention will be better appreciated by reference to the following drawings and Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIG. 1A shows a schematic representation of two STAT dimers bound to adjacent target sites. Interactions between N-domains (N, circled) allow the dimers to bind to each other. Phosphotyrosines are indicated as “Y” with encircled P symbols. DBD: DNA binding domain; SH2: SH2 domain; TAD: transactivation domain.

[0031] FIG. 1B shows the sequence alignment of the conserved N-terminal domain of the STAT family and secondary structure of the N-terminal domain of STAT-4. Human (hSTAT), murine (mSTAT), and Drosophila (DSTAT) proteins are included. The numbering is according to STAT-4. &agr;-Helices &agr;1 to &agr;8 are drawn as cylinders. The blackened part of helix &agr;2 indicates a 3 helix. Invariant residues are highlighted with an asterisk below the alignment. Conserved residues in the hydrophobic core are marked with filled circles above the STAT-4 sequence. The following amino acid exchanges are considered conserved: Q/N, D/E, I/LN, R/K/H, Y/F, S/T. Residues in helices &agr;6 and &agr;7 that contribute to the packing of the coiled-coil are boxed and their position in the helical repeats is indicated (a or d).

[0032] FIG. 2: Tertiary structure of the amino-terminal domain of STAT-4.

[0033] FIG. 2A depicts the overall representation of two monomers (green and gray) in the crystallographic dimer, viewed approximately orthogonal to the molecular 2-fold axis, which is vertical. The ring-shaped N-terminal element is colored red in one monomer.

[0034] FIG. 2B depicts the orthogonal view of one of the N-terminal domains shown in FIG. 2A, showing details of the architecture of the ring-shaped element. Side chains that participate in a charge stabilized hydrogen-bond network are shown in a ball-and-stick representation. The side chain and backbone carbonyl of buried Arg 31 are shown in magenta. For clarity, the indole ring of the invariant residue Trp 4 that seals off this arrangement on the proximal side is drawn with thinner bonds. The blue sphere denotes a buried water molecule. Hydrogen-bonds are indicated by dotted lines. Oxygen, nitrogen, and carbon atoms are colored red, blue and yellow, respectively. Q3-N marks the position of the backbone amide group of residue Gln 3. The light red colored segment of helix &agr;2 highlights its 310 helical conformation. FIGS. 2, 3B, and 3C were created with the program RIBBONS v2.0 [Carson, J. Appl. Cryst. 24:958 (1991)].

[0035] FIG. 3A depicts the surface representation of the N-terminal domain indicating the wedge shaped groove and the dimerization interface. Shown are two monomers of a dimer with the left one rotated 90° around the vertical axis away from the original position in the dimer. Note the hook-like appearance of the monomer with the coiled-coil of helices &agr;6 and &agr;7 pointing out of the planar surface formed by the ring-shaped element comprising the amino-terminal 40 residues. Residues from three separate regions of the N-terminal domain make direct or water mediated contacts in the dimer and are color-coded according to their position. Interface residues at the amino-terminus are in green, those in helices &agr;3 and &agr;4 are in blue and amino acids located in helix &agr;6 are yellow. The position of the critical Trp 37 is highlighted in red. The Figure was created using GRASP [Nicholls et al., Proteins. Struct. Funct. and Genetics 11:281 (1991)].

[0036] FIG. 3B shows a view at the dimerization interface with amino acids represented as ball-and-stick models and the c&agr; backbone as ribbon. The monomer is in the same orientation as the one on the right of panel of FIG. 3A. Side chains are colored according to their position following the same convention as in FIG. 3A; backbone ribbon is colored as in FIG. 2B with the first 40 residues highlighted in red. Note the entirely polar nature of the interface. Leu 33 makes a backbone carbonyl group contact and its position is represented by the filled circle. In the STAT-4 recombinant N-terminal domain used for crystallization, Met 1 was replaced with Gly plus four additional small amino acids, one of which (Gly-1) is visible in the electron density map (see Methods in the EXAMPLE below). In the crystals the amino terminus of Gly-1 is part of the dimer interface, possibly substituting for the native Met 1.

[0037] FIG. 3C shows the close-up stereo view of intermolecular hydrogen bonding network in the dimer. Selected side chains surrounding the conserved Trp 37 (magenta) in helices &agr;4 and &agr;6 of two monomers (green and grey) are shown. Trp 37 makes direct (E 66′) and water mediated contacts (Q 63′). Water molecules are depicted as blue spheres.

[0038] FIG. 4 indicates the importance of the invariant residue Trp 37 for STAT-1 tetramerization and mediation of gene activation.

[0039] FIG. 4A depicts the gel mobility shift that shows that a single point mutation disrupts STAT-1 cooperative DNA-binding. Comparison of tetramer stability between wild type (WT) STAT-1 (lanes 3 and 4) and the W37A mutant (lanes 1 and 2). Radiolabeled DNA containing a tandem binding site was preincubated with equal amounts of active protein of either tyrosine-phosphorylated WT-STAT or the mutant protein followed by a chase with excess (30 fold) unlabeled oligonucleotide for the indicated amount of time. The wild type STAT-1 shows the expected formation and stabilization of 2× (dimeric) complexes relative to the dimeric protein/DNA complex. The W37A mutant does form dimeric complexes, but only very little of the slower migrating 2× (dimeric) species. No increased stability of the 2× (dimeric) complex relative to the dimer is observed. Samples loaded at the later time point (15 minutes) were electrophoresed for shorter times and therefore run higher on the gel. The position of unbound oligonucleotide is marked (free).

[0040] FIG. 4B show the effect of STAT-1 W37A mutation on interferon-&ggr; (IFN-&ggr;) stimulated gene activation in vivo. U3A cells that are lacking STAT-1 [Miller et al., EMBO J. 12:4221 (1993)] were transfected with expression clones containing either wild type or mutant STAT-1 (see Methods of the EXAMPLE, below) along with a luciferase reporter containing a tandem STAT binding site as an enhancer. After stimulation with IFN-&ggr; for 10 hours, luciferase expression was determined spectroscopically (represented as bars). Cells transfected with the wild type protein show an about 2-fold increase in luciferase expression. In contrast, the tetramerization deficient W37A mutant does not markedly increase transcription over background levels from an enhancer with a tandem binding site. Each bar represents ten individual parallel experiments. Error bars denote standard deviation from the mean.

DETAILED DESCRIPTION OF THE INVENTION

[0041] The present invention provides the first three-dimensional structural information regarding the important family of transcription factors known as STATS. More particularly, the present invention provides a crystalline form of the N-terminal cooperative domain (more simply denoted as the N-terminal domain) of a STAT protein of sufficient quality to perform meaningful X-ray crystallographic measurements. In addition, the present invention provides a method of preparing such crystals.

[0042] In addition the present invention provides a method of using the crystals and the crystallographic measurements for drug discovery and development. These methods include procedures for screening drugs that either enhance or inhibit STAT-STAT dimer interactions, which can have a critical effect on the transcription of the specific genes under the control of STAT proteins. For example, antagonists of the STAT N-terminal dimer interaction antagonize STAT functions dependent on this aspect of STAT behavior. Drugs that are antagonists would be useful for the treatment of a variety of disease states, including but not limited to, inflammation, allergy, asthma, and leukemias. On the other hand, drugs that are found to be agonists would stabilize the N-terminal interaction between STAT dimers, thereby enhancing this aspect of the STAT function. Such drugs may therefore have utility in the treatment of anemias, neutropenias, thrombocytopenia, cancer, obesity, viral diseases and growth retardation, or other diseases characterized by a insufficient STAT activity.

[0043] Therefore, if appearing herein, the following terms shall have the definitions set out below.

[0044] As used herein the “&agr;4-tryptophan” is the conserved tryptophan common in all STAT proteins found in the N-terminal domain in an alpha helix defined as &agr;4 in FIG. 1. For mStat4 the &agr;4-tryptophan is W37 of the amino acid sequence. Similarly, the &agr;4-glutamic acid is the conserved glutamic acid common in all STAT proteins found in the N-terminal domain in an alpha helix defined as &agr;4 in FIG. 1. For mStat4 the &agr;4 glutamic acid is E39 of the amino acid sequence.

[0045] As used herein a the term “STAT protein” includes a particular family of transcription factor consisting of the Signal Transducers and Activators of Transcription proteins. These proteins have been defined in International Patent Publication No.s WO 93/19179 (30 September 1993, by James E. Darnell, Jr. et al.), WO 95/08629 (Mar. 30, 1995, by James E. Darnell, Jr. et al.) and United States application having a Ser. No. 08/212,184, filed on Mar. 11, 1994, entitled, “Interferon Associated Receptor Recognition Factors, Nucleic Acids Encoding the Same and Methods of Use Thereof” by James E. Darnell, Jr. et al., all of which are incorporated by reference in their entireties, herein. Currently, there are seven STAT family members which have been identified, numbered STAT 1, 2, 3, 4, 5A, 5B, and 6. STAT proteins include proteins derived from alternative splice sites such as Human STAT1&agr; and STAT1&bgr;, i.e., STAT1&bgr; is a shorter protein than STAT1&agr; and is translated from an alternatively spliced mRNA. Modified STAT proteins and functional fragments of STAT proteins are included in the present invention.

[0046] As used herein the terms “phosphorylated” and “nonphosphorylated” as used in conjunction with or in reference to a STAT protein denote the phosphorylation state of a particular tyrosine residue of the STAT proteins (e.g., Tyr 701 of STAT1). When STAT proteins are phosphorylated, they form homo- or heterodimeric structures in which the phosphotyrosine of one partner binds to the SRC homology domain (SH2) of the other. In their natural environment the newly formed dimer then translocates from the cytoplasm to the nucleus, binds to a palindromic GAS sequence, thereby activating transcription

[0047] The “N-terminal domain” of a STAT protein is used interchangeably herein with the “N-terminal cooperative domain” and refers to the N-terminal portion of a STAT protein involved in STAT protein dimer-dimer interaction at a weak STAT DNA binding site. Preferably the amino acid of the N-terminal domain comprises SEQ ID NO: 1. In one particular embodiment the STAT protein is STAT-4 comprising amino acids 2-123 of SEQ ID NO:2.

[0048] General Techniques for Constructing Nucleic Acids that Express Recombinant STAT Proteins

[0049] In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein “Sambrook et al., 1989”); DNA Cloning. A Practical Approach, Volumes I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins eds. (1985)]; Transcription And Translation [B. D. Hames & S. J. Higgins, eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)]; Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, A Practical Guide To Molecular Cloning (1984); F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

[0050] Therefore, if appearing herein, the following terms shall have the definitions set out below.

[0051] As used herein, the term “gene” refers to an assembly of nucleotides that encode a polypeptide, and includes cDNA and genomic DNA nucleic acids.

[0052] A “vector” is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment. A “replicon” is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo, i.e., capable of replication under its own control.

[0053] A “cassette” refers to a segment of DNA that can be inserted into a vector at specific restriction sites. The segment of DNA encodes a polypeptide of interest, and the cassette and restriction sites are designed to ensure insertion of the cassette in the proper reading frame for transcription and translation.

[0054] A cell has been “transfected” by exogenous or heterologous DNA when such DNA has been introduced inside the cell.

[0055] A “nucleic acid molecule” refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNA molecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; “DNA molecules”), or any phosphoester analogues thereof, such as phosphorothioates and thloesters, in either single stranded form, or a double-stranded helix. Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acid molecule, and in particular DNA or RNA molecule, refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5′ to 3′ direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA). A “recombinant DNA molecule” is a DNA molecule that has undergone a molecular biological manipulation.

[0056] A nucleic acid molecule is “hybridizable” to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength (see Sambrook et al., supra). The conditions of temperature and ionic strength determine the “stringency” of the hybridization. For preliminary screening for homologous nucleic acids, low stringency hybridization conditions, corresponding to a Tm of 55°, can be used, e.g., 5× SSC, 0.1% SDS, 0.25% milk, and no formamide; or 30% formamide, 5× SSC, 0.5% SDS). Moderate stringency hybridization conditions correspond to a higher Tm, e.g., 40% formamide, with 5x or 6× SCC. High stringency hybridization conditions correspond to the highest Tm, e.g., 50% formamide, 5× or 6× SCC. Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of Tm for hybrids of nucleic acids having those sequences. The relative stability (corresponding to higher Tm) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotides in length, equations for calculating Tm have been derived (see Sambrook et al., supra, 9.50-0.51). For hybridization with shorter nucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al., supra, 11.7-11.8). Preferably a minimum length for a hybridizable nucleic acid is at least about 12 nucleotides; preferably at least about 18 nucleotides; and more preferably the length is at least about 27 nucleotides; and most preferably 36 nucleotides.

[0057] In a specific embodiment, the term “standard hybridization conditions” refers to a Tm of 55° C., and utilizes conditions as set forth above. In a preferred embodiment, the Tm IS 60° C.; in a more preferred embodiment, the Tm is 65° C.

[0058] A DNA “coding sequence” is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in a cell in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5′ (amino) terminus and a translation stop codon at the 3′ (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences and synthetic DNA sequences. If the coding sequence is intended for expression in a eukaryotic cell, a polyadenylation signal and transcription termination sequence will usually be located 3′ to the coding sequence.

[0059] Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, terminators, and the like, that provide for the expression of a coding sequence in a host cell. In eukaryotic cells, polyadenylation signals are control sequences.

[0060] A “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3′ direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3′ terminus by the transcription initiation site and extends upstream (5′ direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site (conveniently defined for example, by mapping with nuclease S 1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.

[0061] A coding sequence is “under the control” of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then trans-RNA spliced and translated into the protein encoded by the coding sequence.

[0062] As used herein, the term “homologous” in all its grammatical forms refers to the relationship between proteins that possess a “common evolutionary origin,” including proteins from superfamilies (e.g., the immunoglobulin superfamily) and homologous proteins from different species (e.g., myosin light chain, etc.) (Reeck et al., 1987, Cell 50:667). Such proteins have sequence homology as reflected by their high degree of sequence similarity.

[0063] Accordingly, the term “sequence similarity” in all its grammatical forms refers to the degree of identity or correspondence between nucleic acid or amino acid sequences of proteins that may or may not share a common evolutionary origin (see Reeck et al., supra). However, in common usage and in the instant application, the term “homologous,” when modified with an adverb such as “highly,” may refer to sequence similarity and not a common evolutionary origin.

[0064] The term “corresponding to” is used herein to refer similar or homologous sequences, whether the exact position is identical or different from the molecule to which the similarity or homology is measured. Thus, the term “corresponding to” refers to the sequence similarity, and not the numbering of the amino acid residues or nucleotide bases.

[0065] A gene encoding a STAT protein, whether genomic DNA or cDNA, can be isolated from any animal source, particularly from a mammal. Methods for obtaining the STAT protein gene are well known in the art, as described above (see, e.g., Sambrook et al., 1989, supra).

[0066] A “heterologous nucleotide sequence” as used herein is a nucleotide sequence that is added to a nucleotide sequence of the present invention by recombinant methods to form a nucleic acid which is not naturally formed in nature. Such nucleic acids can encode chimeric and/or fusion proteins. Thus the heterologous nucleotide sequence can encode peptides and/or proteins which contain regulatory and/or structural properties. In another such embodiment the heterologous nucleotide can encode a protein or peptide that functions as a means of detecting the protein or peptide encoded by the nucleotide sequence of the present invention after the recombinant nucleic acid is expressed. In still another such embodiment the heterologous nucleotide can function as a means of detecting a nucleotide sequence of the present invention. A heterologous nucleotide sequence can comprise non-coding sequences including restriction sites, regulatory sites, promoters and the like.

[0067] The present invention also relates to cloning vectors containing genes encoding analogs and derivatives of the STAT protein, including modified STAT proteins of the invention, that have the same or homologous functional activity as STAT protein, and homologs thereof. The production and use of derivatives and analogs related to the STAT protein are within the scope of the present invention.

[0068] STAT protein derivatives and analogs as described above can be made by altering encoding nucleic acid sequences by substitutions, e.g. replacing the &agr;4-tryptophan or &agr;4-glutamic acid with an alanine, or additions or deletions that provide for functionally equivalent or specifically modified molecules.

[0069] Due to the degeneracy of nucleotide coding sequences, other DNA sequences which encode substantially the same amino acid sequence as a nucleic acid encoding a modified STAT protein or an N-terminal STAT protein fragment of the present invention (including the fragment which lacks the &agr;4-tryptophan) may be used in the practice of the present invention. These include but are not limited to allelic genes, homologous genes from other species, which are altered by the substitution of different codons that encode the same amino acid residue within the sequence, thus producing a silent change. Likewise, the modified STAT protein derivatives of the invention include, but are not limited to, those containing, as a primary amino acid sequence, all or part of the amino acid sequence of a STAT protein including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a conservative amino acid substitution. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity, which acts as a functional equivalent, resulting in a silent alteration. Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. Amino acids containing aromatic ring structures are phenylalanine, tryptophan, and tyrosine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0070] Particularly preferred conserved amino acid exchanges are:

[0071] (a) Lys for His or for Arg or vice versa such that a positive charge may be maintained;

[0072] (b) Glu for Asp or vice versa such that a negative charge may be maintained;

[0073] (c) Ser for Thr or vice versa such that a free —OH can be maintained;

[0074] (d) Gln for Asn or vice versa such that a free NH2 can be maintained;

[0075] (e) lie for Leu or for Val or vice versa as roughly equivalent hydrophobic amino acids; and

[0076] (f) Phe for Tyr or vice versa as roughly equivalent aromatic amino acids.

[0077] Non-conserved amino acid substitutions may also be introduced to substitute an amino acid with a particularly preferable property. For example, a Cys may be introduced to provide a potential site for disulfide bridges with another Cys. A His may be introduced as a particular “catalytic” site (i.e., His can act as an acid or base and is the most common amino acid in biochemical catalysis). Pro may be introduced because of its particularly planar structure, which induces &bgr;-turns in the protein's structure.

[0078] The genes encoding STAT proteins, and derivatives and analogs thereof can be produced by various methods known in the art. The manipulations which result in their production can occur at the gene or protein level. For example, the cloned N-terminal domain of a STAT protein gene sequence can be modified by any of numerous strategies known in the art (Sambrook et al., 1989, supra). The sequence can be cleaved at appropriate sites with restriction endonuclease(s), followed by further enzymatic modification if desired, isolated, and ligated in vitro. In the production of the gene encoding a derivative or analog of a STAT protein care should be taken to ensure that the modified gene remains within the same translational reading frame as the STAT protein gene, uninterrupted by translational stop signals, in the gene region where the desired activity is encoded.

[0079] Additionally, the STAT protein-encoding nucleic acid sequence can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new restriction endonuclease sites or destroy preexisting ones, to facilitate further in vitro modification. Any technique for mutagenesis known in the art can be used, including but not limited to, in vitro site-directed mutagenesis (Hutchinson, C., et al., 1978, J. Biol. Chem. 253:6551; Zoller and Smith, 1984, DNA 3:479-488; Oliphant et al., 1986, Gene 44:177; Hutchinson et al., 1986, Proc. Natl. Acad. Sci. U.S.A. 83:710), use of TAB® linkers (Pharmacia), etc. PCR techniques are preferred for site directed mutagenesis (see Higuchi, 1989, “Using PCR to Engineer DNA”, in PCR Technology: Principles and Applications for DNA Amplification, H. Erlich, ed., Stockton Press, Chapter 6, pp. 61-70).

[0080] The identified and isolated gene can then be inserted into an appropriate cloning vector. A large number of vector-host systems known in the art may be used. Possible vectors include, but are not limited to, plasmids or modified viruses, but the vector system must be compatible with the host cell used. Examples of vectors include, but are not limited to, E. coli, bacteriophages such as lambda derivatives, or plasmids such as pBR322 derivatives or pUC plasmid derivatives, e.g., pGEX vectors, pmal-c, pFLAG, etc. The insertion into a cloning vector can, for example, be accomplished by ligating the DNA fragment into a cloning vector which has complementary cohesive termini. However, if the complementary restriction sites used to fragment the DNA are not present in the cloning vector, the ends of the DNA molecules may be enzymatically modified. Alternatively, any site desired may be produced by ligating nucleotide sequences (linkers) onto the DNA termini these ligated linkers may comprise specific chemically synthesized oligonucleotides encoding restriction endonuclease recognition sequences. Recombinant molecules can be introduced into host cells via transformation, transfection, infection, electroporation, etc., so that many copies of the gene sequence are generated. Preferably, the cloned gene is contained on a shuttle vector plasmid, which provides for expansion in a cloning cell, e.g., E. coli, and facile purification for subsequent insertion into an appropriate expression cell line, if such is desired. For example, a shuttle vector, which is a vector that can replicate in more than one type of organism, can be prepared for replication in both E. coli and Saccharomyces cerevisiae by linking sequences from an E. coli plasmid with sequences form the yeast 2 &mgr; plasmid.

[0081] In an alternative method, the desired gene may be identified and isolated after insertion into a suitable cloning vector in a “shot gun” approach. Enrichment for the desired gene, for example, by size fractionation, can be done before insertion into the cloning vector.

Expression of STAT Proteins

[0082] The nucleotide sequence coding for a STAT protein, or functional fragment, including the N-terminal peptide fragment of a STAT protein, derivatives or analogs thereof, including a chimeric protein, thereof, can be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence. Such elements are termed herein a “promoter.” Thus, the nucleic acid encoding a STAT protein of the invention or functional fragment, including the N-terminal peptide fragment of a STAT protein, derivatives or analogs thereof, is operationally associated with a promoter in an expression vector of the invention. Both cDNA and genomic sequences can be cloned and expressed under control of such regulatory sequences. An expression vector also preferably includes a replication origin. The necessary transcriptional and translational signals can be provided on a recombinant expression vector. As detailed below, all genetic manipulations described for the STAT gene in this section, may also be employed for genes encoding a functional fragment, including the N-terminal domain peptide fragment of a STAT protein, derivatives or analogs thereof, including a chimeric protein, thereof.

[0083] Potential host-vector systems include but are not limited to mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); microorganisms such as yeast containing yeast vectors; or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. The expression elements of vectors vary in their strengths and specificities. Depending on the host-vector system utilized, any one of a number of suitable transcription and translation elements may be used.

[0084] A recombinant STAT protein of the invention, may be expressed chromosomally, after integration of the coding sequence by recombination. In this regard, any of a number of amplification systems may be used to achieve high levels of stable gene expression (See Sambrook et al., 1989, supra).

[0085] The cell into which the recombinant vector comprising the nucleic acid encoding STAT protein is cultured in an appropriate cell culture medium under conditions that provide for expression of STAT protein by the cell.

[0086] Any of the methods previously described for the insertion of DNA fragments into a cloning vector may be used to construct expression vectors containing a gene consisting of appropriate transcriptional/translational control signals and the protein coding sequences. These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombination (genetic recombination).

[0087] Expression of STAT protein may be controlled by any promoter/enhancer element known in the art, but these regulatory elements must be functional in the host selected for expression.

[0088] Expression vectors containing a nucleic acid encoding a STAT protein of the invention can be identified by four general approaches: (a) PCR amplification of the desired plasmid DNA or specific mRNA, (b) nucleic acid hybridization, (c) presence or absence of selection marker gene functions, and (d) expression of inserted sequences. In the first approach, the nucleic acids can be amplified by PCR to provide for detection of the amplified product. In the second approach, the presence of a foreign gene inserted in an expression vector can be detected by nucleic acid hybridization using probes comprising sequences that are homologous to an inserted marker gene. In the third approach, the recombinant vector/host system can be identified and selected based upon the presence or absence of certain “selection marker” gene functions (e.g., &bgr;-galactosidase activity, thymidine kinase activity, resistance to antibiotics, transformation phenotype, occlusion body formation in baculovirus, etc.) caused by the insertion of foreign genes in the vector. In another example, if the nucleic acid encoding STAT protein is inserted within the “selection marker” gene sequence of the vector, recombinants containing the STAT protein insert can be identified by the absence of the STAT protein gene function. In the fourth approach, recombinant expression vectors can be identified by assaying for the activity, biochemical, or immunological characteristics of the gene product expressed by the recombinant, provided that the expressed protein assumes a functionally active conformation.

[0089] A wide variety of host/expression vector combinations may be employed in expressing the DNA sequences of this invention. Useful expression vectors, for example, may consist of segments of chromosomal, nonchromosomal and synthetic DNA sequences. Suitable vectors include derivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmids col E1, pCR1, pBR322, pMal-C2, pET, pGEX (Smith et al., 1988, Gene 67:31-40), pMB9 and their derivatives, plasmids such as RP4; phage DNAS, e.g., the numerous derivatives of phage &lgr;, e.g., NM989, and other phage DNA, e.g., M13 and filamentous single stranded phage DNA; yeast plasmids such as the 2 &mgr; plasmid or derivatives thereof; vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to employ phage DNA or other expression control sequences; and the like.

[0090] For example, in a baculovirus expression systems, both non-fusion transfer vectors, such as but not limited to pVL941 (BamH1 cloning site; Summers), pVL1393 (BamH1, SmaI, XhaI, EcoR1, NotI, XmaIII, BglII, and PstI cloning site; Invitrogen), pVL1392 (BglII, PstI, NotI, XmaIII, EcoRI, XbaI, SmaI, and BamH1 cloning site; Summers and Invitrogen), and pBlueBacIII (BamH1, BglII, PstI, NcoI, and HindIII cloning site, with blue/white recombinant screening possible; Invitrogen), and fusion transfer vectors, such as but not limited to pAc70O (BamH1 and KpnI cloning site, in which the BamH1 recognition site begins with the initiation codon; Summers), pAc701 and pAc702 (same as pAc700, with different reading frames), pAc360 (BamH1 cloning site 36 base pairs downstream of a polyhedron initiation codon; Invitrogen(195)), and pBlueBacHisA, B, C (three different reading frames, with BamH1, BglII, PstI, NcoI, and HindIII cloning site, an N-terminal peptide for ProBond purification, and blue/white recombinant screening of plaques; Invitrogen (220)) can be used. Mammalian expression vectors contemplated for use in the invention include vectors with inducible promoters, such as the dihydrofolate reductase (DHFR) promoter, e.g., any expression vector with a DHFR expression vector, or a DHFR/methotrexate co-amplification vector, such as pED (PstI, SalI, SbaI, SmaI, and EcoRI cloning site, with the vector expressing both the cloned gene and DHFR; see Kaufman, Current Protocols in Molecular Biology, 16.12 (1991). Alternatively, a glutamine synthetase/methionine sulfoximine co-amplification vector, such as pEE14 (HindIII, XbaI, SmaI, SbaI, EcoRI, and BclI cloning site, in which the vector expresses glutamine synthase and the cloned gene; Celitech). In another embodiment, a vector that directs episomal expression under control of Epstein Barr Virus (EBV) can be used, such as pREP4 (BamH1, SfiI, XhoI, NotI, NheI, HindIII, NheI, PvuII, and KpnI cloning site, constitutive RSV-LTR promoter, hygromycin selectable marker; Invitrogen), pCEP4 (BamH1, SfiI, XhoI, NotI, NheI, HindIII, NheI, PvuII, and KpnI cloning site, constitutive hCMV immediate early gene, hygromycin selectable marker; Invitrogen), pMEP4 (KpnI, PvuI, NheI, HindIII, NotI, XhoI, SfiI, BamH1 cloning site, inducible methallothionein IIa gene promoter, hygromycin selectable marker: Invitrogen), pREP8 (BamH1, XhoI, NotI, HindIII, NheI, and KpnI cloning site, RSV-LTR promoter, histidinol selectable marker; Invitrogen), pREP9 (KpnI, NheI, HindIII, NotI, XhoI, SfiI, and BamHI cloning site, RSV-LTR promoter, G418 selectable marker; Invitrogen), and pEBVH is (RSV-LTR promoter, hygromycin selectable marker, N-terminal peptide purifiable via ProBond resin and cleaved by enterokinase; Invitrogen). Selectable mammalian expression vectors for use in the invention include pRc/CMV (HindIII, BstXI, NotI, SbaI, and ApaI cloning site, G418 selection; Invitrogen), pRc/RSV (HindIII, SpeI, BstXI, NotI, XbaI cloning site, G418 selection; Invitrogen), and others. Vaccinia virus mammalian expression vectors (see, Kaufman, 1991, supra) for use according to the invention include but are not limited to pSCI 1 (SmaI cloning site, TK- and &bgr;-gal selection), pMJ601 (SalI, SmaI, AflI, NarI, BspMII, BamH1, ApaI, NheI, SacII, KpnI, and HindIII cloning site; TK- and &bgr;-gal selection), and pTKgptF1S (EcoRI, PstI, SalI, AccI, HindII, SbaI, BamH1, and Hpa cloning site, TK or XPRT selection).

[0091] Yeast expression systems can also be used according to the invention to express OB polypeptide. For example, the non-fusion pYES2 vector (XbaI, SphI, ShoI, NotI, GstXI, EcoRI, BstXI, BamH1, SacI, Kpn1, and HindIII cloning sit; Invitrogen) or the fusion pYESH is A, B, C (XbaI, SphI, ShoI, NotI, BstXI, EcoRI, BamH1, SacI, KpnI, and HindIII cloning site, N-terminal peptide purified with ProBond resin and cleaved with enterokinase; Invitrogen), to mention just two, can be employed according to the present invention.

[0092] Once a particular recombinant DNA molecule is identified and isolated, several methods known in the art may be used to propagate it. Once a suitable host system and growth conditions are established, recombinant expression vectors can be propagated and prepared in quantity. As previously explained, the expression vectors which can be used include, but are not limited to, the following vectors or their derivatives: human or animal viruses such as vaccinia virus or adenovirus; insect viruses such as baculovirus; yeast vectors; bacteriophage vectors (e.g., lambda), and plasmid and cosmid DNA vectors, to name but a few.

[0093] Vectors are introduced into the desired host cells by methods known in the art, e g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene gun, or a DNA vector transporter (see, e.g., Wu et al., 1992, J. Biol. Chem. 267:963-967; Wu and Wu, 1988, J. Biol. Chem. 263:14621-14624; Hartmut et al., Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990).

Synthetic Polypeptides

[0094] The term “polypeptide” is used in its broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs, or peptidomimetics. The subunits are linked by peptide bonds. The STAT proteins and more particularly the N-terminal domain fragments thereof, of the present invention may be chemically synthesized.

[0095] More particularly, potential drugs that may be tested in the drug screening assays of the present invention may also be chemically synthesized. Synthetic polypeptides, prepared using the well known techniques of solid phase, liquid phase, or peptide condensation techniques, or any combination thereof, can include natural and unnatural amino acids. Amino acids used for peptide synthesis may be standard Boc (N&agr;-amino protected N&agr;-t-butyloxycarbonyl) amino acid resin with the standard deprotecting, neutralization, coupling and wash protocols of the original solid phase procedure of Merrifield (1963, J. Am. Chem. Soc. 85:2149-2154), or the base-labile N&agr;-amino protected 9-fluorenylmethoxycarbonyl (Fmoc) amino acids first described by Carpino and Han (1972, J. Org. Chem. 37:3403-3409). Both Fmoc and Boc N&agr;-amino protected amino acids can be obtained from Fluka, Bachem, Advanced Chemtech, Sigma, Cambridge Research Biochemical, Bachem, or Peninsula Labs or other chemical companies familiar to those who practice this art. In addition, the method of the invention can be used with other N&agr;-protecting groups that are familiar to those skilled in this art. Solid phase peptide synthesis may be accomplished by techniques familiar to those in the art and provided, for example, in Stewart and Young, 1984, Solid Phase Synthesis, Second Edition, Pierce Chemical Co., Rockford, Ill.; Fields and Noble, 1990, Int. J. Pept. Protein Res. 35:161-214, or using automated synthesizers, such as sold by ABS. Thus, polypeptides of the invention may comprise D-amino acids, a combination of D- and L-amino acids, and various “designer” amino acids (e.g., &bgr;-methyl amino acids, C&agr;-methyl amino acids, and N&agr;-methyl amino acids, etc.) to convey special properties. Synthetic amino acids include ornithine for lysine, fluorophenylalanine for phenylalanine, and norleucine for leucine or isoleucine. Additionally, by assigning specific amino acids at specific coupling steps, &agr;-helices, &bgr; turns, &bgr; sheets, &ggr;-turns, and cyclic peptides can be generated.

[0096] In a further embodiment, subunits of peptides that confer useful chemical and structural properties will be chosen. For example, peptides comprising D-amino acids will be resistant to L-amino acid-specific proteases in vivo. In addition, the present invention envisions preparing peptides that have more well defined structural properties, and the use of peptidomimetics, and peptidomimetic bonds, such as ester bonds, to prepare peptides with novel properties. In another embodiment, a peptide may be generated that incorporates a reduced peptide bond, i.e., R1—CH2—NH—R2, where R1 and R2 are amino acid residues or sequences. A reduced peptide bond may be introduced as a dipeptide subunit. Such a molecule would be resistant to peptide bond hydrolysis, e.g., protease activity. Such peptides would provide ligands with unique function and activity, such as extended half-lives in vivo due to resistance to metabolic breakdown, or protease activity. Furthermore, it is well known that in certain systems constrained peptides show enhanced functional activity (Hruby, 1982, Life Sciences 31:189-199; Hruby et al., 1990, Biochem J. 268:249-262); the present invention provides a method to produce a constrained peptide that incorporates random sequences at all other positions.

[0097] Constrained and Cyclic Peptides.

[0098] A constrained, cyclic or rigidized peptide may be prepared synthetically, provided that in at least two positions in the sequence of the peptide an amino acid or amino acid analog is inserted that provides a chemical functional group capable of crosslinking to constrain, cyclise or rigidize the peptide after treatment to form the crosslink. Cyclization will be favored when a turn-inducing amino acid is incorporated. Examples of amino acids capable of crosslinking a peptide are cysteine to form disulfides, aspartic acid to form a lactone or a lactam, and a chelator such as &ggr;-carboxyl-glutamic acid (Gla) (Bachem) to chelate a transition metal and form a cross-link. Protected &ggr;-carboxyl glutamic acid may be prepared by modifying the synthesis described by Zee-Cheng and Olson (1980, Biophys. Biochem. Res. Commun. 94:1128-1132). A peptide in which the peptide sequence comprises at least two amino acids capable of crosslinking may be treated, e.g., by oxidation of cysteine residues to form a disulfide or addition of a metal ion to form a chelate, so as to crosslink the peptide and form a constrained, cyclic or rigidized peptide.

[0099] The present invention provides strategies to systematically prepare cross-links. For example, if four cysteine residues are incorporated in the peptide sequence, different protecting groups may be used (Hiskey, 1981, in The Peptides: Analysis, Synthesis, Biology, Vol. 3, Gross and Meienhofer, eds., Academic Press: New York, pp. 137-167; Ponsanti et al., 1990, Tetrahedron 46:8255-8266). The first pair of cysteines may be deprotected and oxidized, then the second set may be deprotected and oxidized. In this way a defined set of disulfide cross-links may be formed. Alternatively, a pair of cysteines and a pair of chelating amino acid analogs may be incorporated so that the cross-links are of a different chemical nature.

[0100] Non-Classical Amino Acids that Induce Conformational Constraints.

[0101] The following non-classical amino acids may be incorporated in the peptide in order to introduce particular conformational motifs: 1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Kazmierski et al., 1991, J. Am. Chem. Soc. 113:2275-2283); (2S,3S)-methyl-phenylalanine, (2S,3R)-methyl-phenylalanine, (2R,3 S)-methyl-phenylalanine and (2R,3 R)-methyl-phenylalanine (Kazmierski and Hruby, 1991, Tetrahedron Lett.); 2-aminotetrahydronaphthalene-2-carboxylic acid (Landis, 1989, Ph.D. Thesis, University of Arizona); hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Miyake et al., 1989, J. Takeda Res. Labs. 43:53-76); &bgr;-carboline (D and L) (Kazmierski, 1988, Ph.D. Thesis, University of Arizona); HIC (histidine isoquinoline carboxylic acid) (Zechel et al., 1991, Int. J. Pep. Protein Res. 43); and HIC (histidine cyclic urea) (Dharanipragada).

[0102] The following amino acid analogs and peptidomimetics may be incorporated into a peptide to induce or favor specific secondary structures: LL-Acp (LL-3-amino-2-propenidone-6-carboxylic acid), a &bgr;-turn inducing dipeptide analog (Kemp et al., 1985, J. Org. Chem. 50:5834-5838); &bgr;-sheet inducing analogs (Kemp et al., 1988, Tetrahedron Lett. 29:5081-5082); &bgr;-turn inducing analogs (Kemp et al., 1988, Tetrahedron Lett. 29:5057-5060); &agr;-helix inducing analogs (Kemp et al., 1988, Tetrahedron Lett. 29:4935-4938); y-turn inducing analogs (Kemp et al., 1989, J. Org. Chem. 54:109:115); and analogs provided by the following references: Nagai and Sato, 1985, Tetrahedron Lett. 26:647-650; DiMaio et al., 1989, J. Chem. Soc. Perkin Trans. p. 1687; also a Gly-Ala turn analog (Kahn et al., 1989, Tetrahedron Lett. 30:2317); amide bond isostere (Jones et al., 1988, Tetrahedron Lett. 29:3853-3856); tretrazol (Zabrocki et al., 1988, J. Am. Chem. Soc. 110:5875-5880); DTC (Samanen et al., 1990, Int. J. Protein Pep. Res. 35:501:509); and analogs taught in Olson et al., 1990, J. Am. Chem. Sci. 112:323-333 and Garvey et al., 1990, J. Org. Chem. 56:436. Conformationally restricted mimetics of beta turns and beta bulges, and peptides containing them, are described in U.S. Pat. No. 5,440,013, issued Aug. 8, 1995 to Kahn.

Crystals of the N-terminal Domain Fragment of a STAT Protein

[0103] Crystals of the N-terminal domain fragment of a STAT protein can be grown by a number of techniques including batch crystallization, vapor diffusion (either by sitting drop or hanging drop) and by microdialysis. Seeding of the crystals in some instances is required to obtain X-ray quality crystals. Standard micro and/or macro seeding of crystals may therefore be used. As exemplified below hanging drops of one &mgr;l of the STAT-4 N-terminal domain fragment (20 mg/ml, in 50 mM Hepes/HCl pH 8.0, 150 mM KCl, 2.5 mM CaCl2, 5 mM DTT) was mixed with equal volumes of reservoir buffer containing 0.2 M Na+CH3COO−, 0.1 M Tris/HCL pH 8.0, 17% PEG4000. Hexagonal crystals (0.2×0.2×0.2 mm) were routinely grown overnight at 20° C. Once a crystal of the present invention is grown, X-ray diffraction data can be collected. The Example below used a MAR imaging plate detector for X-ray diffraction data collection though alternative methods may also be used. For example, crystals can be characterized by using X-rays produced in a conventional source (such as a sealed tube or a rotating anode) or using a synchrotron source. Methods of characterization include, but are not limited to, precision photography, oscillation photography and diffractometer data collection. Heavy atom derivatives such as produced with a mercurial, exemplified below, can be performed using Fuji imaging plates. Alternatively, the STAT fragment can be synthesized with selenium-methionine (Se-Met) in place of methionine, and the Se-Met multiwavelength anomalous dispersion data [Hendrickson, Science, 254:51-58 (1991)] can be collected on CHESS F2, using reverse-beam geometry to record Friedel pairs at four X-ray wavelengths, corresponding to two remote points above and below the Se absorption edge (&lgr;1 and &lgr;4) and the absorption edge inflection point (&lgr;2) and peak (&lgr;3). Selenium sites can be located using SHELXS-90 in Patterson search mode (G. M. Sheldrick). Experimental phases (&agr;MAD) can be estimated via a multiple isomorphous replacement/anomalous scattering strategy using MLPHARE (Z. Otwinowski, Southwestern University of Texas, Dallas) with three of the wavelengths treated as derivatives and one (&lgr;2) treated as the parent for example. In either case, data can be processed using HKL, DENZO and SCALEPACK (Z. Otwinowski and W. Minor).

[0104] In addition, X-PLOR, as used in the Example below [Bruger, X-PLOR v. 3.1 Manual, New Haven: Yale University, (1993B)] or Heavy [T. Terwilliger, Los Alamos National Laboratory] may be utilized for bulk solvent correction and B-factor scaling. After density modification and non-crystallographic averaging, the protein is built into a electron density map using the program 0, as exemplified below [Jones et al., Acta Cryst., A47: 110-119 (1991)]. Model building interspersed with positional and simulated annealing refinement [Brünger, 1993B, supra] can permit the an unambiguous trace and sequence assignment of the N-terminal domain fragment of the STAT protein.

Protein-Structure Based Design of Agonists and Antagonists of STAT Proteins

[0105] Once the three-dimensional structure of a crystal comprising a an N-terminal domain fragment of a STAT protein is determined, a potential ligand (antagonist or agonist) is examined through the use of computer modeling using a docking program such as GRAM, DOCK, or AUTODOCK [Dunbrack et al., 1997, supra]. This procedure can include computer fitting of potential ligands to the STAT dimer to ascertain how well the shape and the chemical structure of the potential ligand will complement or interfere with the dimer-dimer interaction. [Bugg et al., Scientific American, December:92-98 (1993); West et al., TIPS, 16:67-74 (1995)]. Computer programs can also be employed to estimate the attraction, repulsion, and steric hindrance of the ligand to the dimer-dimer binding site. Generally the tighter the fit (e.g., the lower the steric hindrance, and/or the greater the attractive force) the more potent the potential drug will be since these properties are consistent with a tighter binding constant. Furthermore, the more specificity in the design of a potential drug the more likely that the drug will not interfer with other properties of the STAT protein or other proteins (particularly proteins present in the nucleus). This will minimize potential side-effects due to unwanted interactions with other proteins.

[0106] Initially a potential ligand could be obtained by screening a random peptide library produced by recombinant bacteriophage for example, [Scott and Smith, Science, 249:386-390 (1990); Cwirla et al., Proc. Natl. Acad. Sci., 87:6378-6382 (1990); Devlin et al., Science, 249:404-406 (1990)] or a chemical library. A ligand selected in this manner could be then be systematically modified by computer modeling programs until one or more promising potential ligands are identified. Such analysis has been shown to be effective in the development of HIV protease inhibitors [Lam et al., Science 263:380-384 (1994); Wlodawer et al., Ann. Rev. Biochem. 62:543-585 (1993); Appelt, Perspectives in Drug Discovery and Design 1:23-48 (1993); Erickson, Perspectives in Drug Discovery and Design 1: 109-128 (1993)].

[0107] Such computer modeling allows the selection of a finite number of rational chemical modifications, as opposed to the countless number of essentially random chemical modifications that could be made, and of which any one might lead to a useful drug. Each chemical modification requires additional chemical steps, which while being reasonable for the synthesis of a finite number of compounds, quickly becomes overwhelming if all possible modifications needed to be synthesized. Thus through the use of the three-dimensional structure disclosed herein and computer modeling, a large number of these compounds can be rapidly screened on the computer monitor screen, and a few likely candidates can be determined without the laborious synthesis of untold numbers of compounds.

[0108] Once a potential ligand (agonist or antagonist) is identified it can be either selected from a library of chemicals as are commercially available from most large chemical companies including Merck, Glaxo Welcome, Bristol Meyers Squib, Monsanto/Searle, Eli Lilly, Novartis and Pharmacia UpJohn, or alternatively the potential ligand may be synthesized de novo. As mentioned above, the de novo synthesis of one or even a relatively small group of specific compounds is reasonable in the art of drug design. The prospective drug can be placed into any standard binding assay described below to test its effect on the dimeric N-terminal domain STAT-STAT interaction.

[0109] When a suitable drug is identified, a supplemental crystal can be grown which comprises a protein-ligand complex formed between an N-terminal domain of the STAT protein and the drug. Preferably the crystal effectively diffracts X-rays allowing the determination of the atomic coordinates of the protein-ligand complex to a resolution of greater than 5.0 Angstroms, more preferably greater than 3.0 Angstroms, and even more preferably greater than 2.0 Angstroms. The three-dimensional structure of the supplemental crystal can be determined by Molecular Replacement Analysis. Molecular replacement involves using a known three-dimensional structure as a search model to determine the structure of a closely related molecule or protein-ligand complex in a new crystal form. The measured X-ray diffraction properties of the new crystal are compared with the search model structure to compute the position and orientation of the protein in the new crystal. Computer programs that can be used include: X-PLOR and AMORE [J. Navaza, Acta Crystallographics ASO, 157-163 (1994)]. Once the position and orientation are known an electron density map can be calculated using the search model to provide X-ray phases. Thereafter, the electron density is inspected for structural differences and the search model is modified to conform to the new structure. Using this approach, it will be possible to use the claimed structure of the N-terminal domain STAT fragment to solve the three-dimensional structures of any such STAT fragment. Other computer programs that can be used to solve the structures of such STAT crystals include QUANTA, CHARMM; INSIGHT; SYBYL; MACROMODE; and ICM.

[0110] For all of the drug screening assays described herein further refinements to the structure of the drug will generally be necessary and can be made by the successive iterations of any and/or all of the steps provided by the particular drug screening assay.

[0111] Phage Libraries for Drug Screening.

[0112] Phage libraries have been constructed which when infected into host E. coli produce random peptide sequences of approximately 10 to 15 amino acids [Parmley and Smith, Gene 73:305-318 (1988), Scott and Smith, Science 249:386-249 (1990)]. Specifically, the phage library can be mixed in low dilutions with permissive E. coli in low melting point LB agar which is then poured on top of LB agar plates. After incubating the plates at 37° C. for a period of time, small clear plaques in a lawn of E. coli will form which represents active phage growth and lysis of the E. coli. A representative of these phages can be absorbed to nylon filters by placing dry filters onto the agar plates. The filters can be marked for orientation, removed, and placed in washing solutions to block any remaining absorbent sites. The filters can then be placed in a solution containing, for example, a radioactive N-terminal peptide fragment of a STAT protein (e.g., a fragment having an amino acid sequence comprising SEQ ID NO: 1). After a specified incubation period, the filters can be thoroughly washed and developed for autoradiography. Plagues containing the phage that bind to the radioactive N-terminal peptide fragment of a STAT protein can then be identified. These phages can be further cloned and then retested for their ability to bind to the N-terminal peptide fragment of a STAT protein as before. Once the phages have been purified, the binding sequence contained within the phage can be determined by standard DNA sequencing techniques. Once the DNA sequence is known, synthetic peptides can be generated which represents these sequences.

[0113] These peptides can be tested, for example, for their ability to: (1) interfere with a dimeric STAT protein binding to a weak STAT DNA binding site; and (2) interfere with a dimeric STAT protein lacking the &agr;4-tryptophan and/or a truncated STAT protein in which the N-terminal domain has been removed (e.g., Statltc, [Vinkemeier et al., EMBO J. 15: 5616 (1996)]) from binding to the same DNA binding site. If the peptide interferes in the first case but does not interfere in the latter case, it may be concluded that the peptide interferes with dimeric N-terminal STAT-STAT interaction.

[0114] The effective peptide(s) can be synthesized in large quantities for use in in vivo models and eventually in humans to modulate STAT signal transduction. It should be emphasized that synthetic peptide production is relatively non-labor intensive, easily manufactured, quality controlled and thus, large quantities of the desired product can be produced quite cheaply. Similar combinations of mass produced synthetic peptides have recently been used with great success [Patarroyo, Vaccine 10:175-178 (1990)].

Binding Assays for Drug Screening Assays

[0115] The drug screening assays of the present invention may use any of a number of assays for measuring the stability of a STAT-STAT dimeric interaction, including N-terminal dimeric STAT fragments and/or a dimeric STAT-STAT-DNA binding interaction. In one embodiment the stability of a preformed DNA-protein complex between a dimeric STAT protein and its corresponding DNA binding site is examined as follows: the formation of a complex between the STAT protein and a labeled oligonucleotide is allowed to occur and unlabelled oligonucleotides are added in vast molar excess after the reaction reaches equilibrium. At various times after the addition of unlabelled competitor DNA, aliquots are layered on a running native polyacrylamide gel to determine free and bound oligonucleotides. In one preferred embodiment the protein is STAT1&agr;, and two different labeled DNAs are used, the natural cfos site, an example of a “weak” site, and the mutated cfos-promotor element, the M67 site [Wagner et al., EMBO J 9:4477 (1990)] an example of a “strong” site as described below. Other examples of weak sites include those in the promoter of the MIG gene, and those in the regulatory region of the interferon-&ggr; gene. Other examples of strong sites include those such as the selected optimum site, S1 [Horvath et al., Genes & Devel. 9:984 (1995)] or the promoter of the IRF-1 gene.

[0116] In a related binding assay, a nucleic acid containing a weak STAT binding site is placed on or coated onto a solid support. Methods for placing the nucleic acid on the solid support are well known in the art and include such things as linking biotin to the nucleic acid and linking avidin to the solid support. Dimeric STAT proteins are allowed to equilibrate with the nucleic acid and drugs are tested to see if they disrupt or enhance the binding. Disruption leads to either a faster release of the STAT protein which may be expressed as a faster off time, and or a greater concentration of released STAT dimer. Enhancement leads to either a slower release of the STAT protein which may be expressed as a slower off time, and/or a lower concentration of released STAT protein.

[0117] The STAT protein may be labeled as described below. For example, in one embodiment radiolabeled STAT proteins are used to measure the effect of a drug on binding. In another embodiment the natural ultraviolet absorbance of the STAT protein is used. In yet another embodiment, a Biocore chip (Pharmacia) coated with the nucleic acid is used and the change in surface conductivity can be measured.

[0118] In yet another embodiment, the affect of a prospective drug (a test compound) on interactions between N-terminal domains of STATs is assayed in living cells that contain or can be induced to contain activated STAT proteins, i.e., STAT protein dimers. Cells containing a reporter gene, such as the heterologous gene for luciferase, green fluorescent protein, chloramphenicol acetyl transferase or &bgr;-galactosidase, operably linked to a promoter comprising two weak STAT binding sites are contacted with a prospective drug in the presence of a cytokine which activates the STAT(s) of interest. The amount (and/or activity) of reporter produced in the absence and presence of prospective drug is determined and compared. Prospective drugs which reduce the amount (and/or activity) of reporter produced are candidate antagonists of the N-terminal interaction, whereas prospective drugs which increase the amount (and/or activity) of reporter produced are candidate agonists. Cells containing a reporter gene operably linked to a promoter comprising strong STAT binding sites are then contacted with these candidate drugs, in the presence of a cytokine which activates the STAT(s) of interest. The amount (and/or activity) of reporter produced in the presence and absence of candidate drugs is determined and compared. Drugs which disrupt interactions between dimeric N-terminal domains of the STATs will not reduce reporter activity in this second step. Similarly, candidate drugs which enhance interactions between dimeric N-terminal domains of STATs will not increase reporter activity in this second step.

[0119] In an analogous embodiment, two reporter genes each operably under the control of one or the other of the two types promoters described above can be comprised in a single host cell as long as the expression of the two reporter gene products can be distinguished. For example, different modified forms of green fluorescent protein can be used as described in U.S. Pat. No. 5,625,048, Issued Apr. 29, 1997, hereby incorporated by reference in its entirety. Although cells that naturally encode the STAT proteins may be used, preferably a cell is used that is transfected with a plasmid encoding the STAT protein. For example transient transfections can be performed with 50% confluent U3A cells using the calcium phosphate method as instructed by the manufacturer (Stratagene). In addition as mentioned above, the cells can also be modified to contain one or more reporter genes, a heterologous gene encoding a reporter such as luciferase, green fluorescent protein or derivative thereof, chloramphenicol acetyl transferase, 13-galactosidase, etc. Such reporter genes can individually be operably linked to promoters comprising two weak STAT binding sites and/or a promoter comprising a strong STAT binding site. Assays for detecting the reporter gene products are readily available in the literature. For example, luciferase assays can be performed according to the manufacturer's protocol (Promega), and p-galactosidase assays can be performed as described by Ausubel et al., [in Current Protocols in Molecular Biology, J. Wiley & Sons, Inc. (1994)].

[0120] In one example, the transfection reaction can comprise the transfection of a cell with a plasmid modified to contain a STAT protein, such as a pcDNA3 plasmid (Invitrogen), a reporter plasmid that contains a first reporter gene, and a reporter plasmid that contains a second reporter gene. Although the preparation of such plasmids is now routine in the art, many appropriate plasmids are commercially available e.g., a plasmid with &bgr;-galactosidase is available from Stratagene.

[0121] The reporter plasmids can contain specific restriction sites in which an enhancer element having a strong STAT binding site or alternatively two tandemly arranged “weak” STAT binding sites can be inserted. In one particular embodiment, thirty-six hours after transfection of the cells with a plasmid encoding STAT-1, the cells are treated with 5 ng/ml interferon-&ggr; Amgen for ten hours. Protein expression and tyrosine phosphorylation (to monitor STAT activation) can be determined by e.g., gel shift experiments with whole cell extracts.

Labels

[0122] Suitable labels include enzymes, fluorophores (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE), Texas red (TR), rhodamine, free or chelated lanthanide series salts, especially Eu3+, to name a few fluorophores), chromophores, radioisotopes, chelating agents, dyes, colloidal gold, latex particles, ligands (e.g., biotin), and chemiluminescent agents.

[0123] When a control marker is employed, the same or different labels may be used for the test and control marker gene.

[0124] In the instance where a radioactive label, such as the isotopes 3H, 14C, 32P, 35S, 36Cl, 51Cr, 57Co, 58Co, 59Fe, 90Y, 125I, 131I, and 186Re are used, known currently available counting procedures may be utilized. In the instance where the label is an enzyme, detection may be accomplished by any of the presently utilized calorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques known in the art.

[0125] Direct labels are one example of labels which can be used according to the present invention. A direct label has been defined as an entity, which in its natural state, is readily visible, either to the naked eye, or with the aid of an optical filter and/or applied stimulation, e.g. U.V. light to promote-fluorescence. Among examples of colored labels, which can be used according to the present invention, include metallic sol particles, for example, gold sol particles such as those described by Leuvering (U.S. Pat. No. 4,313,734); dye sole particles such as described by Gribnau et al. (U.S. Pat. No. 4,373,932 and May et al. (WO 88/08534); dyed latex such as described by May, supra, Snyder (EP-A 0 280 559 and 0 281 327); or dyes encapsulated in liposomes as described by Campbell et al. (U.S. Pat. No. 4,703,017). Other direct labels include a radionucleotide, a fluorescent moiety or a luminescent moiety. In addition to these direct labeling devices, indirect labels comprising enzymes can also be used according to the present invention. Various types of enzyme linked immunoassays are well known in the art, for example, alkaline phosphatase and horseradish peroxidase, lysozyme, glucose-6-phosphate dehydrogenase, lactate dehydrogenase, urease, these and others have been discussed in detail by Eva Engvall in Enzyme Immunoassay ELISA and EMIT in Methods in Enzymology, 70:419-439 (1980) and in U.S. Pat. No. 4,857,453.

[0126] Suitable enzymes include, but are not limited to, alkaline phosphatase, &bgr;-galactosidase, green fluorescent protein and its derivatives, luciferase, and horseradish peroxidase.

[0127] Other labels for use in the invention include magnetic beads or magnetic resonance imaging labels.

[0128] The present invention may be better understood by reference to the following non-limiting Example, which is provided as exemplary of the invention. The following example is presented in order to more fully illustrate the preferred embodiments of the invention. It should in no way be construed, however, as limiting the broad scope of the invention.

EXAMPLE

Structure of the Amino-Terminal Protein Interaction Domain of STAT-4

Introduction

[0129] STATs are a family of transcription factors that are specifically triggered to participate in gene activation when cells encounter cytokines and growth factors. The crystal structure of a conserved domain comprising the first 123 residues of STAT-4 has been determined at 1.45 A. This domain (N-terminal domain) has been implicated in several protein:protein interactions affecting transcription. The N-terminal domain enables STAT dimers to interact and to bind DNA cooperatively, a mechanism important for gene activation and binding site discrimination. The domain consists of 8 helices that are assembled into a hook-like structure. The crystal structure shows the formation of dimers formed by polar interactions across one face of the hook, revealing the nature of the cooperative interactions between STAT dimers. Mutagenesis of an invariant Trp residue that is at the heart of this interface abolishes cooperative dimer:dimer DNA binding by the full length protein in vitro and reduces transcriptional response after cytokine stimulation in vivo.

Materials and Methods

[0130] Expression and Purification of the STAT-4 N-terminal Domain:

[0131] The STAT-4 N-terminal domain was expressed as a C-terminal fusion with glutathione S-transferase (GST). The expression vector was constructed by PCR amplification (Vent DNA-polymerase, New England Biolabs) of the appropriate region of mouse STAT-4 cDNA and cloning of the fragment into the BamHI and EcoRI sites of pGEX2T (Pharmacia). Sequence comparison with the STAT-1 amino-terminal domain led to the decision to terminate the homologous domain in STAT-4 after residue 124. To facilitate cleavage of the GST tag from the recombinant STAT-4, three glycine residues were included after the thrombin cleavage site [Guan and Dixon, Anal. Biochem. 192:262 (1991)]. The resulting protein has four additional amino-terminal amino acids and Met 1 of STAT-4 is replaced with Gly. The construct was verified by dideoxy sequencing. BL21p(lysS) cells were grown and lysed as described [Vinkemeier et al., EMBO J. 15:5616 (1996)]. Soluble protein was incubated with 0.2 Vol. of a 50% (vol./vol.) slurry of glutathione agarose (Pharmacia). The bound protein was washed with cleavage buffer (50 mM Hepes/HCl pH 8.0, 150 mM KCl, 2.5 mM CaCl2, 5 mM DTT). Cleavage with thrombin (˜1U/mg protein; Novagen) was performed overnight at room temperature with the protein bound to the beads. The eluted STAT-4 N-terminal domain fragment (in cleavage buffer) was concentrated to 20 mg/ml with centricon (Amicon) and used for crystallization.

[0132] Crystallization and Data Collection and Processing:

[0133] Initial studies were performed with the STAT- 1 N-terminal domain, but led only to small, needle-like crystals. Hanging drops of one &mgr;l of the STAT-4 N-terminal domain fragment was mixed with equal volumes of reservoir buffer containing 0.2 M Na+CH3COO−, 0.1 M Tris/HCL pH 8.0, 17% PEG4000. Hexagonal crystals (0.2×0.2×0.2 mm) were routinely grown overnight at 20° C. The crystals contain one molecule of the STAT-4 N-terminal domain in the asymmetric unit, and are in the space group P6522 (a=79.51 Å, b=79.51 Å, c=84.68 Å). Crystals were cryo-protected in reservoir solution enriched in PEG to 20% and glycerol to 22.5% prior to flash freezing. Heavy atom derivatives were prepared by soaking crystals for 30 minutes in a saturated solution of parahydroxy-mercunbenzoic acid diluted 1:20 with the cryoprotective solution. Data for the native crystal were collected at the Brookhaven National Laboratory at beam line X25 using a MAR imaging plate detector system. A MAD experiment on a parahydroxy-mercuribenzoic acid derivitized crystal was performed at the Brookhaven National Laboratory on beam line X4A using Fuji imaging plates. Data processing and reduction were carried out with HKL, DENZO, and SCALEPACK (written by Z. Otwinowski and W. Minor).

[0134] Model Building and Refinement.

[0135] Model building was performed using 0 [Jones et al., Acta Crystallogr. A47: 110-119 (1991)]. Bulk solvent correction and anisotropic B-factor scaling was applied during refinement using X-PLOR [Brünger, X-PLOR (Version 3.1) Manual, The Howard Hughes Medical Institute and Department of Molecular Biophysics and Biochemistry, Yale University, 260 Whitney Avenue, New Haven, Conn. 06511, (1992)]. The final refinement statistics are shown in Table 1. Of the five heterologous residues at the amino-terminus the first three residues (GlySerGly) as well as the C-terminal residue Gln 124 are not visible in the electron density map. No amino acids occupy disallowed regions of the Ramachandran plot and 95% fall into the most favored region.

[0136] Preparation, Expression, and Purification of the Mutated STAT-1&agr;:

[0137] Mutated STAT-1&agr; (Trp 37 to Ala) was expressed from pAcSG2 in baculovirus infected insect cells. PCR was used to exchange codon 37 [TGG] with [GCA] in the NcoI/SpeI fragment of human STAT-1 cDNA. Additionally, a 6-His tag was added to the C-terminus. Modifications were confirmed by sequencing. Insect cells were lysed (dounce homogenizer), mutated STAT-1&agr; purified under native conditions on Ni2+ nitrilotriacetic acid (Qiagen) and eluted with 200 mM imidazole in 20 mM Tris/HCl, pH 8.0, 10 mM MgCl2, 50 mM KCl, 5 mM DTT.

[0138] Preparation of EGF-Receptor Kinase and in vitro Phosphorylation of STAT Proteins.

[0139] Tyrosine phosphorylated wild type human STAT-1&agr; was produced as described [Vinkemeier et al., EMBO J. 15:5616 (1996)]. In vitro phosphorylation was done as described [Vinkemeier et al., EMBO J. 15:5616 (1996)]. Human carcinoma A431 cells were grown to 90% confluency in 150 mm diameter plates in Dulbecco's modified Eagle's medium supplemented with 10% bovine calf serum (Hyclone). Cells were washed once with chilled PBS and lysates were prepared in 1 ml ice cold lysis buffer (10mM Hepes/HCl, 150 mM NaCl, 0.5% Triton X-100, 10% Glycerol, 1 mM Na3VO4, 10 mM EDTA, Complete™ protease inhibitors, pH 7.5). After 10 min on ice, the cells were scraped, vortexed and dounce homogenized (5 strokes). The lysates were cleared by centrifugation at 4° C. for 20 min at top speed in an Eppendorf microfuge and stored at −70° C. until needed. Immediately before use 1 volume of the lysate was diluted with 4 volumes of the lysis buffer (“diluted lysate”). EGF-receptor precipitates were obtained by incubating 5 ml of diluted lysate with 50 &mgr;g of an anti-EGF-receptor monoclonal antibody directed against the extracellular domain. After 2 hours of rotating the sample at 4° C., 750 &mgr;l of Protein-A-agarose (50% slurry; Oncogene Science) was added, and the incubation was allowed to proceed, while rotating, for another 1 hour. Agarose beads containing the EGF-receptor immunoprecipitates were then washed 5 times with lysis buffer and finally twice with storage buffer (20% Glycerol, 20 mM Hepes/HCl, 100 mM NaCl, 0.1 mM Na3VO4). Precipitates from 5 ml diluted lysate were dissolved in 0.5 ml storage buffer, flash frozen on dry ice and stored at −70° C. Immediately before an in vitro kinase reaction the Protein-A-agarose bound EGF-receptor from 5 ml dilute lysate was washed once with lx kinase buffer (20 mM Tris/HCl, 50 mM KCl, 0.3 mM Na3VO4, 2 mM DTT, pH 8.0) and then dissolved in 0.4 ml (total volume) of this buffer. Afterwards the washed EGF-receptor precipitate was incubated on ice for 10 min in the presence of a final concentration of mouse EGF of 0.15 ng/&mgr;l. Phosphorylation reactions were carried out in Eppendorf tubes in a final volume of 1 ml. To the pre-incubated kinase preparation the following was added: 60 &mgr;l 10× kinase buffer, 20 &mgr;l 0.1 M DTT, 50 &mgr;l 0.1 M ATP, 4 mg STAT protein (Superdex 200 eluate for STAT1&agr; and STAT1 &bgr;; ammonium sulfate pellets dissolved in [20 mM Tris /HCl, pH 8.0] for STAT1tc), 10 &mgr;l 1M MnCl2 and dH2O to 1 ml. The reaction was allowed to proceed for 15 hours at 4° C. After 3 hours an additional 15 &mgr;l of 0.1 M ATP was added.

[0140] Gel Shift Experiments and Determination of Tetramer Stability:

[0141] Gel shift experiments and determination of tetramer stability were done as described [Vinkemeier et al., EMBO J. 15:5616 (1996)] with an oligonucleotide containing two copies of the STAT recognition element from the c-fos gene [Wagner et al., EMBO J. 9:4477 (1990)] spaced by 10 base pairs (5′-GCCAGTCAGTTCCCGTCAATGCATCAGGTTCCCGTCAATGCAT-3′). Both protein preparations (Tyr-phosphorylated wild type STAT-1&agr; and W37A mutant) were titrated in gel shift experiments with an oligonucleotide containing a single M67 site (5′-GCCGATTTCCCGTAAATCAT-3′) [Wagner et al., EMBO J. 9:4477 (1990)] to assure similar loading of active protein. A 12.5 &mgr;l reaction volume contained DNA binding buffer (20 mM Hepes/HCl, 4% Ficoll, 40 mM KCl, 10 mM MgCl2, 10 mM CaCl2, 1 mM DTT) radiolabelled DNA at a final concentration of 1×10−10 M unless stated otherwise, 50 ng dIdC, 0.2 mg/ml BSA (Boehringer Mannheim), and the indicated amount of purified phosphorylated STAT protein. The reaction volume was mixed and then incubated at room temperature. The time necessary to reach equilibrium was assessed by EMSA [(Stone et al., in Jost, J. P. & Saluz, H. P. (eds.) A Laboratory Guide to In Vitro Studies of Protein-DNA Interactions BioMethods, vol.5:163-195 (1991)]. For all DNA fragments tested, equilibrium turned out to be fully established at the earliest timepoint that can be determined by this technique (30 sec). Therefore incubation periods of 5-15 minutes were chosen. Reaction products were loaded onto a 4% polyacrylamide gel (1.5 mm thick) containing 0.25× Tris-borate-EDTA which had been pre-run at 20V/cm for 2 hours at 4° C. Electrophoresis was continued for 60 minutes at 4° C. Gels were dried and exposed to X-ray film and quantitated by a Molecular Dynamics Phospholmager.

[0142] Transient Transfections:

[0143] Transient transfections were performed on six well plates with 50% confluent U3A cells using the calcium phosphate method as instructed by the manufacturer (Stratagene) with the following modifications. Transfection reactions contained per well 4.5 &mgr;g of either wild type STAT-1&agr; or the W37A mutant in plasmid pcDNA3 (Invitrogen), 4 &mgr;g luciferase reporter plasmid pLuc, and 0.4 &mgr;g &bgr;-galactosidase reporter plasmid (Stratagene).

[0144] The luciferase reporter contained in its BamH1 site as an enhancer element two tandemly arranged “weak” STAT-1 binding sites (5′-GATCAGTTCCCGTCAATCATGATCCAGTTCCCGTCAATGATCCCCGGGATC-3′, binding sites underlined) from the human c-fos promoter. 36 hrs. after transfection, cells were treated with 5 ng/ml interferon-&ggr; (Amgen) for 10 hrs. or left untreated. Luciferase assays were performed according to the manufacturer's protocol (Promega) and &bgr;-galactosidase assays were done as previously described [Ausubel et al., in Current Protocols in Molecular Biology, J. Wiley & Sons, Inc. (1994)]. Protein expression and Tyr-phosphorylation were checked in gel shift experiments with whole cell extracts for both wild type and mutant protein and were comparable. All results shown are luciferase activities normalized against the internal control &bgr;-galactosidase activity.

