Human retinoid X receptor, gamma

Target ID:

RXRG

Aliases:

NR2B3RXRC

Deposition Date:

Tuesday 4th April 2006

Families:

Miscellaneous

Related Diseases:

Chromatin and epigeneticsCancer

Structure type:

apo

Download Coordinates:

2GL8

Authors:

J.R.MIN,A.SCHUETZ,P.LOPPNAU,J.WEIGELT,M.SUNDSTROM,C.H.ARROWSMITH,A.M.EDWARDS,A.BOCHKAREV,A.N.PLOTNIKOV,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Toronto

ICM Datapack:

ICM Datapack

2GL8

tabs

Structure Details

Retinoids are vitamin A-derived metabolites playing important roles in development, cell growth, cell differentiation, apoptosis, and homeostasis.
In fact, they are effective inhibitors of tumor cell growth and are thus used in cancer therapy and prevention. Retinoids transduce their effects by two classes of nuclear receptors: the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs).
Both receptor classes contain alpha, beta and gamma subtypes.

Retinoid X receptor RXR belongs to subfamily two of the nuclear hormone receptor superfamily.
Its ligand-binding domain is capable of 9-cis retinoic acid binding, interaction with transcriptional coregulators, and dimerization.
RXRs can be active as homodimers, but the RXR heterodimers, formed with various other nuclear receptors, are of physiological relevance.

We have solved the crystal structure of the human gamma subtype retinoid X receptor ligand binding domain at 2.4 Å resolution.

Materials and Methods

RXR-gamma

PDB:2GL8

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NM_006917
Entry Clone Source:MGC
SGC Clone Accession:
Tag:N-terminal his tag with integrated thrombin protease site MGSSHHHHHHSSGLVPR*GSHN
Host:E. coli BL21 (DE3) Codon Plus RIL (Stratagen)

Construct

Prelude:
Sequence:ATSGHEDMPVERILEAELAVEPKTESYGDMNMENSTNDPVTNICHAADKQLFTLVEWAKRIPHFSDLTLEDQVILLRAGWNELLIASFSHRSVSVQDGILLATGLHVHRSSAHSAGVGSIFDRVLTELVSKMKDMQMDKSELGCLRAIVLFNPDAKGLSNPSEVETLREKVYATLEAYTKQKYPEQPGRFAKLLLRLPALRSIGLKCLEHLFFFKLIGDTPIDTFLMEMLETPLQIT
Vector:pET28a

Growth

Medium:
Antibiotics:
Procedure:RXR-gamma was expressed in E. coli BL21 (DE3) Codon Plus RIL in Terrific Broth (TB) in the presence of 50 µg/ml of kanamycin. Cell were grown at 37 oC to an OD600 of 1 and induced by isopropyl-1-thio-D-galactopyranoside (IPTG), final concentration 1 mM, and incubated overnight at 12 degC.

Purification

Procedure
The crude extract was cleared by centrifugation. 5 mM imidazole was added to the lysate. The sample was loaded onto 5 ml HiTrap Chelating column (Amersham Biosciences), charged with Ni²⁺. The column was washed with 10 CV of 20 mM Tris-HCl buffer, pH 8.0, containing 250 mM NaCl and 50 mM imidazole, and the protein was eluted with elution buffer (20 mM Tris-HCl, pH 8.0, 250 mM NaCl, 250 mM imidazole). 10 mM DTT was added to RXR-gamma containing fractions. The protein was loaded on Superdex 200 column (26x60) (Amersham Biosciences), equilibrated with 20 mM Tris-HCl buffer, pH 8.0, and 200 mM NaCl, at flow rate 4 ml/min. Thrombin (Sigma) was added to combined fractions containing RXR-gamma and incubated overnight at 4°C. The protein was further purified to homogeneity by anion-exchange chromatography on Resource Q column (1 mL) (Amersham Biosciences), equilibrated with 20 mM Tris-HCl pH 8.0 and eluted with linear gradient of NaCl up to 400 mM concentration (60CV). Purification yield is 12 mg of protein per 1L of culture.

N-terminal his tag was cleaved with thrombin protease (Sigma).

Extraction

Procedure
Cells were harvested by centrifugation at 7,000 rpm. The cell pellets were frozen in liquid nitrogen and stored at -80 °C. For the purification the cell paste was thawed and resuspended in lysis buffer (1× PBS, 0.5 M NaCl, 5% glycerol, 0.1 % CHAPS) with protease inhibitor (0.1 mM phenylmethyl sulfonyl fluoride, PMSF). The cells were lysed by passing through Microfluidizer (Microfluidics Corp.) at 18,000 psi.
Concentration:9.2 mg/ml
Ligand
MassSpec:expected mass = 26980.0 Da, measured mass = 26963.8 Da
Crystallization:Purified RXRG was crystallized using the hanging drop vapor diffusion method at 18 °C by mixing 2 µl of the protein solution with 2 µl of the reservoir solution containing 15 % PEG 3350 and 0.2 M CaCl2.
NMR Spectroscopy:
Data Collection:
Data Processing: