Human cytochrome P450, family 11, subfamily A, polypeptide 1, in complex with 22-hydroxycholesterol

Target ID:

CYP11A1

Deposition Date:

Monday 31st May 2010

Families:

Miscellaneous

Related Diseases:

MetabolismDrug metabolism and toxicology

Structure type:

protein-ligand

Download Coordinates:

3N9Z

Authors:

Strushkevich NV, MacKenzie F, Tempel W, Botchkarev A, Arrowsmith CH, Edwards AM, Bountra C, Weigelt J, Park H

Site:

Toronto

ICM Datapack:

ICM Datapack

3N9Z

tabs

Structure Details

The side-chain cleavage of cholesterol is the first and rate-limiting step in steroid hormone biosynthesis in steroidogenic tissues. Cytochrome P450scc (CYP11A1) is a mitochondrial enzyme that catalyzes the conversion of cholesterol to pregnenolone in three sequential hydroxylation steps. The first hydroxylation occurs at the 22R position to yield 22(R)-hydroxycholesterol, the second hydroxylation occurs at the 20S position to give 20(R),22-(R)-dihydroxycholesterol, and the third oxidative cleavage of the C20-C22 bond of the diol results in the formation of the pregnenolone and isocaproic aldehyde (Burstein et al., 1975; Burstein & Gut, 1976; Hume et al., 1984). The specific stereochemistry of the side chain cleavage reaction suggests high adaptability of the CYP11A1 active site and a unique catalytic mechanism.

Here we present the crystal structure of human CYP11A1 fused with its cognate redox partner, ferredoxin, in complex with the intermediate 22-hydroxycholesterol (See 3NA0 for the second hydroxylation intermediate and 3N9Y for CYP11A1-cholesterol complex.) The structures reveal the interaction mode of cholesterol and its hydroxylated derivatives (intermediates and analog) with the heme and with the surrounding residues. Based on these interactions, we propose the mechanism of hydroxylation and oxidative carbon-carbon bond scission of CYP11A1 during the side-chain cleavage reaction of cholesterol.

A related paper by Mast et al. was published in JBC in 2010.

Materials and Methods

CYP11A1

PDB:3N9Z

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:AT51-H8 (BC032329)
Entry Clone Source:MGC
SGC Clone Accession:
Tag:N-terminal: ferredoxin - MGC: BC010284; C-terminal: 6His-tag
Host:E.coli JM109 (Stratagene)

Construct

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

Growth

Medium:
Antibiotics:
Procedure:CYP11A1 was co-expressed with GroEL/ES in E.coli JM109 in TB medium. Cells were grown at 37°C to an OD₆₀₀ of 1.0 and induced by 0.5mM IPTG and 4mg/ml of arabinose and in the presence of 0.5mM δ-aminolevulinic acid and incubated 48 hours at 26°C.

Purification

Procedure
The lysate was centrifuged at 60 000g for 60min. The supernatant was loaded onto 5ml NiHiTrap column (Amersham Biosciences) equilibrated with Buffer A. The column was washed with buffer A and protein was eluted using a linear gradient of 5-100% Buffer B. The protein was further purified by ion-exchange chromatography on SourceQ column (Amersham Biosciences), equilibrated with 5 mM KPi, pH 7.4, 20% glycerol and 4 mM CHAPS and eluted with linear gradient of Buffer C.

Extraction

Procedure
Collected/resuspended cells in extraction buffer were disrupted in a high-pressure Microfluidizer (Microfluidics Corp.) at 18.000 psi. CHAPS was added to a final concentration 16 mM and lysate was incubated at 4oC for 60min.
Concentration:20 mg/ml
Ligand
MassSpec:
Crystallization:Purified CYP11A1 was crystallized in presence of cholesterol, 22R-hydroxycholesterol, 20S-hydroxycholesterol, and 20R,22R-dihydroxycholesterol using hanging drop vapor diffusion method drop at 18°C by mixing 1µl of the protein solution with 1µl of the reservoir solution containing 10% PEG 8K, 0.2 M calcium acetate, and 0.1 M HEPES pH 7.5.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Burstein S., Gut M. Intermediates in the conversion of cholesterol to pregnenolone: kinetics and mechanism. (1976) Steroids 28:115-31.
Burstein S., Middleditch B.S., Gut M. Mass spectrometric study of the enzymatic conversion of cholesterol to (22R)-22-hydroxycholesterol, (20R,22R)-20,22-dihydroxycholesterol, and pregnenolone, and of (22R)-22-hydroxycholesterol to the lgycol and pregnenolone in bovine adrenocortical preparations. Mode of oxygen incorporation. (1975) J Biol Chem. 250:9028-37.
Hume R., Kelly R.W., Taylor P.L., Boyd G.S. The catalytic cycle of cytochrome P-450scc and intermediates in the conversion of cholesterol to pregnenolone. (1984) Eur J Biochem. 140:583-91.