Human lanosterol 14alpha-demethylase (CYP51) in complex with ketoconazole

Target ID:

CYP51A1

Deposition Date:

Tuesday 30th June 2009

Families:

Miscellaneous

Related Diseases:

Drug metabolism and toxicology

Structure type:

protein-ligand

Download Coordinates:

Superceded by 3LD6

Authors:

Strushkevich NV, Usanov SA, Park H

Site:

Toronto

ICM Datapack:

ICM Datapack

3I3K

tabs

Structure Details

CYP51 is involved in postsqualene part of the cholesterol biosynthesis pathway and catalyses removal of the methyl group at position 14α from sterol precursors. CYP51 is responsible for production of the signaling sterols FF-MAS and T-MAS in oocytes and testis. Ketoconazole is a broad spectrum azole antifungal drug used for the treatment of systemic fungal infections and a known inhibitor of CYP51 and other human P450s. More selective antifungal agents is needed due to the undesired inhibitions of human P450s including human CYP51 and the emergence of azole-resistant fungal strains. To understand structural requirements for inhibitor interactions with CYP51 we solved the crystal structure of human CYP51 in complex with ketoconazole. The human CYP51 has a P450 fold being more similar to human steroid hydroxylases than to Mycobacterium tuberculosis (MT) CYP51. The ketoconazole tightly interacts with the hydrophobic active site with its imidazole nitrogen coordinating the heme iron. The rest of the ketoconazole interacts with aromatic and hydrophobic residues. The ketoconazole adopts an extended conformation from the heme reaching toward the protein surface, not observed in the previously known ketoconazole-bound P450 structures. This extended conformation of ketoconazole suggests where the substrate entry point of human CYP51 is. The CYP51-ketoconazole structure enables the rational design of antifungal compounds less susceptible to human CYP51 and specific modulators of human CYP51.

Materials and Methods

CYP51A1

PDB:3I3K

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:AT52-A8 (BC032322)
Entry Clone Source:MGC
SGC Clone Accession:
Tag:N-terminal: MAKKT;
C-terminal: 6His-tag
Host:E. coli JM109 (Stratagene).

Construct

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

Growth

Medium:
Antibiotics:
Procedure:CYP51A1 was co-expressed with GroEL/ES in E. coli JM109 in TB medium. Cells were grown at 37 degC 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 SourceS column (Amersham Biosciences), equilibrated with buffer 5mM KPi, pH 7.4, 20% glycerol and eluted with linear gradient of Buffer C.

Extraction

Procedure
Collected/resuspended cells (50mM potassium phosphate, 300mM NaCl, 20% glycerol, pH 7.4, 0.4mM PMSF) were disrupted in a high-pressure Microfluidizer (Microfluidics Corp.) at 18.000 psi.
Concentration:20 mg/ml
Ligand
MassSpec:Expected MW is 52550, measured mass is 52551.
Crystallization:Purified CYP51A1 was crystallized in presence of ketoconazole using hanging drop vapor diffusion method drop at 18 °C by mixing 1µl of the protein solution with 1µl of the reservoir solution containing 0.1 M Hepes pH 7.5, 2.5 M Ammonium Sulfate.
NMR Spectroscopy:
Data Collection:
Data Processing: