Abstract
This article reviews work from the author dating back to 1978 and focuses on the structural basis of cytochrome P450 (P450) function using available contemporary techniques. Early studies used mechanism-based inactivators that bound to the protein moiety of hepatic P450s to try to localize the active site. Subsequent studies used cDNA cloning, heterologous expression, site-directed mutagenesis, and homology modeling based on multiple bacterial P450 X-ray crystal structures to predict the active sites of CYP2B enzymes with considerable accuracy. Breakthroughs in engineering and expression of mammalian P450s enabled us to determine our first X-ray crystal structure of ligand-free rabbit CYP2B4. To date, we have solved 11 CYP2B4 and three human CYP2B6 structures, which represent four significantly different conformations. The plasticity of CYP2B4 has been confirmed by deuterium exchange mass spectrometry and is substantiated by molecular dynamics simulations. In addition to major movement of secondary structure elements, more subtle reorientation of active site side chains, especially Phe206, Phe297, and Glu301, contributes to the ability of CYP2B enzymes to bind various ligands. Isothermal titration calorimetry has proven to be a useful tool for studying the thermodynamics of ligand binding to CYP2B4 and CYP2B6, and NMR has enabled study of ligand binding orientation in solution as an adjunct to X-ray crystallography. A major challenge remains to harness the power of the various approaches to facilitate prediction of CYP2B specificity and inhibition.
Footnotes
This work was supported in part by the National Institutes of Health National Institute of Environmental Health Sciences [ES003619].
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.111.039719.
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ABBREVIATIONS:
- P450
- cytochrome P450
- MBI
- mechanism-based inactivator
- WM
- Wistar-Munich
- BBT
- N-benzyl-1-aminobenzotriazole
- SRS
- substrate recognition site
- 4-CPI
- 4-(4-chlorophenyl)imidazole
- PR
- plastic region
- 1-CPI
- 1-(4-chlorophenyl)imidazole
- 1-PBI
- 1-biphenyl-4-methyl-1H-imidazole
- RMSD
- root mean square deviation
- ITC
- isothermal titration calorimetry
- DXMS
- deuterium exchange mass spectrometry
- MD
- molecular dynamics
- BP
- 4-benzylpyridine
- NBP
- 4-(4-nitrobenzyl)pyridine
- tBPA
- tert-butylphenylacetylene
- M1
- 7-hydroxyticlopidine
- M2
- 2-oxoticlopidine
- M3
- 5-(2-chlorobenzyl)thieno[3,2-c]pyridin-5-ium metabolite
- M4
- 5-(2-chlorobenzyl)thieno[3,2-c]pyridin-5-ium metabolite
- M5
- ticlopidine N-oxide
- TSOD
- ticlopidine S-oxide dimer
- M6
- dimerization product of ticlopidine S-oxide.
- Received March 23, 2011.
- Accepted April 18, 2011.
- Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics
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