Human cytochrome P450, family 2, R1 in complex with vitamin D3

Target ID:

CYP2R1

Aliases:

MGC4663

Deposition Date:

Friday 19th January 2007

Families:

Miscellaneous

Related Diseases:

Metabolism

Structure type:

apo

Download Coordinates:

Superceded by 3C6G

Authors:

N.V.STRUSHKEVICH,J.MIN,P.LOPPNAU,W.TEMPEL,C.H.ARROWSMITH,A.M.EDWARDS,M.SUNDSTROM,J.WEIGELT,A.BOCHKAREV,A.N.PLOTNIKOV,S.A.USANOV,G.JONES,H.PARK,STRUCTURAL GENOMICS CONSORTIUM(SGC)

Site:

Toronto

ICM Datapack:

ICM Datapack

2OJD

tabs

Structure Details

Cytochrome P450 (CYP) superfamily of enzymes are ubiquitous heme-containing monooxygenases named for the absorption band at 450 ηm of their reduced carbon monoxide (CO) form. The CYP enzymes are responsible for the metabolism of xenobiotics, including drugs, carcinogens, and environmental chemicals, as well as endogenous compounds such as steroids and fatty acids, thereby participating in the maintenance of the body homeostasis. CYP2R1 belongs to the CYP2 family of proteins, which are frequently involved in the metabolism of foreign compounds. CYPR1 gene deficiency cause inherited rickets due to a defect in vitamin D 25-hydroxylation. CYP2R1 catalyzes the initial step of activation of vitamin D3 (cholecalciferol) to its metabolite 25-hydroxycholecalciferol (the precursor of biologically active 1,25-dihydroxycholecalciferol). CYP2R1 has been shown to be active in the 25-hydroxylation of vitamin D2 (Cheng et al., PNAS 2003), but no xenobiotic substrate for this enzyme has been identified so far. CYP2R1 is expressed in liver and highly conserved across species from Fugu to Human. In contrast, most other members of the CYP2 family show a high degree of divergence, with little conservation.

Here we present the crystal structure of human CYP2R1 in complex with vitamin D3.

Materials and Methods

CYP2R1

PDB:2OJD

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:90199946
Entry Clone Source:LIFESEQ
SGC Clone Accession:
Tag:N-terminal: MAKKT; C-terminal: 4His-tag, no cleavage site
Host:

Construct

Prelude:
Sequence:KQRRPMGFPPGPPGLPFIGNIYSLAASSELPHVYMRKQSQVYGEIFSLDLGGISTVVLNGYDVVKECLVHQSEIFADRPCLPLFMKMTKMGGLLNSRYGRGWVDHRRLAVNSFRYFGYGQKSFESKILEETKFFNDAIETYKGRPFDFKQLITNAVSNITNLIIFGERFTYEDTDFQHMIELFSENVELAASASVFLYNAFPWIGILPFGKHQQLFRNAAVVYDFLSRLIEKASVNRKPQLPQHFVDAYLDEMDQGKNDPSSTFSKENLIFSVGELIIAGTETTTNVLRWAILFMALYPNIQGQVQKEIDLIMGPNGKPSWDDKCKMPYTEAVLHEVLRFCNIVPLGIFHATSEDAVVRGYSIPKGTTVITNLYSVHFDEKYWRDPEVFHPERFLDSSGYFAKKEALVPFSLGRRHCLGEHLARMEMFLFFTALLQRFHLHFPHELVPDLKPRLGMTLQPQPYLICAERR
Vector:pCW-LIC-29

Growth

Medium:
Antibiotics:
Procedure:CYP2R1 was co-expressed with GroEL/ES in E.coli JM109 in TB medium with 100 µg/ml of ampicillin and 35 µg/ml of kanamycin. Cells were grown at 37oC to an OD600 of 1.0. Then 0.5 mM IPTG, 0.5mM ?-aminolevulinic acid and 4mg/ml of arabinose were added and incubation continued 48 hours at 29°C.

Purification

Procedure
Following the incubation the lysate was centrifuged at 60,000g for 60 min. The supernatant was loaded onto 5 ml HiTrap column (Amersham Biosciences), charged with Ni2+ and equilibrated with 50 mM KPi, pH 7.4, containing 300 mM NaCl, 0.2% CHAPS and 20% glycerol (buffer A). The column was washed with 10 CV of buffer A and protein was eluted using a linear gradient of 5-100% Buffer B (buffer A+250mM imidazole). The protein was further purified by ion-exchange chromatography on Source 30S column (Amersham Biosciences), equilibrated with buffer 5 mM KPi, pH 7.4, 20% glycerol, 0.2% CHAPS and eluted with linear gradient of 0-50% Buffer C (50 mM KPi, pH 7.4, 20% glycerol and 1MNaCl).

Extraction

Procedure
Collected/resuspended cells (50 mM potassium phosphate, 300 mM NaCl, 0.2% CHAPS, 20% (v/v) glycerol, pH 7.4, 0.4mM PMSF) were disrupted in a high-pressure Microfluidizer (Microfluidics Corp.) at 18,000 psi. CHAPS was added to final concentration 1% and lysate was incubated at 4°C for 60min.
Concentration:20 mg/ml
Ligand
MassSpec:Expected MW is 54939, measured mass is 54940.5
Crystallization:Purified CYP2R1 was crystallized in presence of vitamin D3 using hanging drop vapor diffusion method drop at 18 °C by mixing 2 µl of the protein solution with 2 µl of the reservoir solution containing 1.1 M Ammonium sulfate, 0.1M ADA, pH 6.5.
NMR Spectroscopy:
Data Collection:
Data Processing: