Crystal structures of the xenobiotic metabolizing cytochrome P450 2B4 have demonstrated markedly different conformations in the presence of imidazole inhibitors or in the absence of ligand. However, knowledge of the plasticity of the enzyme in solution has remained scant. Thus, hydrogen-deuterium exchange mass spectrometry (DXMS) was utilized to probe the conformations of ligand-free P450 2B4 and the complex with 4-(4-chlorophenyl)imidazole (4-CPI) or 1-biphenyl-4-methyl-1H-imidazole (1-PBI). The results of DXMS indicate that the binding of 4-CPI slowed the hydrogen-deuterium exchange rate over the B'- and C-helices and portions of the F-G-helix cassette compared with P450 2B4 in the absence of ligands. In contrast, there was little difference between the ligand-free and 1-PBI-bound exchange sets. In addition, DXMS suggests that the ligand-free P450 2B4 is predominantly open in solution. Interestingly, a new high resolution structure of ligand-free P450 2B4 was obtained in a closed conformation very similar to the 4-CPI complex. Molecular dynamics simulations performed with the closed ligand-free structure as the starting point were used to probe the energetically accessible conformations of P450 2B4. The simulations were found to equilibrate to a conformation resembling the 1-PBI-bound P450 2B4 crystal structure. The results indicate that conformational changes observed in available crystal structures of the promiscuous xenobiotic metabolizing cytochrome P450 2B4 are consistent with its solution structural behavior.
"Though many crystal structures of the soluble domain of cyt-P450 have been reported in the literature, high-resolution structure of the full-length protein is unknown. The N-terminal 60-residues segment containing the hydrophobic domain is usually cleaved off to obtain a single crystal for structural studies by X-ray crystallography44454647. In addition, the full-length protein is quite unstable and highly sensitive to heat. "
[Show abstract][Hide abstract] ABSTRACT: Though the importance of high-resolution structure and dynamics of membrane proteins has been well recognized, optimizing sample conditions to retain the native-like folding and function of membrane proteins for Nuclear Magnetic Resonance (NMR) or X-ray measurements has been a major challenge. While bicelles have been shown to stabilize the function of membrane proteins and are increasingly utilized as model membranes, the loss of their magnetic-alignment at low temperatures makes them unsuitable to study heat-sensitive membrane proteins like cytochrome-P450 and protein-protein complexes. In this study, we report temperature resistant bicelles that can magnetically-align for a broad range of temperatures and demonstrate their advantages in the structural studies of full-length microsomal cytochrome-P450 and cytochrome-b5 by solid-state NMR spectroscopy. Our results reveal that the N-terminal region of rabbit cytochromeP4502B4, that is usually cleaved off to obtain crystal structures, is helical and has a transmembrane orientation with ~17° tilt from the lipid bilayer normal.
"These include two distinct ligand-free states of protein, open and closed, as well as other conformations observed in complex with various inhibitors and drugs (Gay et al., 2010a). The crystal structures of the open ligand free form and two inhibitor-bound complexes were in agreement with the conformational changes observed in solution in recent hydrogen-deuterium exchange mass spectrometry experiments (Wilderman et al., 2010). Furthermore, the flexible regions of 2B4 affected by ligand binding were consistent between the solution studies and X-ray crystal structures. "
[Show abstract][Hide abstract] ABSTRACT: The biochemical, biophysical, and structural analysis of the cytochrome P450 2B subfamily of enzymes has provided a wealth of information regarding conformational plasticity and substrate recognition. The recent X-ray crystal structure of the drug-metabolizing P450 2B6 in complex with 4-(4-chlorophenyl)imidazole (4-CPI) yielded the first atomic view of this human enzyme. However, knowledge of the structural basis of P450 2B6 specificity and inhibition has remained limited. In this study, structures of P450 2B6 were determined in complex with the potent inhibitors 4-benzylpyridine (4-BP) and 4-(4-nitrobenzyl)pyridine (4-NBP). Comparison of the present structures with the previous P450 2B6-4-CPI complex showed that reorientation of side chains of the active site residue Phe206 on the F-helix and Phe297 on the I-helix was necessary to accommodate the inhibitors. However, P450 2B6 does not require any major side chain rearrangement to bind 4-NBP compared with 4-BP, and the enzyme provides no hydrogen-bonding partners for the polar nitro group of 4-NBP within the hydrophobic active site. In addition, on the basis of these new structures, substitution of residue 172 with histidine as observed in the single nucleotide polymorphism Q172H and in P450 2B4 may contribute to a hydrogen bonding network connecting the E- and I-helices, thereby stabilizing active site residues on the I-helix. These results provide insight into the role of active site side chains upon inhibitor binding and indicate that the recognition of the benzylpyridines in the closed conformation structure of P450 2B6 is based solely on hydrophobicity, size, and shape.
"Enzyme Ligand Protein Data Bank Data Code Reference CYP2B4 4-CPI 1SUO Scott et al., 2004 CYP2B4 1-CPI 2Q6N Zhao et al., 2007 CYP2B4 1-PBI 3G5N Gay et al., 2009 CYP2B4 Bifonazole 2BDM Zhao et al., 2006 CYP2B4 Clopidogrel 3ME6 Gay et al., 2010b CYP2B4 Ticlopidine 3KW4 Gay et al., 2010b CYP2B4 tBPA, closed 3R1A Gay et al., 2011 CYP2B4 tBPA, open 3R1B Gay et al., 2011 CYP2B4 3MVR Wilderman et al., 2010 CYP2B4 1PO5 Scott et al., 2003 CYP2B6 4-CPI 3IBD Gay et al., 2010c CYP2B6 BP 3QOA M. B. Shah, J. Pascual, A. G. Roberts, Q. Zhang, C. D. Stout, and J. R. Halpert, manuscript in preparation CYP2B6 NBP 3QUB M. B. Shah, J. Pascual, A. G. Roberts, Q. Zhang, C. D. Stout, and J. R. Halpert, manuscript in preparation "
[Show abstract][Hide abstract] 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.
Drug metabolism and disposition: the biological fate of chemicals 07/2011; 39(7):1113-21. DOI:10.1124/dmd.111.039719 · 3.25 Impact Factor
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