Results:

[0145] Proteolytic digestion of purified STAT-1 showed that the N-terminal 131 residues form a stable domain that is readily cleaved off the intact molecule, indicating that it is an independently folded module [Vinkemeier et al., EMBO J. 15:5616 (1996)]. Sequence alignments show that the N-terminal domain is highly conserved in the STAT family of proteins (FIG. 1). The average sequence identity for this region between mammalian STAT proteins is 40%, and ranges from 51% between STAT-1 and STAT-4 to 20% between STAT-5 and STAT-6. Over the approximately 750 amino acids that span the length of the common core of the STATs only the SH2 domain is more highly conserved [Schindler and Darnell, Annu. Rev. Biochem. 64:621 (1995)]. The N-terminal domain is also found in the Drosophila STAT (dSTAT92E) [Hou et al., Cell 84:411 (1996); Yan et al., Cell 84:421 (1996)], (FIG. 1) and in the recently discovered STAT in Dictyostelium discoideum [Kawata et al., Cell 89:909 (1997)]. Interestingly, the first gene defect established in the DSTAT92 gene is a misspliced variant that produces both normal mRNA and an mRNA encoding only the N-terminal 41 residues. Expression of this fragment has a partial dominant negative effect on transcriptional activation by the wild type protein in cell culture. In the fly it is associated with a weak abnormal phenotype [Yan et al., Proc. Natl. Acad. Sci. USA 93:5842 (1996)].

[0146] As disclosed herein the crystal structure of the N-terminal domain of STAT-4 has been determined by multi-wavelength anomalous diffraction and refined at a resolution of 1.45 Å (R=19.4%, Rfree=22.3%) (Table 1). The STAT-4 N-domain is all helical, with an unusual architecture. Instead of the up-down connectivity of helix bundles or the box-like helical packing of the globin fold, the N-domain is constructed from three distinct structural elements that pack together. The N-terminal 40 residues encompass the first 4 helices (&agr;1-&agr;4), which form a ring-shaped element (colored red in FIGS. 2 and 3B). A small helix (&agr;5) connects this ring to the next structural element, an anti-parallel coiled-coil formed by helices &agr;6 and &agr;7. The heptad repeat of hydrophobic amino acids, characteristic of coiled-coils, is conserved across the STATs (FIG. 1). Finally, the distal surface of the ring-shaped element forms a docking site for the last helix in the structure (&agr;8). The overall appearance of the structure is that of a triangular hook, with the inner surface of the hook being formed by the intersection of the proximal surfaces of the ring-shaped element and the coiled-coil.

[0147] The N-domain of STAT-4 is dimeric in solution, and a two-fold symmetry axis in the crystal generates a dimer with an extensive polar interface that involves one face of the hook. The N-domain has a well defined hydrophobic core that is conserved across the STATs, consistent with a stable and defined fold (FIGS. 1 and 2). However, a notable feature of the N-terminal ring-shaped element is that it is stabilized by polar interactions involving buried charges. The ring is closed off by a helix-dipole interaction between the N-terminal region of helix &agr;1 and the carboxylate group of Glu 39, presented by the C-terminal region of helix &agr;4 (FIG. 2B). Glu 39 forms a hydrogen bond with the amide nitrogen of the Gln 3 residue, and is oriented correctly for this charge dipole interaction by the side chain of Arg 31, which in turn forms a buried ion pair with Glu 112. Glu 112 is positioned by interactions with Tyr 22 and a buried water molecule. Each of the side chains involved is invariant in all STATs (FIG. 1), indicating that the ring-shaped element is conserved in the STAT architecture. 1 TABLE 1 Summary of the Crystallographic Analysis Resolu- Reflections Complete- tion total/ ness Rsym* Sites (Å) unique (%) (%) In/&sgr;† (N) FOM# Native Data 1.45 237647/ 96.4 7.2 19.6 27594 (91.7) (19.2) (4.2) MAD Analysis &lgr;1 = 1.00842Å 2.15 112445/ 98.6 7.7 24.7 2 8854 (95.7) (21.2) (6.1) &lgr;2 = 1.01337Å 2.51 31403/ 100.0 7.3 27.2 2 8992 (100.0) (21.1) (8.1) &lgr;3 = 1.00932Å 2.00 126292/ 96.6 8.4 22.1 2 10731 (95.7) (33.4) (4.5) 0.542 Completeness R-factor R-free Refinement Cut Off Reflections (%) (%)¶ (%)‡ 10-1 45Å |F|/&sgr;(|F|)>2 25285 88.8 18.8 21.6 10-1 45Å all data 26560 93.3 19.4 22.3 r m.s deviation of model bond lengths (Å) 0.015 bond angles (degrees) 1.4 average B-factor (protein) (Å2) 9.6 water molecules 165 average B-factor (water) (Å2) 25 *Rsym = 100 × &Sgr;h&Sgr;iIh,i − Ih|/&Sgr;h&Sgr;iIh,i for the intensity of i observations of reflection h. For Rsym and completeness, the numbers in parenthesis refer to data in the shell of highest resolution. In is the mean intensity of the reflection. †In/&sgr; = mean intensity/mean standard deviation #FOM = overall mean figure of merit [|&Sgr;P(&agr;)ei&agr;/&Sgr;P(&agr;)|], where &agr; is the phase and P(&agr;) is the phase distribution probability ¶R-factor = 100 × &Sgr;|Fobs − Fcalc|/&Sgr;|Fobs|, where Fobs and Fcalc are the observed and calculated structure factors, respectively. ‡R-free = is the same as R-factor, but calculated on the 5% of the reflection data excluded from refinement.

[0148] 2 TABLE II Interactions Between the Two N-terminal Domains of Stat4 sidechain-sidechain contacts; direct Molecule A Å Molecule B Asn5 3.0 Asn5 Trp37 3.2 Glu66 Thr40 3.2 Arg70 3.2 Gln67 Gln41 2.9 Glu66 Asp42 3.3 Lys73 Gln63 3.6 Gln63 Glu66 3.2 Trp37 Gln67 3.2 Thr40 Arg70 3.0 Thr40 Lys73 3.3 Asp42 sidechain-sidechain contacts; water mediated Mole- Mole- cule A Å Water A Å Water B Å Water C Å cule B Gln36 3.0 H2O 984 2.9 H2O 269 2.7 Glu39 Trp37 3.1 H2O 167 3.0 Gln63 Glu39 2.8 H2O 186 3.4 Arg70 2.7 H2O 269 2.8 Arg70 Thr40 2.8 H2O 314 2.8 Gln67 2.8 H2O 314 2.8 H2O 6 2.8 H2O 314 2.8 Thr40 2.8 Glu66 Gln41 2.8 H2O 5212 2.9 Glu66 Asn59 2.9 H2O 212 2.9 Glu66 Gln63 2.7 H2O 901 2.7 Gln63 3.0 H2O 164 3.1 Trp37 Glu66 2.7 H2O 164 3.1 Trp37 2.8 H2O 6 2.8 H2O 314 2.8 Thr40 2.9 H2O 212 2.9 Asn59 2.8 Gln41 Gln67 3.2 H2O 6 2.8 H2O 314 2.8 Thr40 Arg70 2.8 H2O 314 2.8 Thr40 3.4 H2O 186 2.8 Glu39 3.1 H2O 269 2.7 Glu39 sidechain-mainchain contacts; direct Molecule A Å Molecule B Gln36-N 2.9 Gln36 Glu39-O 3.6 Arg70 Arg70 2.6 Glu39-O sidechain-mainchain contacts; water mediated Molecule A Å Water A Å Water B Å Molecule B His32-O 2.9 H2O 1024 3.1 Gln36 Gln36-N 3.2 H2O 41 2.7 Gln36 Gln36 3.1 H2O 1024 2.9 His31-O Trp37-N 2.9 H2O 41 2.7 Gln36 Glu66-O 2.7 H2O 5107 2.8 H2O 5078 2.7 Gln41 The distance in Ångstroms (Å) between the interacting amino acid residues and/or molecules are provided. The water molecules are numbered according to their structure file. The mainchain contacts are indicated by “-N” or “-O” for backbone amido or carboxyl groups respectively.

[0149] A consequence of the utilization of these polar groups in the ring-shaped element is the formation of a compact and potentially specific interaction surface. This structural element forms a relatively flat molecular surface that packs at an angle against another surface presented by the coiled-coil formed by helices &agr;6 and &agr;7. The juxta positioning of the surface of the ring-shaped element with that of the coiled-coil results in a wedge-shaped groove. This groove is lined with hydrophobic residues, with polar residues at the center, and has the natural appearance of a ligand binding site. A possible function for this groove is suggested by the fact that replacement of Arg 31 or Glu 39 in STAT-1 by Ala results in a molecule that is much slower to dephosphorylate after interferon-&ggr; induction when compared to wild type protein [Shuai et al., Mol. Cell. Biol. 16:4932 (1996)]. Mutation of these two residues is likely to drastically alter the conformation of the ring-shaped element (FIG. 2B), suggesting that a phosphatase that controls STAT dephosphorylation might bind to the groove in the N-terminal domain.

[0150] There is one molecule in the asymmetric unit of the STAT-4 crystal, and it is related to another by a two-fold symmetry axis (FIGS. 2A and 3A). There is an extensive interface between the two monomers of the dimer, burying 1,714 A2 of surface area (FIG. 3A). An extended intermolecular hydrogen bonding network is formed at the interface, involving 15 amino acid side chains and 12 water molecules per monomer (FIG. 3B). In addition 5 backbone contacts are also observed in each monomer. 11 of the 15 residues at the interface make direct hydrogen bonding contacts to the other monomer. The water molecules at the interface are very well defined in the electron density map, many of them having low temperature factors (<10 Å2) (FIG. 3C).

[0151] The interface is almost entirely polar, with no involvement of hydrophobic side chains. In contrast to the leucine zipper, wherein hydrophobic residues are utilized to generate the intermolecular interface by the formation of a coiled-coil across the dimer interface [Lupas, Trends in Biochemical Sciences 21:375 (1996)], the coiled-coil in the N-terminal domain is firmly anchored within one N-terminal domain and its role is to serve as an architectural support for the presentation of a number of interacting side chains at the dimer interface and at the potential interaction groove. While hydrophobic interactions are associated with stabilization of folded protein structures and are often found at the core of tight interfaces, polar interactions can provide both stability and specificity in protein-protein interactions [Fersht et al., Nature 314:235 (1985); Xu et al., Journal of Molecular Biology 265:68 (1997)]. In contrast to the residues that constitute the buried core of the N-terminal domain, which are conserved across STATs, the majority of the residues at the dimer interface are not conserved (FIGS. 1 and 3B). This variation could provide specificity in STAT dimer:dimer interactions on DNA. Only two of the residues at the interface are invariant in all STATs: Trp 37, a central anchor residue at the interface, and Glu 39, which also participates in the formation of the ring-shaped element.

[0152] To test the physiological relevance of the dimer that is observed in the crystal structure it was determined if the mutation of the critical Trp 37 residue at the interface would disrupt or reduce oligomerization in vitro and transcriptional activation in vivo. These experiments were carried out using STAT-1, for which a DNA-protein interaction assay had been worked out previously [Vinkemeier et al., EMBO J. 15:5616 (1996)]. In addition, a cell line (U3 Å) lacking STAT-1 is available [Muller et al., EMBO J. 12:4221 (1993)] allowing the introduction of expression vectors encoding mutant STAT-1 molecules. The close similarity between the N-terminal domains of STAT-1 and STAT-4 (51% amino acid identity) ensures that the structural information derived from the STAT-4 crystal structure is an accurate representation of the STAT-1 architecture.

[0153] Conditions under which an oligonucleotide bearing tandem binding sites binds full length STAT-1 as a tetramer have been established; the site contains two “weak” binding sites, spaced 10 base pairs apart [Vinkemeier et al., EMBO J. 15:5616 (1996)]. Competition experiments show that the off time is long for wild type STAT-1 (greater than 15-30 minutes), indicating the formation of a stable complex. In contrast, if an oligonucleotide containing only one “weak” site is used instead, the off time was less than 30 seconds [Vinkemeier et al., EMBO J. 15:5616 (1996)]. The stabilization of STAT-1 on oligonucleotides containing tandem binding sites is not observed if the N-terminal domain is deleted [Vinkemeier et al., EMBO J. 15:5616 (1996)]. This assay was used to test the DNA binding properties of STAT-1 in which Trp 37 was replaced by Ala (W37 Å). Trp 37 plays a central role at the dimer interface, and participates in both direct and water mediated interactions with the other monomer (FIG. 3C).

[0154] The W37A mutant protein binds to the DNA probe with tandem sites, but the interaction is completely displaced by the addition of unlabelled oligonucleotides (FIG. 4A). In contrast, the wild-type protein is resistant to displacement for more than 15 minutes. The same two tandem “weak” binding sites were used to drive transcription from a reporter gene in an interferon-dependent transcriptional assay. U3A cells were co-transfected with the reporter gene and with either wild type or W37A mutant STAT-1. The rather weak transcriptional induction by interferon &ggr; (approximately 2-fold) was abolished by the mutation (FIG. 4B). Activated and dimeric STAT proteins do not form detectable tetrameres in solution in the absence of DNA. It is not known whether this is a consequence of limited binding affinity between N-terminal domains or whether the conformation of the STAT molecule in the absence of DNA impedes further oligomerization. In any case, the presentation of highly polar and unique interaction surfaces by the N-terminal domains of the STATs provides a ready means for generating very specific interactions between adjacent STAT dimers on the DNA, since the hydrogen bonding constraints of the interacting groups places stereochemical constraints on potential partners. While each N-domain dimer is closed, the fact that each STAT dimer presents two N-domains for interaction makes possible the generation of open ended STAT-STAT interactions that are limited only by the nature and number of the adjacent DNA binding sites.

[0155] The present invention is not to be limited in scope by the specific embodiments describe herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

[0156] It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description.

[0157] Various publications are cited herein, the disclosures of which are incorporated by reference in their entireties. The citation of any reference herein should not be construed as an admission that such reference is available as “Prior Art” to the instant application.

Claims

1. A crystal of an N-terminal domain of a STAT protein, wherein the crystal effectively diffracts X-rays for the determination of the atomic coordinates of the N-terminal domain of the STAT protein to a resolution of greater than 5.0 Angstroms.

2. The crystal of claim 1 wherein the crystal effectively diffracts X-rays for the determination of the atomic coordinates of the N-terminus to a resolution of greater than 3.0 Angstroms.

3. The crystal of claim 1 wherein the N-terminal domain comprises the amino acid sequence:

Arg Xaa HXaa Leu Xaa Xaa Trp HXaa Glu Xaa Gln Xaa Trp (SEQ ID NO: 1) wherein: HXaa is either Ile, Leu, Val, Phe, or Tyr.

4. The crystal of claim 3 wherein the N-terminal domain of the STAT protein is contained in a peptide fragment that consists of 100 to 150 amino acids.

5. The crystal of claim 4 wherein the N-terminal domain of the STAT protein comprises amino acids 4-112 of SEQ ID NO:2.

6. The crystal of claim 5 wherein the crystal effectively diffracts X-rays for the determination of the atomic coordinates of the N-terminus to a resolution of 1.45 Angstroms.

7. The crystal of claim 5 having a space group of P6522 and a unit cell of dimensions a=79.5 1 Å, b=79.51 Å, and c=84.68 Å.

8. The crystal of claim 1 having secondary structural elements comprising eight helices (&agr;1-&agr;8) that are assembled into a hook-like structure having an inner and outer surface, wherein:

(a) the first four helices (&agr;1-&agr;4) form a ring-shaped element having a proximal and a distal surface;

(b) helices six (&agr;6) and seven (&agr;7) form an anti-parallel coiled-coil having a proximal and a distal surface;

(c) helix five (&agr;5) connects the ring-shaped element to the anti-parallel coiled-coil; and

(d) helix eight (&agr;8) is wrapped around the distal surface of the ring-shaped element; wherein the inner surface of the hook-like structure is formed by the intersection of the proximal surface of the ring-shaped element with the proximal surface of the antiparallel coiled-coil.

9. A method of using the crystal of claim 1 in a drug screening assay comprising:

(a) selecting a potential drug by performing rational drug design with the three-dimensional structure determined for the crystal, wherein said selecting is performed in conjunction with computer modeling;

(b) contacting the potential drug with a dimeric STAT protein N-terminal domain; and

(c) detecting the binding of the potential drug with the N-terminal domain; wherein a drug is selected that binds to the N-terminal domain.

10. The method of claim 9 wherein contacting the potential drug with a dimeric STAT protein N-terminal domain is performed with a STAT protein fragment containing a STAT protein N-terminal domain comprising SEQ ID NO: 1.

11. The method of claim 10 wherein the STAT protein fragment is labeled.

12. The method of claim 10 wherein the STAT protein fragment is bound to a solid support.

13. A method of using the crystal of claim 1 in a drug screening assay comprising:

(a) selecting a potential drug by performing rational drug design with the three-dimensional structure determined for the crystal, wherein said selecting is performed in conjunction with computer modeling;

(b) contacting the potential drug with two or more dimeric STAT proteins in the presence of a nucleic acid containing at least two adjacent weak binding sites for STAT protein dimers; and

(c) detecting the effect of the potential drug on the binding of the dimeric STAT proteins to each other and/or to the nucleic acid; wherein a potential drug is selected as a candidate drug when it either enhances or diminishes the binding of the dimeric STAT proteins to each other and/or the nucleic acid.

14. The method of claim 13 further comprising:

(d) growing a supplemental crystal containing a protein-drug complex formed between the dimeric N-terminal domain and the candidate drug, wherein the crystal effectively diffracts X-rays for the determination of the atomic coordinates of the protein-ligand complex to a resolution of greater than 5.0 Angstroms;

(e) determining the three-dimensional structure of the supplemental crystal with molecular replacement analysis; and

(f) selecting a drug by performing rational drug design with the three-dimensional structure determined for the supplemental crystal, wherein said selecting is performed in conjunction with computer modeling.

15. A method for identifying a drug that enhances or diminishes the ability of STAT protein dimers to induce the expression of a gene operably under the control of a promoter containing at least two adjacent weak binding sites for STAT protein dimers comprising:

(a) selecting a potential drug by performing rational drug design with the three-dimensional structure determined for the crystal of claim 1, wherein said selecting is performed in conjunction with computer modeling;

(b) measuring the level of expression of a first reporter gene and a second reporter gene contained by a host cell in the presence and absence of the potential drug; wherein the first reporter gene is operably linked to a first promoter containing at least two adjacent weak binding sites for STAT protein dimers, and the second reporter gene is operably linked to a second promoter comprising at least one strong binding site for a STAT protein dimer; wherein the binding of STAT protein dimers to the two adjacent weak binding sites induces the expression of the first reporter gene, and wherein the binding of the STAT protein dimer to the strong binding site induces the expression of the second reporter gene; and wherein the host cell contains STAT protein dimers; and

(c) comparing the level of expression of the first reporter gene with that of the second reporter gene in the presence and absence of the potential drug, wherein when the presence of the potential drug results in an increase in the level of expression of the first reporter gene but not that of the second reporter gene, the potential drug is identified as a drug that enhances the ability of STAT protein dimers to induce the expression of a gene operably under the control of a promoter containing at least two adjacent weak binding sites for STAT protein dimers; and when the presence of a potential drug results in a decrease in the level of expression of the first reporter gene but not that of the second reporter gene the potential drug is identified as a drug that inhibits the ability of STAT protein dimers to induce the expression of a gene operably under the control of a promoter containing at least two adjacent weak binding sites for STAT protein dimers.

16. The method of claim 15 wherein the host cell is a mammalian cell.

17. The method of claim 15 wherein the first reporter gene is contained by a first host cell, and the second reporter gene is contained by a second host cell; and wherein the first host cell and second host cell both contain STAT protein dimers.

18. The method of claim 15 wherein the weak STAT binding sites are selected from the group consisting of sites present in the regulatory regions of the MIG gene, the c-fos gene and the interferon-&ggr; gene.

19. The method of claim 15 further comprising:

(d) growing a supplemental crystal containing a protein-drug complex formed between the dimeric N-terminal domain and the drug, wherein the crystal effectively diffracts X-rays for the determination of the atomic coordinates of the protein-ligand complex to a resolution of greater than 5.0 Angstroms;

(e) determining the three-dimensional structure of the supplemental crystal with molecular replacement analysis; and

(f) selecting a drug by performing rational drug design with the three-dimensional structure determined for the supplemental crystal, wherein said selecting is performed in conjunction with computer modeling.

20. A method for identifying a drug that modulates the ability of adjacent STAT protein dimers to interact and bind to adjacent DNA binding sites comprising:

(a) selecting a potential drug by performing rational drug design with the three-dimensional structure determined for the crystal of claim 1, wherein said selecting is performed in conjunction with computer modeling;

(b) measuring the binding affinity of the STAT protein, or a fragment thereof that comprises the N-terminal domain, to a nucleic acid comprising 2 adjacent weak STAT DNA binding sites in the presence and absence of the potential drug;

(c) measuring the binding affinity of the STAT protein, or the fragment, to a nucleic acid comprising a single strong STAT binding site in the presence and absence of the potential drug; and

(d) comparing the binding affinity measured in step (b) in the presence and absence of the potential drug with the binding affinity measured in step (c) in the presence and absence of the potential drug, wherein a potential drug which causes an increase in the binding affinity measured in step (b) but not in the binding affinity measured in step (c) is identified as a drug that enhances the interaction between adjacent activated STAT dimers, and a potential drug which causes a decrease in the binding affinity measured in step (b) but not in the binding affinity measured in step (c) is identified as a drug that inhibits the interaction between adjacent activated STAT dimers.

21. A method for identifying a drug that modulates the ability of adjacent STAT protein dimers to interact and bind to adjacent DNA binding sites comprising:

(a) measuring the binding affinity of the STAT protein comprising the N-terminal domain, to a nucleic acid comprising two adjacent weak STAT DNA binding sites in the presence and absence of a potential drug;

(b) measuring the binding affinity of a modified form of the STAT protein lacking the &agr;4-tryptophan of N-terminal domain with the nucleic acid in the presence and absence of the potential drug; and

(c) comparing the binding affinity measured in step (a) in the presence and absence of the potential drug with the binding affinity measured in step (b) in the presence and absence of the potential drug, wherein a potential drug which causes an increase in the binding affinity measured in step (a) but not in the binding affinity measured in step (b) is identified as a drug that enhances the interaction between adjacent activated STAT dimers, and a potential drug which causes a decrease in the binding affinity measured in step (a) but not in the binding affinity measured in step (b) is identified as a drug that inhibits the interaction between adjacent activated STAT dimers.

22. A method of making the crystal of claim 1 comprising placing an aliquot of a solution containing the STAT N-terminal domain fragment on a cover slip as a hanging drop above a well containing a reservoir buffer that comprises 0.2 M Na+CH3COO−, 0.1 M Tris/HCL pH 8.0, 17% PEG4000.

23. The method of claim 22 wherein said solution is prepared by combining a preparation of the STAT N-terminal domain fragment that contains 20 mg/ml of the STAT N-terminal domain fragment in 50 mM Hepes/HCl pH 8.0, 150 mM KCl, 2.5 mM CaCl2, and 5 mM DTT with an equal volume of the reservoir buffer; and wherein the aliquot comprises approximately 1 &mgr;l of said solution.

24. The method of claim 23 wherein the STAT N-terminal domain fragment is the STAT-4 N-terminal domain fragment.