Plasticity of Cytochrome P450 2B4 as Investigated by Hydrogen-Deuterium Exchange Mass Spectrometry and X-ray Crystallography

Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California 92093, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 09/2010; 285(49):38602-11. DOI: 10.1074/jbc.M110.180646
Source: PubMed


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.

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    • "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. "
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    Scientific Reports 08/2013; 3:2556. DOI:10.1038/srep02556 · 5.58 Impact Factor
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    • "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. "
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    • "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 "
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