The long-term objective of the proposed research is to elucidate the structural basis for the substrate specificities of cytochromes P450 2B. For decades, these hepatic enzymes have served as a prototype for investigation of the mechanism by which drugs such as phenobarbital and environmental contaminants such as polychlorinated biphenyls activate gene expression. P450 2B enzymes are also very versatile catalysts with a broad range of substrates, including drugs, environmental carcinogens, and steroids. Through extensive prior studies supported by ES03619 more is known at present about the structural determinants of P450 2B specificity than about any other mammalian subfamily. The major accomplishments during the most recent grant period were: solving four X-ray crystal structures of rabbit P450 2B4, pioneering the use of isothermal titration calorimetry (ITC) to study the thermodynamics of P450-ligand interactions in solution and the ensuing conformational changes, and incorporating directed evolution approaches for generating enzymes with enhanced catalytic activity and stability. The P450 2B4 structures represent the greatest diversity of conformations of a single mammalian P450 reported to date, provide a ready explanation for how substrates can gain access to the active site, indicate how ligand binding may facilitate redox partner binding, and are utilized by many other groups to model their results. Above all, the results suggest that P450 2B, and other mammalian P450s, exhibit considerable plasticity and operate by more of an induced fit than the classical lock and key mechanism inferred from the earlier bacterial P450 structures. This concept creates new challenges in predicting cytochrome P450-mediated metabolism but also provides new modalities for engineering novel activities and/or physical properties. Despite the importance of human P450 2B6 in the metabolism of numerous clinically used drugs, insecticides, herbicides, industrial chemicals, and environmental contaminants, structure-function studies have lagged significantly behind those of P450 2B enzymes in rats, rabbits, and dogs. Fortunately, recent advances in heterologous expression and protein engineering now enable a concerted effort to elucidate the structural basis for P450 2B6 function using rigorous biochemical, biophysical, and structural approaches. The central hypothesis is that P450 2B ligand binding affinity and specificity are determined by enzyme plasticity as well as ligand access and binding.
The specific aims are: 1) To elucidate the determinants of affinity and selectivity of 1-aryl- and arylalkylimidazole binding to cytochrome P450 2B4 using X-ray crystallography, isothermal titration calorimetry, time-resolved fluorescence, site-directed mutagenesis, and virtual screening with multiple structures;2) To engineer more functionally diverse or thermostable P450 2B enzymes by site-directed mutagenesis and directed evolution;3) To investigate structure-function relationships of human P450 2B6 wild type and genetic variants. Understanding how hepatic cytochromes P450 recognize different ligands should have important implications for safety assessment of chemicals and drug discovery.
Cytochromes P450 are crucial enzymes found predominantly in the liver that are responsible for breaking down a wide variety of compounds to which humans are exposed, including drugs, environmental contaminants, and industrial chemicals. The proposed research will enable us to understand in detail how P450s bind and metabolize compounds of widely different chemical structure. The results should have important implications for predicting individual response to medications and individual susceptibility to toxic chemicals, and for choosing appropriate animal models for safety evaluation of new compounds.
|Shah, Manish B; Jang, Hyun-Hee; Wilderman, P Ross et al. (2016) Effect of detergent binding on cytochrome P450 2B4 structure as analyzed by X-ray crystallography and deuterium-exchange mass spectrometry. Biophys Chem 216:1-8|
|Liu, Jingbao; Shah, Manish B; Zhang, Qinghai et al. (2016) Coumarin Derivatives as Substrate Probes of Mammalian Cytochromes P450 2B4 and 2B6: Assessing the Importance of 7-Alkoxy Chain Length, Halogen Substitution, and Non-Active Site Mutations. Biochemistry 55:1997-2007|
|Shah, Manish B; Wilderman, P Ross; Liu, Jingbao et al. (2015) Structural and biophysical characterization of human cytochromes P450 2B6 and 2A6 bound to volatile hydrocarbons: analysis and comparison. Mol Pharmacol 87:649-59|
|Jang, Hyun-Hee; Liu, Jingbao; Lee, Ga-Young et al. (2015) Functional importance of a peripheral pocket in mammalian cytochrome P450 2B enzymes. Arch Biochem Biophys 584:61-9|
|Jang, Hyun-Hee; Davydov, Dmitri R; Lee, Ga-Young et al. (2014) The role of cytochrome P450 2B6 and 2B4 substrate access channel residues predicted based on crystal structures of the amlodipine complexes. Arch Biochem Biophys 545:100-7|
|Wilderman, P Ross; Jang, Hyun-Hee; Malenke, Jael R et al. (2014) Functional characterization of cytochromes P450 2B from the desert woodrat Neotoma lepida. Toxicol Appl Pharmacol 274:393-401|
|Shah, Manish B; Jang, Hyun-Hee; Zhang, Qinghai et al. (2013) X-ray crystal structure of the cytochrome P450 2B4 active site mutant F297A in complex with clopidogrel: insights into compensatory rearrangements of the binding pocket. Arch Biochem Biophys 530:64-72|
|Zhang, Haoming; Gay, Sean C; Shah, Manish et al. (2013) Potent mechanism-based inactivation of cytochrome P450 2B4 by 9-ethynylphenanthrene: implications for allosteric modulation of cytochrome P450 catalysis. Biochemistry 52:355-64|
|Shah, Manish B; Kufareva, Irina; Pascual, Jaime et al. (2013) A structural snapshot of CYP2B4 in complex with paroxetine provides insights into ligand binding and clusters of conformational states. J Pharmacol Exp Ther 346:113-20|
|Lee, Sung Chang; Bennett, Brad C; Hong, Wen-Xu et al. (2013) Steroid-based facial amphiphiles for stabilization and crystallization of membrane proteins. Proc Natl Acad Sci U S A 110:E1203-11|
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