Human nuclear receptor subfamily 1, group D, member 2 in complex with heme

Target ID:

NR1D2

Aliases:

BD73EAR-1rHs.37288HZF2RVR

Deposition Date:

Thursday 3rd April 2008

Families:

Miscellaneous

Related Diseases:

MetabolismCancerDrug metabolism and toxicology

Structure type:

protein-ligand

Download Coordinates:

3CQV

Authors:

Pardee K, Xu X, Dong A, Reinking J, Krause H, Schuetz A, Zhang R, Chui H, Arrowsmith CH, Weigelt J, Bountra C, Bochkarev A, Edwards AM

Site:

Toronto

ICM Datapack:

ICM Datapack

3CQV

tabs

Structure Details

Nuclear receptors are intracellular transcription factors that regulate the activity of complex gene networks (Bain et al., 2007; Robinson-Rechavi et al., 2003). Forty-eight nuclear receptors have been identified in human genome through sequence similarity. Nuclear receptors share a common structural organization, consisting of a highly variable N-terminal region, a central conserved DNA-binding domain, and a moderately conserved ligand binding domain.

The human nuclear receptors Rev-erbα and Rev-erbβ use heme as a ligand. These receptors play important roles in circadian rhythm, lipid metabolism, inflammation and diseases such as diabetes, atherosclerosis and cancer. We have found that the Rev-erbs, in the heme-bound state, are redox-sensitive and bind the gases nitric oxide and carbon monoxide. Gas binding occurs via the conserved ligand binding domain of the protein, and de-represses transcription.

This structure shows the ligand binding domain of Rev-erbβ in complex with heme at a resolution of 1.9Å. As previously described for the structure of the apo form of Rev-erb (Woo et al., 2007), the structure comprises a canonical 'α-helix sandwich' fold containing nine α-helices. In this structure, heme is in the oxidized state (Fe III) and is bound to Rev-erbβ through the residues C384 and H568. Comparison of the heme-bound structure to the apo-structure highlights the flexibility of the ligand-binding pocket. While the pocket in the apo-structure was almost completely filled with side chains of hydrophobic residues, heme binding repositions these residues to more distal regions to form the hydrophobic lining of the pocket.

Materials and Methods

NR1D2

PDB:3CQV

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NP_005117
Entry Clone Source:
SGC Clone Accession:
Tag:N-terminal His tag with integrated TEV protease site:
MHHHHHHSSGRENLYFQG
Host:E. Coli BL21-Gold(DE3)pLysS (Stratagen)

Construct

Prelude:
Sequence:SSPPSSDFAKEEVIGMVTRAHKDTFMYNQEQQENVPIDGFSQNENKNSYLCNTGGRMHLVCPMSKSPYVDPHKSGHEIWEEFSMSFTPAVKEVVEFAKRIPGFRDLSQHDQVNLLKAGTFEVLMVRFASLFDAKERTVTFLSGKKYSVDDLHSMGAGDLLNSMFEFSEKLNALQLSDEEMSLFTAVVLVSADRSGIENVNSVEALQETLIRALRTLIMKNHPNEASIFTKLLLKLPDLRSLNNMHSEELLAFKVHP
Vector:pET28-MHL

Growth

Medium:
Antibiotics:
Procedure:Rev-erbβ was expressed in E. Coli BL21-Gold(DE3) in selenomethionine medium in the presence of 50 µg/ml kanamycin, 50 µg/mL chloramphenicol and 12.5 µM hemin (Sigma). Cell were grown at 37 degC to an OD600 of 1.2 and induced by isopropyl-1-thio-D-galactopyranoside (IPTG), final concentration 1 mM, and incubated overnight at 25 °C.

Purification

Procedure
Following centrifugation of sonicated cell lysate, protein was purified from clarified supernatant using Ni-NTA affinity chromatography. Once loaded, the column was washed with 300ml of buffer containing 30 mM imidazole, 500 mM NaCl, 5% glycerol, 0.5 mM TCEP and 50 mM Hepes pH 7.5. Elution of purified Rev-erbβ from the column was done using an equivalent buffer containing 250 mM imidazole. Protein was dialysed overnight into a buffer containing 500 mM NaCl, 0.5 mM TCEP and 50 mM Hepes pH 7.5.

Extraction

Procedure
Cells were harvested by centrifugation at 8,000 rpm. The cell pellets were frozen in liquid nitrogen and stored at -80 °C. In preparation for purification, the cell paste was thawed, resuspended in lysis buffer (5 mM imidazole, 500 mM NaCl, 0.5 mM TCEP, 5% glycerol 50 mM Hepes pH 7.5) and sonicated on ice (3 second intervals) for 5 min.
Concentration:17 mg/ml.
Ligand
MassSpec:
Crystallization:Rev-erbβ 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 1.6 M Ammonium sulfate, 0.1 M Na Hepes pH 7.6, 4% Jeffamine M-600. Partial proteolysis of Rev-erbβ was performed in the crystallization drop by adding a 1:2000 ratio (v/v) of trypsin (1.5 mg/ml) to the protein.
NMR Spectroscopy:
Data Collection:
Data Processing: