title: Crystal structure of a cytochrome P450 2B6 genetic variant in complex with the inhibitor 4-(4-chlorophenyl)imidazole at 2.0-A resolution.
abstract: The structure of the K262R genetic variant of human cytochrome P450 2B6 in complex with the inhibitor 4-(4-chlorophenyl)imidazole (4-CPI) has been determined using X-ray crystallography to 2.0-A resolution. Production of diffraction quality crystals was enabled through a combination of protein engineering, chaperone coexpression, modifications to the purification protocol, and the use of unique facial amphiphiles during crystallization. The 2B6-4-CPI complex is virtually identical to the rabbit 2B4 structure bound to the same inhibitor with respect to the arrangement of secondary structural elements and the placement of active site residues. The structure supports prior P450 2B6 homology models based on other mammalian cytochromes P450 and is consistent with the limited site-directed mutagenesis studies on 2B6 and extensive studies on P450 2B4 and 2B1. Although the K262R genetic variant shows unaltered binding of 4-CPI, altered binding affinity, kinetics, and/or product profiles have been previously shown with several other ligands. On the basis of new P450 2B6 crystal structure and previous 2B4 structures, substitutions at residue 262 affect a hydrogen-bonding network connecting the G and H helices, where subtle differences could be transduced to the active site. Docking experiments indicate that the closed protein conformation allows smaller ligands such as ticlopidine to bind to the 2B6 active site in the expected orientation. However, it is unknown whether 2B6 undergoes structural reorganization to accommodate bulkier molecules, as previously inferred from multiple P450 2B4 crystal structures.
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, 9500 Gilman Dr. #0703, La Jolla, CA 92093, USA. scgay@ucsd.edu
authors.forename: Sean C
authors.initials: SC
authors.lastname: Gay
authors.forename: Manish B
authors.initials: MB
authors.lastname: Shah
authors.forename: Jyothi C
authors.initials: JC
authors.lastname: Talakad
authors.forename: Keiko
authors.initials: K
authors.lastname: Maekawa
authors.forename: Arthur G
authors.initials: AG
authors.lastname: Roberts
authors.forename: P Ross
authors.initials: PR
authors.lastname: Wilderman
authors.forename: Ling
authors.initials: L
authors.lastname: Sun
authors.forename: Jane Y
authors.initials: JY
authors.lastname: Yang
authors.forename: Stephanie C
authors.initials: SC
authors.lastname: Huelga
authors.forename: Wen-Xu
authors.initials: WX
authors.lastname: Hong
authors.forename: Qinghai
authors.initials: Q
authors.lastname: Zhang
authors.forename: C David
authors.initials: CD
authors.lastname: Stout
authors.forename: James R
authors.initials: JR
authors.lastname: Halpert
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D003460:Crystallization; D018360:Crystallography, X-Ray; D065702:Cytochrome P-450 CYP2B6; D004791:Enzyme Inhibitors / Q000737:chemistry*; D006801:Humans; D007093:Imidazoles / Q000737:chemistry*; D008969:Molecular Sequence Data; D010089:Oxidoreductases, N-Demethylating / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics; D017433:Protein Structure, Secondary; D011817:Rabbits
chemical_list: C548678:4-(4-chlorophenyl)imidazole; D004791:Enzyme Inhibitors; D007093:Imidazoles; D001189:Aryl Hydrocarbon Hydroxylases; C585599:CYP2B6 protein, human; D065702:Cytochrome P-450 CYP2B6; D010089:Oxidoreductases, N-Demethylating