Human cytochrome P450, family 7, subfamily A, polypeptide 1

Target ID:

CYP7A1

Deposition Date:

Friday 30th May 2008

Families:

Miscellaneous

Related Diseases:

Metabolism

Structure type:

apo

Download Coordinates:

3DAX

Authors:

Strushkevich NV, Tempel W, Dombrovski L, Dong A, Loppnau P, Arrowsmith CH, Edwards AM, Bountra C, Wilkstrom M, Bochkarev A, Park H

Site:

Toronto

3DAX

tabs

Structure Details

Hepatic conversion of cholesterol to bile acids is a major route for the elimination of cholesterol from the mammalian body. Normal bile acid formation occurs via two major pathways starting with either 7-hydroxylation or 27-hydroxylation of cholesterol. The 7-hydroxylation is catalyzed by hepatic microsomal cholesterol 7-hydroxylase (CYP7A1). The CYP7A1 is a rate-limiting enzyme in the classical bile acid biosynthesis pathway and therefore plays an important role in maintaining cholesterol homeostasis. Expression of the cholesterol 7-hydroxylase gene is highly regulated. Multiple mechanisms are involved in the control of CYP7A1 transcription and a variety of transcription factors and nuclear receptors participate in regulatory networks. Various components of diet and drugs can modulate catalytic activity of CYP7A1 affecting cholesterol metabolism and absorption.

Here we present the structure of the substrate-free CYP7A1. CYP7A1 exhibits a typical P450 fold whereas catalytically important residues near the heme are unique and N289 that is proposed to participate in substrate positioning is found above the heme. The kink of I-helix, together with the tilted B'-helix and the long B-C loop, may be part of the access channel. CYP7A1 is in a closed conformation with aromatic residues forming the roof of the active site. The meander region of CYP7A1 is similar to CYP8A1 that does not require a redox protein partner for catalysis. Together, the CYP7A1 structure represents the first structure of P450s involved in sterol ring hydroxylation and provides the basis for investigating the topology of the active site to design new regulators of cholesterol homeostasis.

Materials and Methods

CYP7A1

PDB:3DAX

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NP_000771
Entry Clone Source:Origene
SGC Clone Accession:
Tag:N-terminal: MAKKTSS;
C-terminal: 5His-tag, no cleavage site
Host:E.coli JM109 (Stratagene).

Construct

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

Growth

Medium:
Antibiotics:
Procedure:CYP7A1 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
Column 1: 5ml NiHiTrap column (Amersham Biosciences)

Following the incubation 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 Source 30S column (Amersham Biosciences), equilibrated with buffer 5mM KPi, pH 7.4, 20% glycerol, 7mM sodium chlorate and eluted with linear gradient of Buffer C.

Extraction

Procedure
Collected/resuspended cells with Lysis buffer were disrupted in a high-pressure Microfluidizer (Microfluidics Corp.) at 18.000 psi. The sodium chlorate was added to final concentration 23mM and lysate was incubated at 4°C for 60min.
Concentration:20 mg/ml.
Ligand
MassSpec:Expected MW is 56257, measured mass is 56255.
Crystallization:Purified CYP7A1 was crystallized in presence of cholesterol 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 Potassium chloride, 0.1 M tri-Sodium citrate pH 5.5, 20 % PEG 400.
NMR Spectroscopy:
Data Collection:
Data Processing: