WO2007121462A2

WO2007121462A2 - CRYSTAL STRUCTURE OF LXR-ß AND LXR-α

Info

Publication number: WO2007121462A2
Application number: PCT/US2007/066854
Authority: WO
Inventors: Jodi K. Muckelbauer; Vidhyashankar Ramamurthy
Original assignee: Bristol-Myers Squibb Company
Priority date: 2006-04-18
Filing date: 2007-04-18
Publication date: 2007-10-25
Also published as: WO2007121462A3

Abstract

Provided herein are crystalline forms comprising an LXR-ß and an LXR-α ligand binding domain complexed with one or more molecules. In certain embodiments, the LXR-ß or LXR-α ligand binding domain is complexed with a ligand, such as epoxycholesterol, and a co-activator peptide, such as TIF2 or GRIP1B, which may be used in methods for screening for compounds that interact and/or modulate LXR-ß or LXR-α.

Description

CRYSTAL STRUCTURE OF LXR-β AND LXR-α

FIELD OF THE INVENTION

[0001] This present invention relates generally to crystalline forms comprising an LXR-β and an LXR-α ligand binding domain complexed with one or more molecules.

BACKGROUND OF THE INVENTION

[0002] Liver X Receptors ("LXR") LXR-α and LXR-β, are transcription factors belonging to the nuclear hormone receptor superfamily. The alpha and beta subtypes are encoded by separate genes and share about 78% amino acid identity in the DNA- binding and ligand-binding domains (Janowski, B. A., et al., Nature (1996) 383, 728- 731). LXR-α is expressed in organs and/or tissues such as liver, small intestine, spleen, kidney, adrenal gland, adipose, and macrophages, whereas LXR-β is expressed ubiquitously (Peet, D. J., et al., Cell (1998) 93, 693-704; Lehmann, J. M., et al., J. Biol. Chem. (1997) 272, 3137-3140).

[0003] LXRs regulate cholesterol homeostasis by functioning as intracellular receptors for oxygenated cholesterol metabolites, known as oxysterols (Repa, J. J., et al., Science (2000) 289, 1524-1529; Schwartz, K., et al., Biochem. Biophys. Res. Commun. (2000) 274, 794-802; Venkateswaran, A., et al., Proc. Natl. Acad. ScL U.S.A. (2000) 97, 12097-12101). Endogenous ligands of LXRs include oxidized derivatives of cholesterol such as 22(R)-hydroxycholesterol, 24(S),25- epoxycholesterol, and 27-hydroxycholesterol.

[0004] Upon ligand binding, a conformational change in the LXR leads to recruitment of coactivator proteins to the ligand binding domain (LBD), which stimulates expression of target genes. The genes expressed include ATP-binding- cassette transporters (e.g., ABCAl, ABCGl), and apolipoprotein E (Laffitte, B. A., et al., Proc. Natl. Acad. ScL U.S.A. (2001) 98, 507-512; Venkateswaran, A., et al., J. Biol. Chem. (2000) 275, 14700-14707; Costet, P., et al., J. Biol. Chem. (2000) 275, 28240-28245). LXR activation may lead to an increase in high density lipoprotein (HDL) particle number and a decrease in atherosclerotic lesions, thus making LXRs attractive therapeutic targets for treatment of disorders such as dyslipidemia and atherosclerosis (Hoerer, S., et al., J. MoI. Biol. (2003) 334, 853-861). [0005] While LXR crystals have been generated (PCT Application No. WO 2004/058819 and U.S. Patent Application Publication No. 20070060740), there remains a need to determine three-dimensional structures of LXRs in complex with ligands to understand better the molecular interactions between the ligand-binding pocket(s) of LXRs and LXR-ligands. There is also a need to design and identify potent and selective modulators of LXR- receptors (e.g., LXR-α and LXR-β) for therapeutic applications.

SUMMARY OF THE INVENTION In one aspect, the invention provides a crystalline form comprising an LXR-β ligand binding domain complexed with one or more molecules. In one embodiment, the LXR-β ligand binding domain is complexed with a ligand, such as an epoxycholesterol, and a co-activator peptide, such as TIF2. In another embodiment, a crystalline form is provided that diffracts X-rays to a resolution of about 2.0 Angstroms (A), has a unit cell size comprising the dimensions of a=103.7 A, b=l 10.1 A, c=60.5 A, and β = 119.7°, belongs to the space group C2, and comprises an atomic structure according to the structure coordinates of Table 5. In another embodiment, the LXR-β ligand binding domain comprises the amino acid sequence of SEQ ID NO: 1, and TIF2 comprises the amino acid sequence of SEQ ID NO: 12. In another embodiment, the epoxycholesterol is selected from the group comprising 22(R)- hydroxycholesterol, 24(S),25 -epoxycholesterol and 27-hydroxycholesterol. In a further embodiment, the LXR- β ligand binding domain polypeptide is encoded by a nucleic acid of SEQ ID NO: 10 and sequences deviating from SEQ ID NO: 10 due to the degeneracy in the genetic code. In another aspect, the invention provides a crystalline form comprising an

LXR-α ligand binding domain complexed with one or more molecules. In one embodiment, the LXR-α ligand binding domain is complexed with a ligand such as an an epoxycholesterol derivative of formula (I), and a co-activator peptide, such as GRIPlB. In another embodiment, a crystalline form is provided that diffracts X-rays to a resolution of about 2.9 Angstroms (A), has a unit cell size comprising the dimensions of a=b=71.2 A and c=143.2 A, belongs to the space group P4₃22, and comprises an atomic structure according to the structure coordinates of Table 6. In another embodiment, the LXR-α ligand binding domain comprises the amino acid sequence of SEQ ID N0:9 and GRIPlB comprises the amino acid sequence of SEQ ID NO: 13. In another embodiment, the epoxycholesterol is selected from the group comprising 22(R)-hydroxycholesterol, 24(S),25 -epoxycholesterol and 27- hydroxycholesterol. In a further embodiment, the LXR- β ligand binding domain polypeptide is encoded by a nucleic acid of SEQ ID NO: 11 and sequences deviating from SEQ ID NO: 11 due to the degeneracy in the genetic code. [0006] In another aspect, the invention provides methods of identifying modulators of LXR-β activity. In one embodiment, the method includes (a) providing the structure coordinates of an LXR-β ligand binding domain in complex with epoxycholesterol and TIF2; (b) using the three-dimensional structure of an LXR-β ligand binding domain and one or more modeling techniques to design or select a modulator; (c) providing the modulator; and (d) physically contacting the modulator with an LXR-β ligand binding domain, wherein a modulator of LXR-β activity is identified. In one embodiment, the methods may further comprise (e) altering the modulator identified in step (b); and (f) contacting the altered modulator of step (e) with an LXR-β ligand binding domain and determining the ability of the altered modulator to modulate LXR-β activity. The modulator may be designed de novo or designed from a known modulator. The one or more modeling techniques may include graphic molecular modeling and computational chemistry techniques. [0007] In another aspect, the invention provides methods of identifying modulators of LXR-α activity. Specific embodiments include: (a) providing the structure coordinates of an LXR-α ligand binding domain in complex with an epoxycholesterol derivative of formula (I) and GRIPlB; (b) using the three- dimensional structure of an LXR-α ligand binding domain and one or more structural techniques to design or select a modulator; (c) providing the modulator; and (d) physically contacting the modulator with an LXR-α ligand binding domain, wherein a modulator of LXR-α activity is identified. In another embodiment, the methods may further comprise (e) altering the modulator identified in step (b); and (f) contacting the altered modulator of step (e) with an LXR-α ligand binding domain and determining the ability of the altered modulator to modulate LXR-α activity. The modulator may be designed de novo or designed from a known modulator. The one or more modeling techniques may include graphic molecular modeling and computational chemistry techniques.

[0008] In another aspect, the invention provides methods of screening a plurality of compounds for a modulator of LXR-β comprising: (a) providing a library of test samples; (b) contacting a crystal comprising an LXR-β ligand binding domain in complex with epoxycholesterol and TIF2 with each test sample; (c) detecting an interaction between a test sample and the crystalline LXR-β ligand binding domain in complex with epoxycholesterol and TIF2; (d) identifying a test sample that interacts with the crystalline LXR-β ligand binding domain in complex with epoxycholesterol and TIF2; and (e) isolating a test sample that interacts with the crystalline LXR-β ligand binding domain in complex with epoxycholesterol and TIF2; whereby a plurality of compounds is screened for a modulator of LXR-β. [0009] In another aspect, the invention provides methods of screening a plurality of compounds for a modulator of LXR-α comprising: (a) providing a library of test samples; (b) contacting a crystal comprising an LXR-α ligand binding domain in complex with an epoxycholesterol derivative of formula (I) and GRIPlB with each test sample; (c) detecting an interaction between a test sample and the crystalline LXR-α ligand binding domain in complex with an epoxycholesterol derivative of formula (I) and GRIPlB; (d) identifying a test sample that interacts with the crystalline LXR-α ligand binding domain in complex with an epoxycholesterol derivative of formula (I) and GRIPlB; and (e) isolating a test sample that interacts with the crystalline LXR-α ligand binding domain in complex with an epoxycholesterol derivative of formula (I) and GRIPlB; whereby a plurality of compounds is screened for a modulator of LXR-α. [0010] In another aspect, the invention provides methods of identifying an LXR-β modulator comprising: (a) inputting structure coordinates describing an LXR-β ligand binding domain in complex with epoxycholesterol and TIF2 to a computerized modeling system; and (b) modeling ligands for the binding pocket of the LXR-β ligand binding domain. [0011] In another aspect, the invention provides methods of identifying an LXR- α modulator comprising: (a) inputting structure coordinates describing an LXR-α ligand binding domain in complex with an epoxycholesterol derivative of formula (I) and GRIPlB to a computerized modeling system; and (b) modeling ligands for the binding pocket of the LXR-α ligand binding domain.

[0012] In another aspect, the invention provides methods for identifying an agent that interacts with an active site of an LXR-β ligand binding domain. Such methods comprise the steps of: (a) obtaining a crystallized complex comprising an LXR-β ligand binding domain in complex with epoxy cholesterol and TIF2; (b) determining the structure coordinates of the amino acids in the crystallized complex; (c) generating a three-dimensional model of the LXR-β ligand binding domain complex using the structure coordinates of the amino acids, wherein the ± root mean square deviation from the backbone atoms of said amino acids of not more than about 1.5 A; (d) determining an active site of the LXR-β ligand binding domain complex from the three-dimensional model; and (e) performing computer-aided fitting analyses to identify an agent which interacts with the active site. The identified agent can be an inhibitor or an activator of LXR-β activity. In some embodiments, the ± root mean square deviation from the backbone atoms of the amino acids is not more than about 1.0 A or not more than about 0.5 A. In some embodiments, the identified agent can be contacted with the LXR-β ligand binding domain complex to determine the effect the agent has on LXR-β activity. [0013] In another aspect, the invention provides methods for identifying an agent that interacts with an active site of an LXR-α ligand binding domain. Such methods comprise the steps of: (a) obtaining a crystallized complex comprising an LXR-α ligand binding domain in complex with an epoxycholesterol derivative of formula (I) and GRIPlB; (b) determining the structure coordinates of the amino acids in the crystallized complex; (c) generating a three-dimensional model of the LXR-α ligand binding domain complex using the structure coordinates of the amino acids, wherein the ± root mean square deviation from the backbone atoms of said amino acids of not more than about 1.5 A; (d) determining an active site of the LXR-α ligand binding domain complex from the three-dimensional model; and (e) performing computer- aided fitting analyses to identify an agent which interacts with the active site. The identified agent can be an inhibitor or an activator of LXR-α activity. In some embodiments, the ± root mean square deviation from the backbone atoms of the amino acids is not more than about 1.0 A or not more than about 0.5 A. In some embodiments, the identified agent can be contacted with the LXR-α ligand binding domain complex to determine the effect the agent has on LXR-α activity. [0014] In another aspect, the invention provides crystallizable compositions comprising an LXR-β ligand binding domain complexed with epoxycholesterol and TIF2. In one embodiment, the composition comprises an LXR-β ligand binding domain of SEQ ID NO: 1 and TIF2 of SEQ ID NO: 12 and mutants thereof, wherein said mutants comprise one or more sequence substitutions. [0015] In another aspect, the invention provides crystallizable compositions comprising an LXR-α ligand binding domain complexed with an epoxycholesterol derivative of formula (I) and GRIPlB. In one embodiment, the composition comprises an LXR-α ligand binding domain of SEQ ID NO: 9 and GRIPlB of SEQ

ID NO: 13 and mutants thereof, wherein said mutants comprise one or more sequence substitutions.

[0016] In another aspect, the invention provides methods for designing compounds that modulate LXR-β activity, comprising: (a) generating a computer readable model of a binding site of an LXR-β ligand binding domain in complex with epoxycholesterol and TIF2; and (b) using the model to design a compound having a structure and charge distribution compatible with the binding site, wherein the compound comprises a functional group that interacts with the binding site to modulate LXR-β activity.

[0017] In another aspect, the invention provides methods for designing compounds that modulate LXR-α activity, comprising: (a) generating a computer readable model of a binding site of an LXR-α ligand binding domain in complex with an epoxycholesterol derivative of formula (I) and GRIPlB; and (b) using the model to design a compound having a structure and charge distribution compatible with the binding site, wherein the compound comprises a functional group that interacts with the binding site to modulate LXR-α activity.

[0018] In another aspect, the invention provides methods for identifying compounds that bind to an LXR-β ligand binding domain comprising: (a) providing a set of structure coordinates defining the three-dimensional structure of a crystal of LXR-β ligand binding domain in complex with epoxycholesterol and TIF2, or coordinates having a root mean square deviation therefrom, with respect to at least 50% of Ca atoms, of not more than about +1.5 A, in computer readable form; and (b) selecting a compound using the structure coordinates, wherein selecting is performed in conjunction with computer modeling, to identify a compound that binds to the LXR-β ligand binding domain. [0019] In another aspect, the invention provides methods for identifying compounds that bind to an LXR-α ligand binding domain comprising: (a) providing a set of structure coordinates defining the three-dimensional structure of a crystal of LXR-α ligand binding domain in complex with an epoxycholesterol derivative of formula (I) and GRIPlB, or coordinates having a root mean square deviation therefrom, with respect to at least 50% of Ca atoms, of not more than about +1.5 A, in computer readable form; and (b) selecting a compound by performing drug design with the structure coordinates, wherein selecting is performed in conjunction with computer modeling, to identify a compound that binds to the LXR-α ligand binding domain. [0020] In another aspect, the invention provides compounds, modulators, and agents identified or designed by any of the aforementioned methods, as well as pharmaceutical compositions comprising any compound, modulator, or agent so identified or designed. [0021] In another aspect, the invention provides methods for preparing a crystalline form comprising an LXR-β ligand binding domain complexed with epoxycholesterol and TIF2. Such methods comprise: (a) mixing an LXR-β ligand binding domain, epoxycholesterol and TIF2 with a crystallization solution to form a mixture; (b) streak-seeding drops of the mixture of step (a); (c) vapor equilibrating the seeded drops in a closed container against the crystallization solution to obtain a crystalline form of the complex and to produce an equilibrated crystal drop solution; (d) replacing the equilibrated crystal drop solution with a cryoprotectant; and (e) flash-freezing the crystal. The crystallization solution may comprise one or more precipitants selected from the group consisting of 15-30% PEG10,000, PEG3350, and Jeffamine ED-2001 pH 7.0; one or more salts selected from the group consisting of 0.1-0.2M ammonium acetate, sodium formate, magnesium chloride, and sodium chloride; and one or more buffers selected from the group consisting of 0.1M Bis-tris, pH 5.5 and 6.5 and 0.1M Hepes, pH 7.0 and 7.5. In one embodiment, the crystallization solution comprises 15-30% PEG3350 and 0.1M Hepes, pH 7.2-7.5. In some embodiments, the seeded drops are equilibrated by hanging drop method in step (c); in some embodiments, the cryoprotectant comprises crystallization solution and about 20% glycerol in step (d); and in other embodiments, the crystal is flash-frozen in liquid nitrogen in step (e).

[0022] In another aspect, the invention provides methods for preparing a crystalline form comprising an LXR-α ligand binding domain complexed with an epoxycholesterol derivative of formula (I) and GRIPlB. Such methods comprise: (a) mixing an LXR-α ligand binding domain, an epoxycholesterol derivative of formula (I) and GRIPlB with a crystallization solution to form a mixture; (b) streak-seeding drops of the mixture of step (a); (c) vapor equilibrating the seeded drops in a closed container against the crystallization solution to obtain a crystalline form of the complex and to produce an equilibrated crystal drop solution; (d) replacing the equilibrated crystal drop solution with a cryoprotectant; and (e) flash-freezing the crystal. In one embodiment, the crystallization solution comprises 1.4-2. OM lithium sulfate, 2%-8% (±)-2-methyl-2,4-pentanediol and 0.1M imidazole, pH 6.5. In another embodiment, the seeded drops are equilibrated by hanging drop method in step (c); in some embodiments, the cryoprotectant comprises crystallization solution and about 25% glycerol in step (d); and in other embodiments, the crystal is flash-frozen in liquid nitrogen in step (e).

[0023] In another aspect, the invention provides methods of modeling a three- dimensional structure of an LXR-β ligand binding domain in complex with epoxycholesterol and TIF2, from a template comprising the X-ray structure of an LXR-β ligand binding domain in complex with epoxycholesterol and TIF2, comprising: (a) selecting an X-ray structure of an LXR-β ligand binding domain as a starting model for the LXR-β ligand binding domain; (b) manipulating the starting model for the LXR-β ligand binding domain as a rigid body to superimpose its backbone atoms onto corresponding backbone atoms of a three-dimensional template structure comprising an LXR-β ligand binding domain in complex with epoxycholesterol and TIF2, to form a manipulated model; (c) making a copy of the TIF2 from the template structure to form a model of TIF2 bound to a template LXR-β ligand binding domain; (d) merging the model of the TIF2 into the manipulated model to form a modified model; (e) removing one or more amino acids from the modified model; and (f) optimizing side-chain conformations. The X-ray structure of an LXR-β ligand binding domain may be a structure formulated by homology modeling and the optimizing may comprise varying distance constraints. In some embodiments, the LXR-β ligand binding domain is a mutant or homologue of the template LXR-β ligand binding domain. In other embodiments, the LXR-β ligand binding domain is a different crystal form of the template LXR-β ligand binding domain. [0024] In another aspect, the invention provides methods of modeling a three- dimensional structure of an LXR-α ligand binding domain in complex with an epoxy cholesterol derivative of formula (I) and GRIPlB, from a template comprising the X-ray structure of an LXR-α ligand binding domain in complex with an epoxycholesterol derivative of formula (I) and GRIPlB, comprising: (a) selecting an X-ray structure of an LXR-α ligand binding domain as a starting model for the LXR- α ligand binding domain; (b) manipulating the starting model for the LXR-α ligand binding domain as a rigid body to superimpose its backbone atoms onto corresponding backbone atoms of a three-dimensional template structure comprising an LXR-α ligand binding domain in complex with an epoxycholesterol derivative of formula (I) and GRIPlB, to form a manipulated model; (c) making a copy of the GRIPlB from the template structure to form a model of GRIPlB bound to a template LXR-α ligand binding domain; (d) merging the model of the GRIPlB into the manipulated model to form a modified model; (e) removing one or more amino acids from the modified model; and (f) optimizing side-chain conformations. The X-ray structure of an LXR-α ligand binding domain may be a structure formulated by homology modeling and the optimizing may comprise varying distance constraints. In some embodiments, the LXR-α ligand binding domain is a mutant or homologue of the template LXR-α ligand binding domain. In other embodiments, the LXR-α ligand binding domain is a different crystal form of the template LXR-α ligand binding domain. [0025] In another aspect, the invention provides a method of screening comprising: (a) providing a complexed crystalline form having more than one components; (b) soaking said crystalline form in buffer; (c) adding a test compound to said soaking crystalline form; and (d) evaluating whether said test compound displaced a component of said complexed crystalline form. [0026] In another aspect, the invention provides a method of screening comprising: (a) providing a crystalline form comprising LXR- β complexed with epoxycholesterol and TIF2 crystals; (b) soaking said crystalline form in buffer; (c) adding a test compound to said soaking crystalline form; and (d) evaluating whether said test compound displaced expoxycholesterol from said crystalline form. [0027] In another aspect, the invention provides a method of screening comprising: (a) providing a crystalline form comprising LXR-α complexed with epoxycholesterol and GRIPlB crystals; (b) soaking said crystalline form in buffer; (c) adding a test compound to said soaking crystalline form; and (d) evaluating whether said test compound displaced expoxycholesterol from said crystalline form. [0028] In another aspect, the invention provides a method of identifying a modulator of LXR-β or LXR-α comprising: (a) providing the structure coordinates of the LXR-β or LXR-α polypeptide provided in one of Tables 5-6 defining a three- dimensional structure of the LXR-β or LXR-α polypeptide; (b) using the three- dimensional structure to design or select a test compound by computer modeling; (c) synthesizing or acquiring the test compound; and (d) determining the ability of the test compound to modulate a biological activity of the LXR-β or LXR-α polypeptide, wherein a difference in the biological activity of the LXR-β or LXR-α polypeptide observed in the presence and absence of the test compound indicates the test compound is a modulator of the LXR-β or LXR-α polypeptide. [0029] In one embodiment, the step of using the three-dimensional structure to design or select a test compound by computer modeling comprises: (a) identifying chemical entities or fragments with the potential to bind the LXR-β or LXR-α polypeptide; and (b) assembling the identified chemical entities or fragments into a single molecule to provide the structure of the test compound. In another embodiment, the biological activity is an activity associated with a transcription factor or an intracellular receptor. In another embodiment, the activity is regulating cholesterol homeostasis. In another embodiment, the test compound is labeled. In another embodiment, the label is a fluorescent label. In another embodiment, the label emits a signal upon binding LXR-β or LXR-α.

[0030] In another aspect, the invention provides a method of identifying a modulator of an LXR-β or LXR-α polypeptide comprising: (a) obtaining a crystal of a complex comprising the LXR-β or LXR-α polypeptide and a ligand; (b) obtaining the structure coordinates of the crystal; (c) using the structure coordinates and one or more molecular techniques to identify a compound that modulates LXR-β or LXR-α activity; (d) assaying properties of the compound by administering it to a cell or cell extract of LXR-β or LXR-α; and (e) detecting at least one LXR-β or LXR-α activity, wherein an increase or a decrease in the at least one LXR-β or LXR-α activity indicates that the compound is a modulator of LXR-β or LXR-α. [0031] In one embodiment, the property is an inhibitory property or an activating property. In another embodiment, the at least one LXR-β or LXR-α activity is transcription factor activity or intracellular receptor activity. In another embodiment, the at least one LXR-β or LXR-α activity is regulating cholesterol homeostasis.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIG. 1 shows the amino acid sequence of residues 214-461 of the human LXR-β ligand binding domain including an N-terminal Hisβ tag. [0033] FIG. 2 shows the structure of 24(S)-25-epoxycholesterol.

[0034] FIG. 3 shows the X-ray crystal structure of a human LXR-β LBD dimer complexed with epoxycholesterol and TIF2 co-activator peptide. LXR-β LBD Ca backbone is shown as a ribbon, TIF2 co-activator peptide is shown in stick and epoxycholesterol is shown as space filling spheres. [0035] FIG. 4 shows the multiple alignment of LXR-α (NR1H3_HUMAN) (SEQ ID NO:2) with its structural homologs from PDB. In each case, the ligand binding domain of the receptor was used. IUHL B: LXRα in heterodimer with RXRβ, B chain (SEQ ID NO:3); 1PQ6 B: LXRβ complexed with GW3965, B chain (SEQ ID NO:4); 1MVC_A: Retinoid X-receptor alpha, A chain (SEQ ID NO:5); 2LBD: Retinoic acid receptor gamma (SEQ ID NO:6); 1IE9 A: Vitamin D nuclear receptor, A chain (SEQ ID NO:7); INA V_A: Thyroid hormone receptor beta, A chain (SEQ ID NO:8). [0036] FIG. 5 shows the amino acid sequence of residues 205-447 of the LXR-α ligand binding domain including an N-terminal Hisβ tag (SEQ ID NO:9).

[0037] FIG. 6 shows the structure of an epoxycholesterol derivative of formula

(I)- [0038] FIG. 7 shows the X-ray crystal structure of human LXR-α LBD complexed with an epoxycholesterol derivative of formula (I) and GRIPlB co- activator peptide. LXR-α LBD Ca backbone is shown as a ribbon, GRIPlB co- activator peptide is shown in stick and the epoxycholesterol derivative is shown as space filling spheres. [0039] FIG. 8 shows the nucleic acid sequence of the ligand binding domain of LXR-β (SEQ ID NO: 10).

[0040] FIG. 9 shows the nucleic acid sequence of the ligand binding domain of LXR-α (SEQ ID NO: 11).

DESCRIPTION OF TABLES

[0041] Table 1 is a table showing the data statistics for the LXR-β ligand binding domain in complex with epoxycholesterol and TIF2.

[0042] Table 2 is a table showing the refinement statistics for the LXR-β ligand binding domain in complex with epoxycholesterol and TIF2. [0043] Table 3 is a table showing the data statistics for the LXR-α ligand binding domain in complex with epoxycholesterol derivative of formula (I) and GRIPlB.

[0044] Table 4 is a table showing the refinement statistics for the LXR-α ligand binding domain in complex with epoxycholesterol derivative of formula (I) and

GRIPlB. [0045] Table 5 is a table showing structure coordinates describing the structure of an LXR-β ligand binding domain in complex with epoxycholesterol and TIF2.

[0046] Table 6 is a table showing structure coordinates describing the structure of an LXR-α ligand binding domain in complex with epoxycholesterol derivative of formula (I) and GRIPlB.

DETAILED DESCRIPTION OF THE INVENTION [0047] Described herein is a detailed three-dimensional structure of an LXR-β ligand binding domain. In one embodiment the LXR-β ligand binding domain is in complex with an epoxycholesterol ligand and a TIF2 coactivator peptide. Also described herein is a detailed three-dimensional structure of an LXR-α ligand binding domain. In one embodiment the LXR-α ligand binding domain is in complex with an epoxycholesterol derivative of formula (I):

and GRIPlB coactivator peptide. Further provided are: 1) methods of using the three-dimensional crystal structure to identify and design compounds that can modulate LXR-β or LXR-α activity; and 2) methods of using the three-dimensional crystal structure to determine the structure of an unknown LXR-β or LXR-α crystal. The three-dimensional structure of the ligand binding domain of LXR-β and LXR-α disclosed herein reveals several unique structural features heretofor unidentified, which can be exploited in a modulator design process. Thus, as described herein, the present invention encompasses not only the three-dimensional structure of the ligand binding domain of LXR-β and LXR-α (described by the structure coordinates presented in Tables 5-6), but also various uses of the structure including screening methods and modulator design methods.

DEFINITIONS

[0048] The articles "a" and "an" are used herein to refer to one or to more than one (e.g., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element. [0049] As used herein, the term "about," when referring to a value or to an amount of mass, weight, time, volume, concentration, length, or percentage is meant to encompass variations of ±20% or less (e.g., ±15%, ±10%, ±7%, ±5%, ±4%, ±3%, ±2%, ±1%, or ±0.1%) from the specified amount, as such variations are appropriate. [0050] As used herein, the terms "binding site," "ligand binding site," and "ligand binding domain" are used interchangeably and mean a region of a molecule or molecular complex that, as a result of its shape, favorably associates with a ligand, cofactor, ion, etc. In one aspect of the present invention, the LXR-β ligand binding domain comprises amino acids 214-461 (SEQ ID NO: 1; Figure 1) of human LXR-β. In another aspect of the present invention, the LXR-α ligand binding domain comprises amino acids 205-447 (SEQ ID NO:9; Figure 5) of human LXR-α. [0051] As used herein, the term "biological activity" or "activity" means any observable effect flowing from an LXR-β or LXR-α polypeptide or any full length or truncated LXR-β or LXR-α polypeptide. Representative, but non-limiting, examples of biological activity in the context of the present invention include an activity associated with a transcription factor or intracellular receptor. In one embodiment, the activity is regulation of cholesterol homeostasis. [0052] As used herein the term "complementary" means a nucleic acid sequence that is base paired, or is capable of base-pairing, according to the standard Watson- Crick complementarity rules. Depending on the context, the term can also refer to a favorable spatial arrangement between the surface of a ligand and the surface of its binding site. [0053] As used herein, the terms "dock" and "perform a fitting operation," in all their grammatical forms, mean the computational placement of a chemical entity (e.g., a ligand or modulator (or a candidate ligand or modulator), such as a small organic molecule) within a space at least partially enclosed by the protein structure (e.g., a ligand binding site) so that structural and chemical feature complementarity between chemical entity and binding site components (e.g., binding contacts) can be assessed in terms of interactions typical of protein/ligand complexes. Such placement could be conducted manually or automatically and either approach can employ software designed for such purpose (e.g., INSIGHT II and modules therein, available from Accelrys, San Diego, California, USA or Maestro™ and Glide™, available from Schrδdinger, LLC, New York, New York). [0054] As used herein, the terms "fusion protein" and "fusion polypeptide" refer to a chimeric protein as that term is known in the art and may be constructed using methods known in the art. In many examples of fusion proteins, there are two different polypeptide sequences, and in certain cases, there may be more. The sequences can be linked in frame. A fusion protein can include a domain which is found (albeit in a different protein) in an organism which also expresses the first protein. In various embodiments, the fusion polypeptide can comprise one or more amino acid sequences linked to a first polypeptide. In the case where more than one amino acid sequence is fused to a first polypeptide, the fusion sequences can be multiple copies of the same sequence, or alternatively, may be different amino acid sequences. The fusion polypeptides can be fused to the N-terminus, the C-terminus, or the N- and C-terminus of the first polypeptide. Exemplary fusion proteins include polypeptides comprising a glutathione S-transferase tag (GST-tag), histidine tag (His- tag), an immunoglobulin domain or an immunoglobulin binding domain. [0055] As used herein, the term "identity" refers to the subunit sequence match between two polymeric molecules, e.g., between two nucleic acid molecules, e.g., two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then they are identical at that position. The "similarity" between two sequences is a direct function of the number of matching or identical positions, e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two compound sequences are identical then the two sequences share 50% similarity, if 90% of the positions, e.g., 9 of 10, are identical, the two sequences share 90% similarity. By way of example, the DNA sequences 3ΑTTGCC5' and 3'TATGGC share 50% similarity. Amino acid or nucleotide sequences which share common structural domains that have at least 40% similarity, preferably 50%, preferably 60% similarity, more preferably 70%, even more preferably 80%, even more preferably 85%, even more preferably 90%, even more preferably 95%, even more preferably 96%, preferably 97%, even more preferably 98%, even more preferably 99% similarity across the amino acid sequences of the domains and contain at least one and preferably two structural domains or motifs, are defined herein as "homologous". Furthermore, amino acid or nucleotide sequences which share at least 40% similarity, preferably 50%, preferably 60% similarity, more preferably 70%, even more preferably 80%, even more preferably 90%, even more preferably 95%, even more preferably 96%, preferably 97%, even more preferably 98%, even more preferably 99% similarity and share a common functional activity are defined herein as homologous.

[0056] As used herein, "percent identity" or "percent similarity" is used synonymously with "sequence identity." The determination of percent identity between two nucleotide or amino acid sequences can be accomplished using a mathematical algorithm. For example, a mathematical algorithm useful for comparing two sequences is the algorithm of Karlin and Altschul (1990, Proc. Natl. Acad. Sci. USA 87:2264-2268), modified as in Karlin and Altschul (1993, Proc. Natl. Acad. Sci. USA 90:5873-5877). This algorithm is incorporated into the NBLAST and XBLAST programs of Altschul, et al. (1990, J. MoI. Biol. 215:403-410), and can be accessed, for example at the National Center for Biotechnology Information (NCBI) world wide web site having the universal resource locator "www.ncbi.nlm.nih.gov/BLAST/". BLAST nucleotide searches can be performed with the NBLAST program (designated "blastn" at the NCBI web site), using the following parameters: gap penalty = 5; gap extension penalty = 2; mismatch penalty = 3; match reward = 1; expectation value 10.0; and word size = 11 to obtain nucleotide sequences homologous to a nucleic acid described herein. BLAST protein searches can be performed with the XBLAST program (designated "blastx" at the NCBI web site) or the NCBI "blastp" program, using the following parameters: expectation value 10.0, BLOSUM62 scoring matrix to obtain amino acid sequences homologous to a protein molecule described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997, Nucleic Acids Res. 25:3389-3402). Alternatively, PSI-Blast or PHI-Blast can be used to perform an iterated search which detects distant relationships between molecules (Id.) and relationships between molecules which share a common pattern. When utilizing BLAST, Gapped BLAST, PSI-Blast, and PHI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov. Another example of computing percent identity is by comparing sequence information using the GAP computer program, available from the University of Wisconsin Geneticist Computer Group. The GAP program utilizes the alignment method of Needleman et al, (1970) J. MoI Biol. 48: 443, as revised by Smith et al, (1981) Adv. Appl. Math. 2:482. Briefly, the GAP program defines similarity as the number of aligned symbols (e.g., nucleotides or amino acids) that are similar, divided by the total number of symbols in the shorter of the two sequences. The preferred parameters for the GAP program are the default parameters. See, e.g., Schwartz et al (eds.), (1979), Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 357-358, and Gribskov et al, (1986) Nucl. Acids. Res. 14: 6745.

[0057] The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent sequence identity, typically exact matches are counted.

[0058] As used herein, the terms "lattice parameter," "lattice parameters," "unit cell dimension," and "unit cell dimensions refer to the spacing between unit cells in various directions. There may exist a variation in the lattice parameter on any cell axis at any cell point within any given structure. For example, for the lattice parameters for the crystal structures of the present invention may vary from 2-5% for any axis.

[0059] As used herein, the terms "LXR-β gene," "LXR-α gene," "LXR-β or LXR-α gene," and "recombinant LXR-β gene," "recombinant LXR-α gene," or "recombinant LXR-β or LXR-α gene" mean a nucleic acid molecule comprising an open reading frame encoding an LXR-β or LXR-α polypeptide of the present invention, including both exon and (optionally) intron sequences. [0060] As used herein, the term "substantially identical" means at least 75% sequence identity between nucleotide or amino acid sequences. Sequence similarity is calculated based on a reference sequence, which can be a subset of a larger sequence, such as a conserved motif, coding region, flanking region, etc. In the context of nucleic acids, a reference sequence will usually be about 18 nucleotides (nt) long, more usually about 30 nt long, and can extend to the complete sequence that is being compared. Algorithms for sequence analysis are known in the art, such as BLAST, described in Altschul et al, (1990) J. MoI Biol. 215: 403-10. [0061] As used herein, nucleic acid and protein sequences are "substantially identical" to specific sequences disclosed herein if the sequences have between about 70% and 80%, preferably between about 81% to about 90% or even more preferably between about 91% and 99.99% sequence identity with the corresponding sequence of the protein or nucleic acid sequences described herein.

[0062] As used herein, the terms "LXR-β or LXR-α gene product", "LXR-β or LXR-α protein", "LXR-β or LXR-α polypeptide", "LXR-β or LXR-α ligand binding domain polypeptide" and "LXR-β or LXR-α peptide" are used interchangeably and mean a polypeptide having an amino acid sequence that is substantially identical to a wild type LXR-β or LXR-α amino acid sequence from an organism of interest and which is biologically active in that it comprises all or a part of the amino acid sequence of an LXR-β or LXR-α polypeptide, preferably the ligand binding domain, or cross-reacts with antibodies raised against an LXR-β or LXR-α polypeptide, or retains all or some of the biological activity (e.g., transcription factor activity and/or the ability to regulation cholesterol homeostasis) of the native amino acid sequence or protein. [0063] As used herein, the terms "LXR-β or LXR-α gene product", "LXR-β or LXR-α protein", "LXR-β or LXR-α polypeptide", "LXR-β or LXR-α ligand binding domain polypeptide" and "LXR-β or LXR-α peptide" also include analogs of an LXR-β or LXR-α polypeptide. By "analog" it is intended that a DNA or amino acid sequence can contain alterations relative to the sequences disclosed herein, yet still retain all or some of the biological activity of those sequences. Those of ordinary skill in the art will appreciate that other analogs as yet undisclosed or undiscovered can be used to design and/or construct an LXR-β or LXR-α analog. The terms "LXR- β or LXR-α gene product", "LXR-β or LXR-α protein", "LXR-β or LXR-α polypeptide", "LXR-β or LXR-α ligand binding domain polypeptide" and "LXR-β or LXR-α peptide" also include fusion, chimeric or recombinant LXR-β or LXR-α polypeptides and proteins comprising sequences of the present invention. [0064] As used herein, the terms "isolated" and "purified" are used interchangeably and refer to material (e.g., a nucleic acid or a protein) that has been removed from its original environment, e.g., the natural environment, if it is naturally occurring. The terms, therefore, refer to an object species that is the predominant species present (e.g., on a molar basis it is more abundant than any other individual species in the composition). [0065] As used herein, the term "isomorphous replacement" means a method of the introduction of non-naturally occurring, well ordered, x-ray scatterers into a crystal. These x-ray scatterers are often heavy metal atoms, and if the additions do not change the structure of the molecule or of the crystal cell, the resulting crystals are isomorphous. Isomorphous replacement experiments are usually performed by diffusing different heavy-metal metals into the channels of a pre-existing protein crystal. Growing the crystal from protein that has been soaked in a solution containing the heavy atom is also possible (Petsko, G. A., 1985. Methods in Enzymology, Vol. 114. Academic Press, Orlando, pp. 147-156). The phrase "heavy atom derivatization" is synonymous with the term "isomorphous replacement" and these terms are used synonymously herein.

[0066] As used herein, the term "ligand" means any molecule that is known or suspected to associate with another molecule. The term "ligand" encompasses inhibitors, activators, agonists, antagonists, natural substrates and analogs of natural substrates.

[0067] As used herein the terms "modulate" and grammatical derivations thereof refer to an increase, decrease, or other alteration of any and/or all chemical and biological activities or properties mediated by a given DNA sequence, RNA sequence, polypeptide, peptide or molecule. The definition of "modulate" as used herein encompasses agonists and/or antagonists of a particular activity, DNA, RNA, or protein. The term "modulation" therefore refers to both upregulation (e.g., activation or stimulation) and downregulation (e.g., inhibition or suppression) of a response by any mode of action. A "modulator of LXR-β or LXR-α" is a molecule that causes an increase, decrease, or other alteration of any and/or all chemical and biological activities or properties of LXR-β or LXR-α. Samples or assays that are treated with a potential modulator are compared to control samples without the potential modulator, to examine the extent of inhibition or activation of LXR-β or LXR-α activity. [0068] As used herein, the term "molecular replacement" means a method of determining a three-dimensional structure of a compound (e.g., a protein) that involves generating a preliminary model of a wild-type or mutant protein that has been crystallized whose structure coordinates are unknown, by orienting and positioning a molecule whose structure coordinates are known (e.g., a homolog of LXR-β or LXR-α, e.g., LXR-β or LXR-α polypeptide, as disclosed herein) within the unit cell of the unknown crystal so as best to account for the observed diffraction pattern of the unknown crystal. Phases can then be calculated from this model and combined with the observed amplitudes to give an approximate Fourier synthesis of the structure whose coordinates are unknown. This, in turn, can be subject to any of the several forms of refinement to provide a final, accurate structure of the unknown crystal (see, e.g., Lattman, (1985) Method Enzymol. 115:55-77; Rossmann (ed), The Molecular Replacement Method, Gordon & Breach, New York, New York, USA, (1972)). Commonly used computer software packages for molecular replacement are CNX, X-PLOR (Brunger, (1992) Nature 355: 472-475), AMoRe (Navaza, (1994) Acta. Cryst. 50: 157-163), the CCP4 package, the MERLOT package (Fitzgerald, (1988) J. Appl. Cryst., Vol. 21, pp. 273-278), XTALVIEW (McRee et al, (1992) J. MoI. Graphics 10: 44- 46) and EPMR. The quality of the model may be analyzed using a program such as PROCHECK or 3D-Profiler (Laskowski et al., (1993) J. Appl. Cryst. 26:283-291; Luthy et al., Nature 356: 83-85, 1992; and Bowie et al., Science 253: 164-170, 1991).

[0069] Using the structure coordinates of LXR-β or LXR-α provided by the present invention, in conjunction with appropriate commercially available software (e.g., AMoRe, Navaza, (1994) Acta. Cryst. 50: 157-163), molecular replacement can be used to determine the structure coordinates of a crystalline mutant or homolog of an LXR-β or LXR-α polypeptide, a structure known or suspected to be similar to the LXR-β or LXR-α structure of the present invention or of a different crystal form of an LXR-β or LXR-α polypeptide. [0070] As used herein, the term "mutant" encompasses fusion, chimeric and recombinant polypeptides and proteins comprising sequences of the present invention. In the context of the present invention, the term "mutant" encompasses a polypeptide otherwise falling within the definition of a polypeptide as set forth herein, but having an amino acid sequence which differs from that of the wildtype polypeptide, by way of one or more deletions, substitutions, or insertions.

[0071] As used herein, a "polypeptide having biological activity" refers to a polypeptide exhibiting activity substantially similar, but not necessarily identical to, an activity of an LXR-β or LXR-α polypeptide of the present invention as measured in a particular biological assay. Representative biological activities include the ability to catalyze the dephosphorylation of phosphotyrosine residues in a protein or peptide; the ability to remove a phosphate from a non-protein tyrosine phosphatase substrate such as DiFMUP or pNPP; to interact with proteins, peptides, sugars, lipids, co-factors or any other biomolecules. A substantially similar biological activity means that the polypeptides carry out a similar function, e.g., a similar enzymatic reaction or a similar physiological process, etc. For example, two homologous proteins may have a substantially similar biological activity if they are involved in a similar enzymatic reaction, e.g., they are both phosphatases which catalyze dephosphorylation of a substrate polypeptide, however, they may dephosphorylate different regions on the same protein substrate or different substrate proteins altogether. Alternatively, two homologous proteins may also have a substantially similar biological activity if they are both involved in a similar physiological process, e.g., transcription. For example, two proteins may be transcription factors, however, they may bind to different DNA sequences or bind to different polypeptide interactors. Substantially similar biological activities may also be associated with proteins carrying out a similar structural role, for example, two membrane proteins. [0072] As used herein, the term "root mean square deviation" means the square root of the arithmetic mean of the squares of the deviations. It is a way to express the deviation or variation from a trend or object, e.g. in the present invention pairs of atoms. In the present invention, for example, "root mean square deviation" describes the variation in the backbone of a mutant or homologous protein from the backbone of LXR-β or LXR-α or a binding pocket portion thereof, as defined by the structure coordinates of LXR-β or LXR-α described in Tables 5-6 herein.

[0073] As used herein, the term "space group" means the arrangement of symmetry elements of a crystal.

[0074] As used herein, the term "stringent hybridization conditions" refers to an overnight incubation at 42°C in a solution comprising 50% formamide, 5x SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x

Denhardt's solution, 10% dextran sulfate, and 20 μg/mL denatured, sheared salmon sperm DNA, followed by washing the filtrates in 0. Ix SSC at about 65°C. [0075] As used herein, the terms "structural coordinates," "structure coordinates," and "atomic coordinates" include any set of structure coordinates for LXR-β or LXR- α, including those describing an LXR-β or LXR-α mutant, fusion protein, etc., and fragments thereof. Structural coordinates that have a root mean square deviation (RMSD) preferably no more than about 3.0 A, more preferably no more than about 2.0 A, even more preferably less than about 1.0 A when superimposed on the polypeptide backbone Ca atoms defined by the structural coordinates listed in Tables 5-6 herein are considered identical. [0076] In one embodiment, the LXR-β ligand binding domain complex is a crystal according to the structure coordinates of Table 5. In another embodiment, the LXR-α ligand binding domain is a crystal according to the structure coordinates of Table 6. The structure coordinates refer to coordinates derived from mathematical equations related to the patterns obtained from diffraction of a beam of X-rays by the atoms (scattering centers) of an LXR-β or LXR-α ligand binding domain in a complexed crystal form. The diffraction data are used to calculate an electron density map of the repeating unit of the crystal. The electron density maps are then used to establish the positions of the individual atoms of an LXR-β or LXR-α ligand binding domain complex. [0077] Those of ordinary skill in the art will understand that a set of structure coordinates for an LXR-β or LXR-α ligand binding domain polypeptide complex is a relative set of points that define a shape in three dimensions and that slight variations in the individual coordinates will have little effect on overall shape. For example, variations in coordinates can be generated because of mathematical manipulations of the coordinates. If such variations are within an acceptable standard of error as compared to the original coordinates, the resulting three-dimensional shape is considered to be the same. Thus, in one embodiment, any molecule or molecular complex that has a root mean square deviation (RMSD) of conserved residue backbone atoms (N, Ca, C, O) of less than about 1.5 A, about 1.0 A or about 0.5 A when superimposed on the relevant backbone atoms described by the coordinates listed in Table 5 or Table 6 are considered identical.

[0078] Various computational analyses are available to determine whether a molecule or a molecular complex or a portion thereof is sufficiently similar to all or parts of an LXR-β or LXR-α ligand binding domain complex to be considered the same. Such analyses may be carried out in current software applications, for example, CNX and QUANTA (Accelrys, Inc.).

Properties of Crystals of the Present Invention

[0079] The three-dimensional structure of an LXR-β ligand binding domain in complex with 24(S)-25-epoxycholesterol (Figure 2) and TIF2, a co-activator peptide comprising the amino acid sequence of GVSPKKKENALLRYLLDKDDTKD (SEQ ID NO: 12), were obtained from crystals of the LXR-β ligand binding domain complex. The structure coordinates calculated for the three-dimensional structure of the LXR-β ligand binding domain complex are set forth in Table 5. Data statistics for the structure are set forth in Table 1 and refinement statistics are set forth in Table 2. The asymmetric unit contains an LXR-β:epoxycholesterol:TIF2 dimer (Figure 3) and initial electron density maps showed clear density for ligand and co-activator peptide for each monomer. In certain embodiments, the protein crystals described herein diffract to a resolution of about 2.0 A.

TABLE 1 Data Statistics

^a R-sym = ∑_h ∑i|I(h) - <I(h)_!>| / ∑_h ∑il(h)_l5 where <I(h);> is the average intensity of reflection h, ∑_h is the sum over all reflections, and ∑_λ is the sum of all measurements of reflection h.

TABLE 2 Refinement Statistics

^a R- value = ∑_h||F_obs| - |F_calc|| / ∑_h|F_obs|, where F_obs and F_cai_c are the observed and calculated structure factor amplitudes, respectively, for reflection h.

^b R- free = R- factor for a 7% subset of reflections not used in the refinement.

[0080] The three-dimensional structure of an LXR-α ligand binding domain in complex with epoxy cholesterol derivative of formula (I) (Figure 6) and GRIPlB, a co-activator peptide comprising the amino acid sequence of KEKHKILHRLLQDS

(SEQ ID NO: 13), was obtained from crystals of the LXR-α ligand binding domain complex. The structure coordinates calculated for the three-dimensional structure of the LXR-α ligand binding domain complex are set forth in Table 6. Data statistics for the structure are set forth in Table 3 and refinement statistics are set forth in Table 4. The asymmetric unit contains one copy of LXR-α, epoxycholesterol derivative and GRIPlB co-activator peptide (Figure 7). Initial electron density maps showed clear density for ligand and co-activator peptide. In certain embodiments, the protein crystals diffract to a resolution of about 2.9 A.

TABLE 3 Data Statistics

^a R-sym = ∑_h ∑i|I(h) - <I(h)₁>| / ∑_h EJ(Ii)₁, where <I(h);> is the average intensity of reflection h, ∑_h is the sum over all reflections, and E₁ is the sum of all measurements of reflection h.

TABLE 4 Refinement Statistics

Method of Forming LXR-β and LXR-α Crystals

[0081] Any crystallization technique known to those skilled in the art may be employed to obtain the crystals described herein, including, but not limited to, batch crystallization, vapor diffusion (either by sitting drop or hanging drop) and micro dialysis. Seeding of the crystals in some instances may be required to obtain X-ray quality crystals. Standard micro and/or macro seeding of crystals may therefore be used. In one embodiment, the crystals are obtained using the hanging drop vapor diffusion method. In another embodiment, the crystals obtained may have a unit cell size comprising the dimensions of a=103.7 A, b=l 10.1 A, c=60.5 A, and β = 119.7°, a space group of C2, and structure coordinates as set forth in Table 5. In another embodiment, the crystals obtained may have a unit cell size comprising the dimensions of a=b=71.2 A and c=143.2 A, a space group of P4₃22, and structure coordinates as set forth in Table 6. [0082] The formation of LXR-β and LXR-α crystals can depend on a number of different parameters, including pH, temperature, protein concentration, the nature of the solvent and precipitant, as well as the presence of ligands. Prior to the instant invention, it was not known what conditions were useful for forming an LXR-β or LXR-α crystal suitable for X-ray diffraction analysis. [0083] The native, analog, derivative and mutant co-crystals, and fragments thereof, disclosed in the present invention can be obtained by a variety of techniques, including batch, liquid bridge, vapor diffusion, and free interface diffusion. Seeding of the crystals can be useful in obtaining X-ray quality crystals. Standard micro and/or macroseeding of crystals can therefore be used in the context of the present invention. In one embodiment, hanging or sitting drop methods are used for the crystallization of LXR-β and LXR-α polypeptides and fragments thereof. [0084] In an example of a hanging drop method, a drop comprising an amount of LXR-β or LXR-α polypeptide is mixed with an equal volume of reservoir buffer and grown at about 20⁰C until crystals form. General guidance and methods for forming crystals are known in the art (MacPherson, Crystallization of Biological Macromolecules, Cold Spring Harbor Press, Cold Spring Harbor, New York, USA (1999), incorporated herein by reference) and can be employed in the context of the present invention to form crystals comprising LXR-β or LXR-α, and/or fragments thereof.

Acquisition and Processing of Diffraction Data

[0085] Once a crystal comprising an LXR-β or LXR-α polypeptide of the present invention is available, X-ray diffraction data can be collected. Crystals can be prepared for diffraction using known methodology (see, e.g., Buhrke et ah, A

Practical Guide for the Preparation of Specimens for X-ray Fluorescence and X-ray Diffraction Analysis, Wiley-VCH, New York, New York, USA (1998); and Rodgers (1994) Structure 2, 1135-1140 and/or Garmen & Schneider (1997) J. Appl. Crystallogr. 30, 211-237, both of which are incorporated herein by reference). [0086] Examples of area electronic detectors for acquiring diffraction data include charge coupled device detectors, multi-wire area detectors and phosphoimager detectors (Amemiya, (1997) Methods in Enzymology, Vol. 276. Academic Press, San Diego, pp. 233-243; Westbrook & Naday, (1997) Methods in Enzymology, Vol. 276. Academic Press, San Diego, pp. 244-268; 1997. & Kahn & Fourme, Methods in Enzymology, Vol. 276. Academic Press, San Diego, pp. 268-286).

[0087] In one embodiment, a suitable system for diffraction data collection might include a Bruker AXS Proteum R system, equipped with a copper rotating anode source, Confocal Max-Flux ^R optics and a SMART 6000 charge coupled device detector. Collection of x-ray diffraction patterns are well documented by those skilled in the art (See, for example, Ducruix and Geige, 1992, IRL Press, Oxford, England). In another embodiment, a suitable system for diffraction collection might include a Rigaku FR-E copper rotating anode source with Rigaku Confocal MicroMax^® optics and a Rigaku Saturn92 charge coupled device detector. In another embodiment, a suitable system for diffraction collection might include a Rigaku FR-E copper rotating anode source with Rigaku Confocal Max-Flux HR^® optics and a Rigaku R- axis IV++ image plate detector. In a further embodiment, a suitable system for diffraction collection might include an Advanced Photon Source beamline ID 17 with a Area Detector System Corporation Q210 mosaic (2x2) charge coupled device detector.

[0088] To collect diffraction data from the crystals described herein, the crystals may be flash-frozen in the crystallization buffer employed for growing the crystals. The crystallization buffer may include one or more precipitants such as 15-30%

PEG10,000, PEG3350, and Jeffamine ED-2001 pH 7.0; one or more salts such as 0.1- 0.2M ammonium acetate, sodium formate, magnesium chloride, and sodium chloride; and one or more buffers such as 0. IM Bis-tris, pH 5.5 and 6.5 and 0. IM Hepes, pH 7.0 and 7.5. In a specific, non-limiting example, the crystallization buffer contains 15-30% PEG3350 and 0. IM Hepes, pH 7.2-7.5. Cryoprotectants (e.g., glycerol, ethylene glycol, low molecular weight PEGs, alcohols, etc.) may be added to the crystallization solution in order to achieve glass formation upon flash freezing, provided the cryoprotectant is compatible with preserving the integrity of the crystals. The flash-frozen crystals are maintained at a temperature of less than -110° C. or less than -150° C. during the collection of the crystallographic data by X-ray diffraction. [0089] Any method known to those skilled in the art may be used to process the X-ray diffraction data. One approach is to employ an isomorphous replacement technique. As used herein, heavy atom derivative or derivatization refers to the method of producing a chemically modified form of a protein or protein complex crystal wherein said protein is specifically bound to a heavy atom within the crystal. Isomorphous replacement techniques require the introduction of new, well ordered, x- ray scatterers into the crystal. These additions are often heavy metal atoms, so that they make a significant difference in the diffraction pattern. In practice a crystal is soaked in a solution containing heavy metal atoms or salts, or organometallic compounds, e.g., lead chloride, gold cyanide, thimerosal, lead acetate, uranyl acetate, mercury chloride, gold chloride, etc., which can diffuse through the crystal and bind specifically to the protein. The location(s) of the bound heavy metal atom(s) or salts can be determined by X-ray diffraction analysis of the soaked crystal. [0090] Isomorphous replacement experiments are often performed by diffusing heavy metal atoms into the channels of a pre-existing protein crystal. Alternatively, a crystal can be formed from protein that has been soaked in a solution containing the heavy atom (see, e.g., Petsko, Methods in Enzymology, Vol. 114. Academic Press, Orlando, pp. 147-156 (1985)). Alternatively, the heavy atom may also be reactive and attached covalently to exposed amino acid side chains (such as the sulfur atom of cysteine) or it may be associated through non-covalent interactions. [0091] This data collected is used to generate MIR phase information which is used to construct the three-dimensional structure of the crystallized LXR-β or LXR-α ligand binding domain described herein. Thereafter, an initial model of the three- dimensional structure may be built using the program O (Jones et al, 1991, Acta Crystallogr. AM: 110-119). The interpretation and building of the structure may be further facilitated by use of the program CNS (Brunger et al, 1998, Acta Crystallogr. D54:905-921).

[0092] Another approach is to employ anomalous scattering. Anomalous scattering occurs with all atoms, but the effect is strongest in heavy atoms. Anomalous scattering, therefore, requires the incorporation of a heavy atom. One method for preparing a protein for anomalous scattering involves replacing the methionine residues in whole or in part with selenium containing seleno-methionine. Soaks with halide salts and other non-reactive ions can also be employed (see Dauter & Wlodawer, Acta Crystallogr D , 57: 239-49 (2001)). A related anomalous acattering approach that can be employed is multiple anomalous scattering (MAD) (see Hendrickson & Ogata, Methods in Enzymology 276, 494- 523 (1997)). [0093] X-PLOR (Brunger, (1992) X-PLOR, Version 3.1. A System for X-ray Crystallography and NMR, Yale University Press, New Haven, Connecticut; Accelrys, San Diego, California) or HEAVY (Terwilliger, Los Alamos National Laboratory, Los Alamos, New Mexico) can be utilized for bulk solvent correction and B-factor scaling. After density modification and non-crystallographic averaging, the protein can be built into a electron density map using appropriate computer software, such as O (Jones et al, (199 'I) Acta Cryst. A47: 110-119). [0094] Additional data collection methods, as well as general crystallographic methods, will be known to those of ordinary skill in the art upon consideration of the present disclosure (see, e.g., McRee, Practical Protein Crystallography, (2^nd ed.) Academic Press, San Diego, California, USA (1999), incorporated herein by reference). [0095] The method of molecular replacement broadly refers to a method that involves generating a preliminary model of the three-dimensional structure of an LXR-β or LXR-α polypeptide whose structural coordinates were previously unknown. Molecular replacement is achieved by orienting and positioning a molecule whose structural coordinates are known within the unit cell of the unknown crystal so as to best account for the observed diffraction pattern of the unknown crystal. Phases can then be calculated from this model and combined with the observed amplitudes to give an approximate Fourier synthesis of the structure whose coordinates are unknown. This in turn can be subject to any of several forms of refinement to provide a final, accurate structure. References describing molecular replacement methods include Lattman, Method Enzymol . 115: 55-77 (Rossmann (ed.) 1985) and The Molecular Replacement Method (Gordon & Breach, New York 1972). Using the structure coordinates of an LXR-β or LXR-α ligand binding domain, molecular replacement methods can be used to determine the structure coordinates of a mutant or homologue of an LXR-β or LXR-α ligand binding domain, or of a different crystal form of the LXR-β or LXR-α ligand binding domain.

Determining a Three-dimensional Structure of the Present Invention [0096] Certain embodiments include methods of modeling a three-dimensional structure of an LXR-β or LXR-α ligand binding domain in complex with epoxycholesterol and TIF2, from a template comprising the X-ray structure of an

LXR-β or LXR-α ligand binding domain in complex with epoxycholesterol and TIF2, comprising: (a) selecting an X-ray structure of an LXR-β or LXR-α ligand binding domain as a starting model for the LXR-β or LXR-α ligand binding domain; (b) manipulating the starting model for the LXR-β or LXR-α ligand binding domain as a rigid body to superimpose its backbone atoms onto corresponding backbone atoms of a three-dimensional template structure comprising an LXR-β or LXR-α ligand binding domain in complex with epoxycholesterol and TIF2, to form a manipulated model; (c) making a copy of the TIF2 from the template structure to form a model of TIF2 bound to a template LXR-β or LXR-α ligand binding domain; (d) merging the model of the TIF2 into the manipulated model to form a modified model; (e) removing one or more amino acids from the modified model; and (f) optimizing side- chain conformations. In a further embodiment, the X-ray structure of an LXR-β or LXR-α ligand binding domain is a structure formulated by homology modeling. In another embodiment, the optimization of side-chain conformations comprises varying distance constraints. [0097] After acquiring X-ray diffraction data from a crystal comprising or LXR-β or LXR-α polypeptide, the three-dimensional structure of the polypeptide can be determined by analyzing the diffraction data. Such an analysis can be employed whether the polypeptide is a wildtype polypeptide or a fragment thereof, or a mutant, derivative or analog of an LXR-β or LXR-α polypeptide. [0098] X-ray diffraction data can be used to determine a structure by employing available software packages, to integrate the data such as HKL2000, with its component programs DENZO and SCALEPACK; (Otwinowski, Z & Minor, W., (1997) p. 307-326. in Carter and Sweet (ed.), Methods Enzymol, Macromolecular Crystallography part A, vol. 276. Academic Press, Inc., New York, NY; D*TREK (Rigaku), MOSFLM (Leslie, A.G. W. (1992) Joint CCP4+ESF-EAMCB Newsletter on Protein Crystallography, No. 26), XDS (Kabsch, W. (1993) J. Appl. Crystallogr. 26: 795-800.), to phase the data either by MIR, SIR, MIRAS, SIRAS, and MAD such as the CCP4 package (SERC Collaborative Computing Project No. 4, Daresbury Laboratory, UK, 1979); SHARP (GlobalPhasing, Ltd), PHASER, refinement programs such as CNX (Brunger, A. T., P. D. Adams, G. M. Clore, W. L. DeLano, P. Gros, R. W. Grosse-Kunstleve, J.-S. Jiang, J. Kuszewski, M. Nilges, N. S. Pannu, R. J. Read, L. M. Rice, T. Simonson, and G. L. Warren. 1998. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. Sect D 54:905-921.), BUSTER/TNT (GlobalPhasing Ltd), REFMAC (CCP4), and molecular graphics programs capable of displaying electron density and manipulating structure coordinates such as QUANTA (Accelrys, 2005 & preceding), COOT (Emsley, P. & Cowtan, K. (2004) Acta Crystallogr. Sect D 60:2126-2132 ), O (Jones et al. (1991) Acta Cryst. A 47, 110-119); CHAIN (J. Sack (1988) J. MoI. Graphics 6: 224-225), MIFIT (Rigaku: http://www.moleculariamges.com/MIFit.html).

[0099] The present invention therefore provides a method for determining the three-dimensional structure of a crystallized LXR-β or LXR-α polypeptide, optionally in complex with a ligand, and optionally in complex with, or in further complex with, at least one ligand to a resolution of about 3.0 A or better. In one embodiment, the method comprises: (a) crystallizing an LXR-β or LXR-α polypeptide; and (b) analyzing the crystallized complex to determine a three-dimensional structure of the LXR-β or LXR-α polypeptide in complex with a ligand or a phosphate mimetic.

Generation of LXR-β and LXR-α ligand binding domain polypeptides [00100] Those of ordinary skill in the art are familiar with recombinant methods for the cloning, expression, and purification of crystallizable LXR-β and LXR-α ligand binding domain polypeptides and compositions thereof for subsequent X-ray crystallography studies (See e.g., Sambrook et ah, Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, 2nd ed., Cold Springs Harbor, New York (1989)). Other references describing molecular biology and recombinant DNA techniques include, for example, DNA Cloning 1: Core Techniques, (D. N. Glover, et al, eds., IRL Press, 1995); DNA Cloning 2: Expression Systems, (B. D. Hames, et al. , eds., IRL Press, 1995); DNA Cloning 3: A Practical Approach, (D. N. Glover, et al., eds., IRL Press, 1995); DNA Cloning 4: Mammalian Systems, (D. N. Glover, et al, eds., IRL Press, 1995); Oligonucleotide Synthesis (M. J. Gait, ed., IRL Press, 1992); Nucleic Acid Hybridization: A Practical Approach, (S. J. Higgins and B. D. Hames, eds., IRL Press, 1991); Transcription and Translation: A Practical Approach, (S. J. Higgins & B. D. Hames, eds., IRL Press, 1996); R. I. Freshney, Culture of Animal Cells: A Manual of Basic Technique, 4th Edition (Wiley-Liss, 1986); and B. Perbal, A Practical Guide To Molecular Cloning, 2nd Edition, (John Wiley & Sons, 1988); and Current Protocols in Molecular Biology (Ausubel et ah, eds., John Wiley & Sons), which is regularly and periodically updated.

[00101] Suitable vectors for expression of LXR-β or LXR-α polypeptide constructs are, for example, bacterial or yeast plasmids, wide host range plasmids and vectors derived from combinations of plasmid and phage or virus DNA. Vectors derived from chromosomal DNA are also included. Furthermore, an origin of replication and/or a dominant selection marker can be present in vectors having nucleic acids encoding Applicants' LXRs. Such vectors are suitable for infecting, transfecting, and/or transforming a host cell. Exemplary plasmid vectors for expression 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 (Amersham-Pharmacia, Piscataway, N.J.), pET vectors (Novagen, Madison, Wis.), pmal-c vectors (Amersham-Pharmacia, Piscataway, N.J.), pFLAG vectors (Chiang and Roeder, 1993, Pept. Res. 6:62-64), and baculovirus vectors (Invitrogen, Carlsbad, Calif; Pharmingen, San Diego, Calif.).

[00102] LXR-β and LXR-α polypeptide constructs may be expressed in any cell suitable for use as a host cell for recombinant DNA expression, including any eukaryotic or prokaryotic host cell. Suitable host cells transformed with the DNA constructs can be fermented and subjected to conditions which facilitate the expression of the heterologous DNA, leading to the formation of large quantities of the desired protein.

[00103] The recombinant LXR-β and LXR-α polypeptides of the invention are purified prior to being crystallized. Selection of an appropriate purification procedure for the chimeric polypeptides present in the host cell extract or culture medium is routine to one skilled in the art, and may be based on the properties of the polypeptides, such a size, charge and function. Methods of purification include centrifugation, electrophoresis, chromatography, dialysis or a combination thereof. As known in the art, electrophoresis may be utilized to separate the proteins in the sample based on size and charge. Electrophoretic procedures are well known to the skilled artisan, and include isoelectric focusing, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), agarose gel electrophoresis, and other known methods of electrophoresis. [00104] The purification step may be accomplished by a chromatographic fractionation technique, including size fractionation, fractionation by charge and fractionation by other properties of the polypeptides being separated. As known in the art, chromatographic systems include a stationary phase and a mobile phase, and the separation is based upon the interaction of the polypeptides to be separated with the different phases. Column chromatographic procedures may be utilized. Such procedures include partition chromatography, adsorption chromatography, size- exclusion chromatography, ion-exchange chromatography and affinity chromatography. An affinity tag may also be engineered into the desired polypeptide for purification purposes. For example, the DNA constructs may encode 6-Histidine tags (His6 tags) to facilitate protein purification on nickel affinity columns. [00105] In a specific, non-limiting example, a full-length cDNA encoding LXR-β or LXR-α may be subcloned from a cDNA preparation by the polymerase chain reaction (PCR), using at least one primer design based on known, homologous, or obtained protein sequence, and inserted into an expression vector. Standard deletion mutagenesis techniques then may be used to remove those regions of the LXR-β or LXR-α cDNA not encoding the ligand binding domain. A specific, non-limiting example of an LXR-β ligand binding domain comprises amino acids 214-461 (SEQ ID NO: 1; Figure 1) of human LXR-β ligand binding domain. A specific, non-limiting example of an LXR-α ligand binding domain comprises amino acids 205-447 (SEQ ID NO:9; Figure 5) of human LXR-α ligand binding domain. [00106] In certain embodiments, an LXR-β or LXR-α ligand binding domain is expressed as a His-tagged protein by subcloning a DNA sequence encoding residues 214-461 of the human LXR-β ligand binding domain or residues 204-447 of the human LXR-α into the pETIOlDTOPO vector (Invitrogen, Carlsbad, CA) and over- expressing the His-tagged fusion protein expressed from the resulting vector in E. coli at 20° C; the soluble protein then may be purified by nickel-agarose affinity and anion exchange chromatography. Polypeptides that are Structurally Homologous or Structurally Equivalent to an LXR-β and LXR-α Polypeptide of the Present Invention

[00107] The LXR-β and LXR-α structural coordinates set forth herein can be used to aid in obtaining structural information about a crystallized molecule or molecular complex that is structurally homologous to an LXR-β or LXR-α polypeptide. The present invention allows a determination of at least a portion, if not all, of the three- dimensional structure of a molecule or a molecular complex that contains one or more structural features that are similar to structural features of an LXR-β or LXR-α polypeptide. These molecules and molecular complexes are referred to herein as "structurally homologous" to LXR-β or LXR-α. Thus, the present invention also provides LXR-β and LXR-α polypeptides that are structurally homologous to the polypeptides of SEQ ID NOs: 1 and 9.

[00108] Compounds that are structurally homologous can be formulated to mimic key portions of an LXR-β or LXR-α structure. Such compounds are structural homologs. The generation of a structurally homologous protein can be achieved by the techniques of modeling and chemical design known to those of skill in the art and described herein. Modeling and chemical design of LXR-β or LXR-α structural equivalents can be based on the structure coordinates of a crystalline LXR-β or LXR- α polypeptide of the present invention. It will be understood that all such structurally homologous constructs fall within the scope of the present invention.

[00109] Structural homologs can include, for example, regions of amino acid identity, conserved active site or binding site motifs, and similarly arranged secondary structural elements (e.g., α-helices and β-sheets). Structural similarity can be determined by aligning the residues of two amino acid sequences to optimize the number of identical amino acids along the lengths of their sequences; gaps in either or both sequences are permitted in making the alignment in order to optimize the number of identical amino acids, although the amino acids in each sequence must nonetheless remain in their proper order. In one embodiment, two amino acid sequences are compared using the BLASTP program of the BLAST 2 search algorithm, (as described by Tatusova et al, (1999) FEMS Microbiol. Lett. 174:247- 50. See also Altschul et al, (1986) Bull. Math. Bio. 48: 603-616 and Henikoff & Henikoff, (1992) Proc. Natl. Acad. ScL U.S.A. 89: 10915-10919). The default values for all BLAST 2 search parameters can be used.

[00110] In a comparison of two amino acid sequences using the BLAST search algorithm, the percentage of structurally identical amino acids is referred to as "sequence similarity." In one embodiment of the present invention, a structurally homologous molecule comprises a protein that has a sequence similarity of at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% with a native or recombinant LXR-β or LXR-α amino acid sequence (e.g., SEQ ID NOs: 1 or 9). Percent sequence similarity is calculated as: (the total number of identical matches) multiplied by (the length of the longer sequence plus the number of gaps introduced into the longer sequence in order to align the two sequences)x 100%. [00111] Structurally homologous proteins and polypeptides are generally defined as having one or more amino acid substitutions, deletions or additions from a native or recombinant LXR-β or LXR-α amino acid sequence (e.g., SEQ ID NOs: 1 or 9). These changes are preferably of a minor nature and preferably comprise conservative amino acid substitutions and other substitutions that do not significantly affect the folding, activity or structure of the protein or polypeptide; small deletions, typically of one to about 30 amino acids; and small amino- or carboxyl-terminal extensions, such as an amino- terminal methionine residue, a small linker peptide of up to about 20-25 residues, or a small extension that facilitates purification (e.g., an affinity tag). See, in general, Ford et ah, (1991) Protein Express. Purif. 2: 95-107, incorporated herein by reference.

[00112] In one embodiment, the LXR-β or LXR-α polypeptide may comprise a sequence that has sequence similarity with the amino acid sequence of SEQ ID NOs: 1 or 9 or is encoded by a nucleotide sequence that has sequence similarity with SEQ ID NOs: 10 or, respectively.

[00113] Thus, the present invention also provides for homologs of LXR-β and LXR-α polypeptides. Homologs can differ from naturally occurring polypeptides or peptides by conservative amino acid sequence substitutions or by modifications which do not affect sequence, or by both. The present invention extends to peptides which are derivatives of the LXR-β or LXR-α polypeptides, such peptides may have a sequence which has at least about 30%, or 40%, or 50%, or 60%, or 70%, or 75%, or 80%, or 85%, or 90%, 95%, 98% or 99% sequence similarity to the sequence of the LXR-β or LXR-α polypeptide (SEQ ID NOs: 1 or 9). Thus, a peptide homologous to any one of the LXR-β or LXR-α polypeptides of the invention may include 1, 2, 3, 4, 5 or greater than 5 amino acid alterations. [00114] For example, conservative amino acid changes may be made, which although they alter the sequence of the polypeptide or peptide, do not normally alter its function. Conservative amino acid substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; phenylalanine, tyrosine.

[00115] Modifications (which do not normally alter primary sequence) include in vivo, or in vitro chemical derivatization of polypeptides, e.g., acetylation, or carboxylation. Also included are modifications of glycosylation, e.g., those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g., by exposing the polypeptide to enzymes which affect glycosylation, e.g., mammalian glycosylating or deglycosylating enzymes. Also embraced are sequences which have phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine. [00116] Also included are polypeptides which have been modified using ordinary molecular biological techniques so as to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as a therapeutic agent. Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, e.g. , D-amino acids or non-naturally occurring synthetic amino acids. The peptides of the invention are not limited to products of any of the specific exemplary processes listed herein. [00117] In another embodiment, an LXR-β or LXR-α polynucleotide hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOs: 10 or 11, respectively. As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences have at least 60% sequence identity to each other typically remain hybridized to each other. Preferably, the conditions are such that sequences have at least about 70%, more preferably at least about 80%, even more preferably at least about 85% or 90% sequence identity to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. A preferred, non-limiting example of stringent hybridization conditions are hybridization in 6x sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2x SSC, 0.1% SDS at 50⁰C. Another example of stringent hybridization conditions are hybridization in 6x sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2x SSC, 0.1% SDS at 55°C. A further example of stringent hybridization conditions are hybridization in 6x sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2x SSC, 0.1% SDS at 60⁰C. Preferably, stringent hybridization conditions are hybridization in 6x sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2x SSC, 0.1% SDS at 65°C.

[00118] An LXR-β or LXR-α polynucleotide encoding an LXR-β or LXR-α polypeptide which is homologous to the polypeptide of SEQ ID NOs: 1 and 9, can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NOs: 10 or 11 such that one or more amino acid substitutions, additions or deletions are introduced into the encoded polypeptide. Mutations can be introduced into SEQ ID NOs: 10 or 11, by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. Thus, a predicted nonessential amino acid residue in an LXR-β or LXR-α polypeptide is preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of an LXR-β or LXR-α polynucleotide coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for LXR-β or LXR-α biological activity to identify mutants that retain activity. Following mutagenesis of SEQ ID NOs: 10 or 11 the encoded polypeptide can be expressed recombinantly and the activity of the polypeptide can be determined.

[00119] In a preferred embodiment, a mutant LXR-β or LXR-α polypeptide can be assayed for its ability to remove a phosphate from a tyrosine phosphorylated peptide or protein or to remove a phosphate from a non-protein tyrosine phosphatase substrate such as DiFMUP or pNPP.

Structural Equivalents

[00120] In another aspect, the present invention encompasses structural equivalents of LXR-β or LXR-α polypeptides. In the context of the present invention, any molecule or complex or portion thereof, that has a root mean square deviation of conserved residue backbone Ca atoms of less than about 3.0 A, when superimposed on the relevant backbone Ca atoms described by the reference structure coordinates of a polypeptide of the invention, is considered "structurally equivalent" to the reference molecule. That is to say, the crystal structures of those portions of the two molecules are substantially identical, within acceptable error. In one embodiment of the present invention, any molecule or complex or portion thereof, that has a root mean square deviation of the binding site of a ligand binding domain of LXR-β comprises amino acids 214-461 (SEQ ID NO: 1; Figure 1) of human LXR-β that has a root mean square deviation of conserved residue backbone Ca atoms of less than about 5.0 A, when superimposed on the relevant backbone Ca atoms described by the reference structure coordinates of a polypeptide of the invention, is considered "structurally equivalent" to the reference molecule. In another embodiment of the present invention, any molecule or complex or portion thereof, that has a root mean square deviation of the binding site of a ligand binding domain of LXR-α comprises amino acids 205-447 (SEQ ID NO:9; Figure 5) of human LXR-α.

[00121] Various computational analyses can be used to determine whether a molecule (or a binding pocket portion thereof) is "structurally equivalent," in terms of its three-dimensional structure, to all or part of an LXR-β or LXR-α polypeptide or its binding pocket(s). Such analyses can be performed by various software applications, for example the Molecular Similarity application of QUANTA (Molecular Simulations Inc., San Diego, California, USA) version 5.0, and as described in the accompanying User's Guide. The Molecular Similarity application permits comparisons between different structures, different conformations of the same structure, and different parts of the same structure.

Functional Equivalents [00122] The present invention also encompasses functional equivalents of LXR-β and LXR-α polypeptides. A functional equivalent, as used herein, means a polypeptide having an amino acid sequence that is substantially identical to an LXR-β or LXR-α amino acid sequence (e.g., SEQ ID NOs: 1 or 9 and/or a polypeptide encoded by SEQ ID NOs: 10 and 11, respectively) and retains at least one activity of the naturally-occurring form of the protein (e.g., the ability to regulate cholesterol homeostasis). Fragments of an LXR-β or LXR-α polypeptide that exhibit such activity are encompassed by the term "functional equivalent." [00123] Thus, structurally similar proteins and peptides can be formulated to mimic the key structural regions of an LXR-β or LXR-α polypeptide. The generation of a functional equivalent can be achieved by the techniques of modeling and chemical design known to those of skill in the art. It will be understood that all such structurally similar constructs fall within the scope of the present invention.

Machine-Readable Data Storage Media and Computer Systems of the Present Invention

[00124] In one aspect of the present invention, a three-dimensional structure of an LXR-β and LXR-α polypeptide have been determined and the corresponding structure coordinates form an aspect of the present invention. In order to employ the structure coordinates provided herein, it is often desirable to convert them into a graphical three-dimensional representation of the LXR-β or LXR-α polypeptide they describe. This can be done by employing commercially and freely-available software, in conjunction with a computer that is capable of generating a three-dimensional graphical representation of a molecule, or a portion thereof, from a set of structure coordinates provided on a machine-readable data storage medium. [00125] As used herein, "machine-readable media" refers to any media that can be read and accessed directly by a computer. Such media include, but are not limited to: hard disk devices, CD devices, digital video disk devices, floppy disk devices, removable hard disk devices, magneto-optic disk devices, magnetic tape devices, flash memory devices, bubble memory devices, holographic storage devices, and any other mass storage peripheral device.

[00126] Generally, then, the present invention provides a machine-readable data storage medium comprising a data storage material encoded with machine readable data comprising all or a part of a set of structure coordinates of an LXR-β or LXR-α polypeptide (Tables 5-6). A number of programs can be used to process the machine- readable data of this invention. Examples of suitable programs are described herein.

Identifying And Designing Modulators

[00127] The present invention, which comprises, in part, the structure coordinates of Tables 5-6, has broad-based utility and can be employed in many applications. Representative applications include modulator design, mutant design and screening operations. [00128] The crystal structures described herein are useful for identifying modulators of LXR-β or LXR-α. For example, a three-dimensional structure of an LXR-β or LXR-α ligand binding domain complexed with one or more molecules (e.g., epoxycholesterol and TIF2), a substrate or modulator binding site of the LXR-β or LXR-α ligand binding domain complex can be used, along with computer-aided modeling techniques, to design and/or select for a potential modulator of LXR-β or LXR-α based on the predicted ability of the modulator to bind to a binding site. A modulator can be designed de novo or from a known modulator. Modeling techniques suitable for use include graphic molecular modeling and computational chemistry techniques. [00129] An additional embodiment provides for synthesizing and testing the designed or selected modulator for its ability to modulate the activity of LXR-β or LXR-α. A modulator can be an agent that inhibits or activates LXR-β or LXR-α activity. For example, a potential modulator may be contacted with an LXR-β or LXR-α ligand binding domain, and the activity of the LXR-β or LXR-α may be measured and compared to wild-type activity. As another specific, non-limiting example, the designed or selected potential modulator may be synthesized and introduced into an in vivo or in vitro model system and then the activity of the LXR-β or LXR-α may be monitored. A potential modulator identified in this manner may be altered, and the altered modulator then contacted with an LXR-β or LXR-α ligand binding domain to determine the ability of the altered modulator to modulate LXR-β or LXR-α activity. A modulator can be essentially any compound, including, a small- molecule, a peptide, a protein, a nucleic acid (including siRNA, anti-sense RNA, catalytic DNA or RNA, DNAzymes, ribozymes) and antibodies and antibody fragments. The virtual models, atomic structure, methods and compositions are useful in the drug discovery of further, as yet unidentified inhibitors or modulators of LXR-β or LXR-α, and in the design or redesign of modulators of LXR-β or LXR-α activity.

[00130] Also contemplated are molecules which comprise binding sites and/or active sites of an LXR-β or LXR-α ligand binding domain complex. Such molecules may be used to screen test compounds, for example compounds in a combinatorial library, for binding to the ligand binding domain and/or for suitability as ligands. A binding site of an LXR-β or LXR-α ligand binding domain can also be referred to as a binding cavity or a binding pocket. Further, a ligand of an LXR-β or LXR-α ligand binding domain encompasses essentially any molecule that can bind to, or modulate the binding of, the LXR-β or LXR-α ligand binding domain, including a substrate or a modulator. [00131] Further provided are methods for designing a modulator of LXR-β or LXR-α, comprising the steps of (i) producing a computer readable model of a molecule comprising a region (e.g., a binding site) of a ligand binding domain of LXR-β or LXR-α; and (ii) using the model to design a test compound having a structure and charge distribution compatible with (e.g., able to be accommodated within) the region of the LXR-β or LXR-α ligand binding domain, wherein the test compound can comprise a functional group that may interact with and modulate LXR-β or LXR-α activity. [00132] The structure coordinates as set forth in Table 5 of an LXR-β ligand binding domain in complex with epoxycholesterol and TIF2, or the structure coordinates as set forth in Table 6 of an LXR-α ligand binding domain in complex with an epoxycholesterol derivative of formula (I) and GRIPlB may be used in conjunction with computer modeling using a docking program such as GRAM,

DOCK, HOOK or AUTODOCK (Dunbrack et al, 1997, Folding & Design 2:27-42) to identify potential modulators. This procedure can include computer fitting of potential modulators to a model of an LXR-β ligand binding domain (including models of regions of an LXR-β ligand binding domain, such as, for example, a binding site) to ascertain how well the shape and the chemical structure of the potential modulator will complement the binding site or to compare the potential modulators with the binding of substrate or known inhibitor molecules in the binding site. [00133] Alternatively, or in addition to, designing modulators, random screening of a library of test samples for compounds that interact with and/or bind to a site/region of interest (e.g., a binding site) of an LXR-β or LXR-α ligand binding domain may be used to identify useful compounds. Such libraries may include small molecule libraries, peptide libraries, and phage libraries. Interactions refer to detectable interactions between molecules, including binding interactions, such as between a protein and another protein or between a protein and a nucleic acid. Screening may be virtual, so that small molecule databases are computationally screened for chemical entities or compounds that can bind to or otherwise interact with a virtual model of a binding site of an LXR-β or LXR-α ligand binding domain. Alternatively, screening can be against actual molecular models of an LXR-β or LXR-α ligand binding domain. Further, antibodies can be generated that bind to a site of interest of an LXR-β or LXR-α ligand binding domain. After candidate compounds that can bind to an LXR-β or LXR-α ligand binding domain are identified, the compounds can then be tested to determine whether they can modulate LXR-β or LXR-α activity. [00134] Reference will now be made to specific examples illustrating the constructs and methods above. It is to be understood that the examples are provided to illustrate preferred embodiments and are not otherwise limiting to the subject matter described and claimed herein.

EXAMPLES

EXAMPLE 1

Construct Design, Cloning, Expression, Purification and Crystallization of LXR-β (214-461)

Construct Design

[00135] The parent construct LXR-β (214-461)-pET-DEST-NT2 expresses a gene encoding a protein of -35 KDa of which ~7 KDa is not part of the LXR-β ligand binding domain (LBD). In order to obtain the -28 KDan LXR-β LBD, the parent construct was cleaved with thrombin after expression and purification. In crystallization trials, the protein from the parent construct yielded crystals that diffracted to only about 8 A. The thrombin cleavage reaction to remove the ~ 7 KDa was poor in terms of yield and reproducibility. In order to increase yield and reproducibility, a minimal construct of LXR-β LBD with an N-terminal His-tag was constructed. Such a construct simplified the purification process and did not involve any proteolytic cleavage reaction that may potentially decrease the protein yield. At the same time it did not significantly jeopardize obtaining diffraction quality crystals since it is not substantially different from the native LXR-β LBD.

Cloning [00136] A construct of the human LXR-β LBD containing amino acids 214-461 with an N-terminal His-tag was cloned into the E.coli expression vector pET 10 IDTOPO (Invitrogen, Carlsbad, California). The parent construct, LXR- β(E214-E461)-pET-DEST-NT2 was used as template in PCR reactions. The primers used in the PCR reactions are described below: Lb214-46 IfI :

5'-CACCATGCATCATCATCATCATCACGAAGGAGAGGGTGTC-S ' (SEQ ID NO: 14) Lb214-461rl : 5'-TTACTCGTGGACGTCCCAGAT-S ' (SEQ ID NO: 15)

[00137] The resulting PCR product was cloned into pET 10 IDTOPO by topoisomerase-mediated directional cloning as per the manufacturer's instructions. The resulting expression vector was confirmed by DNA sequencing of the inserted region.

Expression

[00138] The resulting recombinant plasmid pET 10 lDTOPOLXR-b(214-461) was transformed into E.coli BL21 Star(DE3) cells and positive transformants were selected overnight at 37 ⁰C. on a Luria Broth (LB) plate containing 100 micrograms/mL ampicillin. A single colony was selected and grown overnight at 27 ⁰C. in 10 mL LB with 100 micrograms/mL ampicillin, and then used to inoculate 1 liter of Terrific Broth (Mediatech, Inc, Herndon, Virginia) augmented with Overnight Express Autoinduction System 1 (EMD Biosciences, Madison, Wisconsin) at 100 micrograms/mL. The cells were grown at 37 ⁰C. until they reached an optical density at 600 nm of 3 to 4, then the temperature was reduced to 20 ⁰C. The cells were then grown overnight and harvested by centrifugation at 8000g for 20 min. The cell pellet was stored frozen at -80 ⁰C. Additional supplies of cells were grown using identical reagents in a 1OL fermentor.

Purification

[00139] All steps in the purification were performed at 4 ⁰C. 230 grams of cells were resuspended in IL lysis buffer (50 mM sodium phosphate, 300 mM sodium chloride, 10 mM imidazole, pH 8.0), containing 2 grams of lysozyme, three tablets of "Complete EDTA-free" protease inhibitor (Roche Diagnostics, Mannheim,

Germany), DNase I and 2 mM magnesium chloride. The suspension was mixed for 30 minutes and then subjected to further lysis with two passes at 700 MPa through a cell homogenizer (NIRO SOAVI, Parma, Italy), followed by centrifugation for 30 min. at 2800Og to clarify the resultant solution.

[00140] The soluble protein fraction recovered was subjected to a batch Ni-NTA purification by mixing with 30 mL of Ni-NTA superflow resin (Qiagen, Valencia, California) for Ih. The suspension was then packed equally onto two columns. The columns were each washed with 300 mL of wash buffer (50 mM sodium phosphate, 300 mM sodium chloride, 20 mM imidazole, pH 8.0), and then eluted with 6x25 mL of elution buffer (50 mM sodium phosphate, 300 mM sodium chloride, 300 mM imidazole, pH 8.0). Eluates containing the LXR-β (214-461) were combined and dialyzed against 4 liters of 50 mM sodium phosphate, 300 mM sodium chloride, pH 8.0 overnight with stirring.

[00141] The dialyzed eluate from above was subjected to further purification on a 28 mL Ni-NTA superflow column. The column was washed with 126 mL wash buffer, and then eluted with a 22 column volume 20-300 mM imidazole gradient. The peak fractions containing the LXR-β were pooled, dialyzed against 2x4L of 10 mM Tris, 150 mM NaCl, 5 mM DTT, 0.1 mM EDTA, 5% Glycerol, pH 8.0, concentrated to ~lmg/mL and stored frozen in ~13 mL aliquots. When needed, aliquots were thawed and subjected to further purification on a Hiprep 26/60 Sephacryl S-100 High Resolution column (GE Healthcare, Pittsburgh, Pennsylvania) equilibrated in at least 1.5 column volumes. The peak fractions were pooled.

Crystallization and Soaking

[00142] An aliquot of pure protein was complexed with a four- fold molar excess of epoxycholesterol (Figure 2) and concentrated to 13 mg/mL. 50 mM of epoxycholesterol solubilized in 100% ethanol was then added to 1 mM and 10 mM of TIF2 co-activator peptide (GVSPKKKENALLRYLLDKDDTKD (SEQ ID NO: 12)) solubilized in water was added to 1 mM. This complex mixture was used in hanging drop crystallization trials mixing 1 microliter of protein solution with 1 microliter of precipitant solution. The LXR-β/epoxycholesterol/TIF2 crystals were obtained at 4 ⁰C from a number of conditions containing different precipitants, including 15-30% PEG10,000, PEG3350, Jeffamine ED-2001 pH 7.0, different salts including 0.1-0.2M ammonium acetate, sodium formate, magnesium chloride, sodium chloride, and different buffers including 0.1M Bis-tris, pH 5.5 and 6.5 and O. IM Hepes, pH 7.0 and 7.5 and took 2-3 days to grow. For routine use, crystals of LXR- β/epoxycholesterol/TIF2 were obtained at 4 ⁰C. from 15-30% PEG3350, 0.1M Hepes, pH 7.2-7.5. Crystals were frozen in liquid nitrogen after transferring to a cryo buffer containing well solution and 20% glycerol.

[00143] In order to enable soaking of other compounds to replace epoxycholesterol in LXR-β/epoxycholesterol/TIF2 crystals, 2-4 of these crystals were soaked at 4 ⁰C. in 20 microliter of hanging drop containing 15-30% PEG3350, 0. IM Hepes, pH 7.2- 7.5 and 2-4 mM of the compound of interest. The crystals were allowed to soak for 2 to 9 days in order for the compound of interest to successfully replace epoxycholesterol.

EXAMPLE 2 Structure Determination of LXR-β(214-461) with 24(S)-25-Epoxycholesterol and TIF2 Co-activator Peptide

[00144] Data from a single crystal were collected in a nitrogen cryostream (-170 ⁰C.) on an FR-E X-ray generator (wavelength = 1.541A) with a Saturn92 CCD detector and Micromax Confocal optics. The images were processed and scaled using HKL2000 (Otwinowski, Z. et al. (1997) Processing of X-Ray Diffraction Data Collected in Oscillation Mode. Methods in Enzymology, Macromolecular

Crystallography, Part A; CW. Carter, Jr. and R.M. Sweet, eds.; Academic Press; New York, Vol. 276, pp 307-326). The data statistics are shown in Table 1 and the refinement statistics are shown in Table 2. Phases for a C2 space group (cell dimensions a = 103.7A, b = 110. lA, c = 60.5A, α = γ = 90°, β = 119.7°) were obtained with the molecular replacement program EPMR (Kissinger, CR. et al.

(1999) Rapid automated molecular replacement by evolutionary search. Acta Cryst., 199, D55, 484-491) using coordinates 1P8D (Williams S. et al. (2003) X-ray Crystal Structure of the Liver X Receptor β Ligand Binding Domain. J. Biol. Chem., 278, 27138-27143) as an initial model. The asymmetric unit contains an LXR- β:epoxycholesterol:TIF2 dimer (Figure 3) and initial electron density maps showed clear density for ligand and co-activator peptide for each monomer. 24(S)-25- epoxycholesterol and TIF2 co-activator peptide (residues NALLRYLLDK of SEQ ID NO: 12) were built into the LXR-β model with iterative cycles of refinement, model building and solvent additions using the programs CNX and QUANTA (Accelrys, Inc.), respectively. No electron density was observed for LXR-β:A residues 255-258 and LXR-β:B residues 257-259, therefore, these residues were omitted from the final model.

TABLE 5

Structure coordinate data for LXR-β ligand binding domain in complex with epoxycholesterol and TIF2

- Ill -

EXAMPLE 3 Design of Expression Construct LXRα (Q205-E447)

[00145] The expression construct for the ligand binding domain (LBD) of LXR-α was designed using a combination of molecular modeling and sequence alignment analysis. Structure-based sequence alignments were calculated using a protein structure threading algorithm (Proceryon), using structures of the related nuclear hormone receptors (NHRs) VDR (Vitamin D receptor), THR (Thyroid hormone receptor), and RARgamma (Retinoic acid receptor-gamma) as templates. The resulting sequence alignments were examined with particular attention to the N- terminal starting and C-terminal stopping points for both the expression construct used to obtain those structures, and to the starting and stopping points for observed density in those structures. A consensus homology model was constructed for LXR- α based on the published structures of RARgamma (2LBD.pdb), THR(lBSX.pdb), and PPARg (lT7G.pdb). The resulting model suggested that the start of Helix 1 of the LXR-α LBD is likely to begin at S207 (SPEQLGM).

[00146] In addition, sequence-only alignments (calculated without consideration of secondary structure propensities) were also calculated to compare LXR-α to the LBD of the related NHR, ratFXR (Farnesoid X-Activated Receptor), and with RARgamma and VDR. Again the N-terminal starting and C-terminal stopping residues of the expression constructs used for successful expression of those NHR-LBDs were considered in the design of the LXR-α LBD. Analysis of these alignments resulted in a suggested N-terminal start site of Q205 for LXR-α, and expression through the natural C-terminus of E447. [00147] Additionally, it was observed that both LXR-α and LXR-β contain short Ser- and Pro-rich segments immediately upstream of the start of their LBDs as predicted in the above sequence alignments and homology modeling. This region is likely to be flexible and poorly structured, and thus not desirable to be included in the expression construct. In the LXR-α, this Ser/Pro-rich region is shorter than in LXR- β, and ends just before Q205, which is in turn just two amino acids before the predicted start of Helix 1 of the LBD.

[00148] Taken together, these analyses led to the design of the expression construct LXR-α (Q205-E447), encoding the LBD of LXR-α as defined by amino acid residues encompassing Q205 through E477, numbered consistently with the NCBI RefSeq entry NP_005684.

[00149] A sequence alignment of the ligand-binding domain of LXR-α (Swiss-prot accession code NR1H3_HUMAN) with its representative structural homologs from the PDB is shown in Figure 4. The residue numbers refer to the position of the amino acid in the full length sequence of LXR-α in NR1H3_HUMAN (SEQ ID NO:2), the first sequence in the multiple alignment. All the homologs belong to the same structural class of all-alpha helical proteins. The helical regions are indicated by the boxes in the multiple alignment shown in the figure.

[00150] As seen from Figure 4, the part of the LXR-α sequence that contains residues S207 through W443 defines the structural fold. This part of the sequence must therefore be minimally retained for structural integrity. At the N-terminal end, it is seen that Q205 and L206 are conserved in both LXR-α and LXR-β, whereas there is no residue conservation further to the left of Q205 between the two isoforms. It was therefore decided to start the construct at Q205 from the N-terminus. Continuing beyond W443 at the C-terminal end, D444 through E447 are conserved between the two isoforms and these were therefore retained in the construct design. The complete construct that was thus considered was Q205 through E447.

EXAMPLE 4

Cloning of Expression Construct LXR-α (Q205-E447)-pET-DEST-NT2

A. Construction of LXR-α (Q205-E447)-pET-DEST-NT2 Expression Vector

[00151] The "PCR Cloning System with Gateway Technology" kit (available from Invitrogen Corp., Carlsbad, California) was used to first create an LXR-α (Q205-E447) entry clone. A plasmid containing the gene encoding amino acids (163- 447) of LXR-α was used as template for PCR (polymerase chain reaction) amplification the LXR-α (Q205-E447) region. In order to include the attBl and attB2 recombination sequences used for Gateway cloning, a two-step PCR method was employed. In the first PCR step, specific oligonucleotide primers designed for the LXR-α (Q205-E447) region plus adjoining sequences (thrombin cleavage site on the 5' end and stop codons on the 3' end) were employed, using an annealing temperature at 55°C:

Forward primer:

5-tggttccgcgtggtagcCAGCTCAGCCCGGAACAACTG-3' (SEQ ID NO: 16) Reverse primer:

S-cgcgggtcagtcagttattaTTCGTGCACATCCCAGATCTCAGA-S' (SEQ ID NO: 17). [00152] In the second PCR step, the product of the first PCR reaction was used as template for amplification using primers containing the complete attB 1 and attB2 recombination sequences (uppercase, underlined), and priming on the sequences added in the 5' ends of the first step primers (lowercase letters). Annealing temperature was again 55⁰C:

Forward primer:

5 ' -GGGGACAAGTTTGTACAAAAAAGCAGGCTtggttccgcgtggtagc-3 ' (SEQ ID NO: 18)

Reverse primer:

5 ' -GGGACCACTTTGTACAAGAAAGCTGGGTcgcgggtcagtcagttatta-3 ' (SEQ ID

NO: 19)

[00153] The amplified LXR-α (Q205-E447)-attB PCR product was used to create an entry clone using the gateway BP recombination reaction. The desired entry clone, LXR-α (Q205-E447)-pDONR221 was confirmed by DNA sequencing of the inserted region, and was used in turn to create an E. coli expression vector using the gateway LR recombination reaction. The destination vector (pET-DEST-NT2) contains the T71ac promoter, a ribosome-binding site, and Hisβ-tag coding sequence followed by the S-tag coding sequence at 5 ' end. The destination plasmid, LXR-α (Q205-E447)-pET-DEST-NT2 was confirmed by PCR amplification of the inserted region.

B. Design and Construction of pETlOlDTOPOLXRα (Q205-E447)

Expression Vector

[00154] The parent construct LXR-α (Q205-E447)-pET-DEST-NT2 expresses a protein of -35 KDa of which ~7 KDa is not part of the LXR-α LBD. In order to obtain the -28 KDan LXR-α LBD, the parent construct was cleaved with thrombin after expression and purification. Use of this construct presented certain issues. One was that the required cleavage with thrombin significantly reduced the protein yield, thereby making it unamenable for crystallization. The second issue was that the parent construct is significantly different from the native LXR-α LBD and may affect the results drastically. In order to increase yield and reproducibility, a minimal construct of LXR-α LBD with an N-terminal His-tag was constructed. Such a construct simplified the purification process and did not involve any proteolytic cleavage reaction that may potentially decrease the protein yield. At the same time it did not significantly jeopardize obtaining diffraction quality crystals since it is not substantially different from the native LXR-α LBD.

[00155] The LXR-α (Q205-E447) region was further sub-cloned into an E. coli expression vector encoding a simple, non-cleavable, N-terminal Hisβ-tag, pET 10 IDTOPO (Invitrogen, Carlsbad, California). The parent construct, LXR-α

(Q205-E447)-pET-DEST-NT2 was used as template for PCR amplification using the following primers:

Forward primer La205-447fl: 5 ' -CACCATGCATCATCATCATCATCACCAGCTCAGCCCGGAA-S ' (SEQ ID NO:20)

Reverse primer La205-447rl : 5 '-TTATTCGTGCACATCCCAGAT-S ' (SEQ ID N0:21)

[00156] The resulting PCR product was cloned into pET 101 DTOPO by topoisomerase-mediated directional cloning as per the manufacturer's instructions. The resulting expression vector was confirmed by DNA sequencing of the inserted region.

EXAMPLE 5

Expression of LXR-α (Q205-E447) Protein

[00157] The resulting recombinant plasmid pET101DTOPOLXRα(205-447) was transformed into E. coli BL21 Star(DE3) cells and positive transformants were selected overnight at 37 ⁰C. on a Luria Broth (LB) plate containing 100 micrograms/mL ampicillin. A single colony was selected and grown overnight at

27 ⁰C. in 10 mL LB with 100 micrograms/mL ampicillin, and then used to inoculate 1 liter of Terrific Broth (Mediatech, Inc, Herndon, Virginia) augmented with Overnight Express Autoinduction System 1 (EMD Biosciences, Madison, Wisconsin) and 100 micrograms/mL. The cells were grown at 37 ⁰C. until they reached an optical density at 600 nm of 3 to 4, then the temperature was reduced to 20 ⁰C. The cells were then grown overnight and harvested by centrifugation at 8000g for 20 min. The cell pellet was stored frozen at -80 ⁰C. Additional supplies of cells were grown using identical reagents in a 1OL fermentor.

EXAMPLE 6 Purification of LXR-α (Q205-E447) Protein [00158] All steps in the purification were performed at 4 ⁰C. 235 grams of cells were resuspended in IL lysis buffer (50 mM sodium phosphate, 300 mM sodium chloride, 10 mM imidazole, pH 8.0), containing 2 grams of lysozyme, three tablets of "Complete EDTA-free" protease inhibitor (Roche Diagnostics, Mannheim, Germany), DNase I and 2 mM magnesium chloride. The suspension was mixed for 30 minutes and then subjected to further lysis with two passes at 700 MPa through a cell homogenizer (NIRO SOAVI, Parma, Italy), followed by centrifugation for 30 minutes at 2800Og to clarify the resultant solution.

[00159] The soluble protein fraction recovered was subjected to a batch Ni-NTA purification by mixing with 30 mL of Ni-NTA superflow resin (Qiagen, Valencia, California) for Ih. The suspension was then packed equally onto two columns. Each was washed with 300 mL of wash buffer (50 mM sodium phosphate, 300 mM sodium chloride, 20 mM imidazole, pH 8.0), and then eluted with 6x25 mL of elution buffer (50 mM sodium phosphate, 300 mM sodium chloride, 300 mM imidazole, pH 8.0). Eluates containing the LXRα (205-447) were combined and dialyzed against 4 liters of 50 mM sodium phosphate, 300 mM sodium chloride, pH 8.0 overnight with stirring.

[00160] The dialyzed eluate from above was subjected to further purification on a 28 mL Ni-NTA superflow column. The column was washed with 126 mL wash buffer, and then eluted with a 22 column volume 20-300 mM imidazole gradient. The peak fractions containing the LXRα were pooled, dialyzed against 2x4L of 10 mM Tris, 150 mM NaCl, 5 mM DTT, 0.1 mM EDTA, 5% glycerol, pH 8.0, concentrated to ~lmg/mL and stored frozen in ~13 mL aliquots. When needed, aliquots were thawed and subjected to further purification on a Hiprep 26/60 Sephacryl S-IOO High Resolution column (GE Healthcare, Pittsburgh, Pennsylvania) equilibrated in at least 1.5 column volumes. Peak fractions were pooled.

EXAMPLE 7

Crystallization and Soaking of LXR-α (Q205-E447) Protein

[00161] An aliquot of pure protein was complexed with four-fold molar excess of an epoxycholesterol derivative of formula (I) (Figure 6) and concentrated to 13 mg/mL. 10OmM of the epoxycholesterol derivative solubilized in 100% ethanol was then added to 1 mM, and 10 mM GRIPl co-activator peptide (KEKHKILHRLLQDS (SEQ ID NO: 13)) solubilized in water was added to 1 mM. This complex mixture was used in hanging drop crystallization trials mixing 1 microliter of protein solution with 1 microliter of precipitant solution. The LXR-α/epoxy cholesterol derivative/GRIPl crystals were obtained at 4 ⁰C. in 1.4-2.0M lithium sulfate, 2%-8% (±)-2-methyl-2,4-pentanediol and 0. IM imidazole, pH 6.5. Crystals were observed in ~5 days. Crystals were frozen in liquid nitrogen after transferring to a cryo buffer containing well solution and 25% glycerol.

[00162] In order to enable soaking of other compounds to replace epoxycholesterol in LXR-α/epoxycholestero I/GRIP 1 crystals, 2-4 of these crystals may be soaked at 4 ⁰C. in 20 microliter of hanging drop containing 15-30% PEG3350, 0. IM Hepes, pH 7.2-7.5 and 2-4 mM of the compound of interest. The crystals may then soak for 2 to 9 days in order for the compound of interest to successfully replace epoxycholesterol.

EXAMPLE 8 Structure Determination of LXR-α (Q205-E447) Protein with Epoxycholesterol

Derivative and GRIPlB Co-activator Peptide

[00163] Data from a single crystal were collected in a nitrogen cryostream (-170⁰C.) at IMCA-CAT beamline ID- 17, Advanced Photon Source, Argonne National Laboratories at a wavelength of lA on an ADSC Q210 detector. The images were processed and scaled using HKL2000 (Otwinowski, Z. et al. (1997) Processing of X-Ray Diffraction Data Collected in Oscillation Mode. Methods in Enzymology, Macromolecular Crystallography, Part A; CW. Carter, Jr. and R.M. Sweet, eds.; Academic Press; New York, Vol. 276, pp 307-326) and data statistics are shown in Table 2. Phases for a P4₃22 space group (cell dimensions a = b = 71.2A, c = 143.2A, α = β = γ = 90°) were obtained by molecular replacement with the CCP4 version of AMoRe (Kissinger, CR. et al. (1999) Rapid automated molecular replacement by evolutionary search. Acta Cryst., 199, D55, 484-491) using coordinates of LXR-α from IUHL (Williams S. et al. (2003) X-ray Crystal Structure of the Liver X Receptor β Ligand Binding Domain. J. Biol. Chem., 278, 27138-27143) as an initial model. The asymmetric unit contains one copy of LXR-α, epoxycholesterol derivative and GRIPlB co-activator peptide (Figure 7). Initial electron density maps showed clear density for ligand and co-activator peptide. The epoxycholesterol derivative and GRIPlB co-activator peptide (residues HKILHRLLQD of SEQ ID NO: 13) were built into the LXR-α model with iterative cycles of refinement and model building using the programs CNX and QUANTA (Accelrys, Inc.), respectively. No electron density was observed for LXR-α:A residues 233-247, therefore, these residues were omitted from the final model. The refinement statistics are shown in Table 4.

TABLE 6 Structure coordinate data for LXR-α ligand binding domain in complex with

Claims

CLAIMSWe claim:

1. A crystalline form comprising a liver X receptor (LXR) β ligand binding domain polypeptide complexed with an epoxycholesterol and a TIF2 polypeptide.

2. The crystalline form of claim 1, wherein the crystalline form has lattice parameters of a=103.7 A, b= 110.1 A, c=60.5 A, and β = 119.7°.

3. The crystalline form of claim 1, wherein the crystalline form has symmetry consistent with the space group C2.

4. The crystalline form of claim 1, wherein the crystalline form is described by the coordinates of Table 5.

5. The crystalline form of claim 1, wherein the crystalline form is such that the three-dimensional structure can be determined to a resolution of at least 2.0 A.

6. The crystalline form of claim 1, wherein the epoxycholesterol is selected from the group comprising 22(R)-hydroxy cholesterol, 24(S),25- epoxycholesterol and 27-hydroxycholesterol.

7. The crystalline form of claim 1, wherein the LXR- β ligand binding domain polypeptide comprises the amino acid sequence of SEQ ID NO: 1.

8. The crystalline form of claim 1, wherein the TIF2 polypeptide comprises the amino acid sequence of SEQ ID NO: 12.

9. A crystalline form comprising an LXR-α ligand binding domain polypeptide complexed with an epoxycholesterol and a GRIPlB polypeptide.

10. The crystalline form of claim 9 wherein the crystalline form has lattice parameters of a=b=71.2 A and c=143.2 A.

11. The crystalline form of claim 9, wherein the crystalline form has symmetry consistent with the space group P4₃22.

12. The crystalline form of claim 9, wherein the crystalline form is described by the coordinates of Table 6.

13. The crystalline form of claim 9, wherein the crystalline form is such that the three-dimensional structure can be determined to a resolution of at least 2.9 A.

14. The crystalline form of claim 9, wherein the epoxycholesterol is selected from the group comprising 22(R)-hydroxy cholesterol, 24(S),25- epoxycholesterol and 27-hydroxycholesterol.

15. The crystalline form of claim 9, wherein the LXR-α ligand binding domain polypeptide comprises the amino acid sequence of SEQ ID NO:9.

16. The crystalline form of claim 9, wherein GRIPlB comprises the amino acid sequence of SEQ ID NO: 13.

17. A crystalline form comprising a LXR-β ligand or LXR- α binding domain complexed with one or more molecules.

18. A method of identifying a modulator of LXR-β activity, comprising the steps of: (a) providing the structure coordinates of a LXR-β ligand binding domain in complex with an epoxycholesterol and TIF2; (b) using the three- dimensional structure of a LXR-β ligand binding domain and one or more modeling techniques to design or select a modulator; (c) providing the modulator; and (d) physically contacting the modulator with a LXR-β ligand binding domain, wherein a modulator of LXR-β activity is identified.

19. The method of claim 18, further comprising (e) altering the modulator identified in step (b); and (f) contacting the altered modulator of step (e) with an LXR-β ligand binding domain and determining the ability of the altered modulator to modulate LXR-β activity.

20. A method of identifying a modulator of LXR-α activity, comprising the steps of: (a) providing the structure coordinates of a LXR-α ligand binding domain in complex with an epoxycholesterol and GRIPlB; (b) using the three- dimensional structure of a LXR-α ligand binding domain and one or more modeling techniques to design or select a modulator; (c) providing the modulator; and (d) physically contacting the modulator with a LXR-α ligand binding domain, wherein a modulator of LXR-α activity is identified.

21. The method of claim 20, further comprising (e) altering the modulator identified in step (b); and (f) contacting the altered modulator of step (e) with a LXR- α ligand binding domain and determining the ability of the altered modulator to modulate LXR-α activity.