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Title: Analysis of Cytochrome P450 CYP119 Ligand-dependent Conformational Dynamics by Two-dimensional NMR and X-ray Crystallography

Abstract

Defining the conformational states of cytochrome P450 active sites is critical for the design of agents that minimize drug-drug interactions, the development of isoform-specific P450 inhibitors, and the engineering of novel oxidative catalysts. In this paper, we used two-dimensional 1H,15N HSQC chemical shift perturbation mapping of 15N-labeled Phe residues and x-ray crystallography to examine the ligand-dependent conformational dynamics of CYP119. Active site Phe residues were most affected by the binding of azole inhibitors and fatty acid substrates, in agreement with active site localization of the conformational changes. This was supported by crystallography, which revealed movement of the F-G loop with various azoles. Nevertheless, the NMR chemical shift perturbations caused by azoles and substrates were distinguishable. The absence of significant chemical shift perturbations with several azoles revealed binding of ligands to an open conformation similar to that of the ligand-free state. In contrast, 4-phenylimidazole caused pronounced NMR changes involving Phe-87, Phe-144, and Phe-153 that support the closed conformation found in the crystal structure. The same closed conformation is observed by NMR and crystallography with a para-fluoro substituent on the 4-phenylimidazole, but a para-chloro or bromo substituent engendered a second closed conformation. An open conformation is thus favored in solution with manymore » azole ligands, but para-substituted phenylimidazoles give rise to two closed conformations that depend on the size of the para-substituent. Finally, the results suggest that ligands selectively stabilize discrete cytochrome P450 conformational states.« less

Authors:
 [1];  [1];  [2];  [3];  [1];  [1]
  1. Univ. of California, San Francisco, CA (United States)
  2. XenoTech, LLC, Lenexa, KA (United States)
  3. Univ. of Kansas Medical Center, Kansas City, KA (United States)
Publication Date:
Research Org.:
Univ. of California, San Francisco, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
OSTI Identifier:
1349037
Grant/Contract Number:  
AC02-05CH11231; AC02-76SF00515
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Journal of Biological Chemistry
Additional Journal Information:
Journal Volume: 290; Journal Issue: 16; Journal ID: ISSN 0021-9258
Publisher:
American Society for Biochemistry and Molecular Biology
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Conformational Change; Cytochrome P450; Molecular Dynamics; Nuclear Magnetic Resonance (NMR); X-ray Crystallography

Citation Formats

Basudhar, Debashree, Madrona, Yarrow, Kandel, Sylvie, Lampe, Jed N., Nishida, Clinton R., and de Montellano, Paul R. Ortiz. Analysis of Cytochrome P450 CYP119 Ligand-dependent Conformational Dynamics by Two-dimensional NMR and X-ray Crystallography. United States: N. p., 2015. Web. doi:10.1074/jbc.M114.627935.
Basudhar, Debashree, Madrona, Yarrow, Kandel, Sylvie, Lampe, Jed N., Nishida, Clinton R., & de Montellano, Paul R. Ortiz. Analysis of Cytochrome P450 CYP119 Ligand-dependent Conformational Dynamics by Two-dimensional NMR and X-ray Crystallography. United States. doi:10.1074/jbc.M114.627935.
Basudhar, Debashree, Madrona, Yarrow, Kandel, Sylvie, Lampe, Jed N., Nishida, Clinton R., and de Montellano, Paul R. Ortiz. Tue . "Analysis of Cytochrome P450 CYP119 Ligand-dependent Conformational Dynamics by Two-dimensional NMR and X-ray Crystallography". United States. doi:10.1074/jbc.M114.627935. https://www.osti.gov/servlets/purl/1349037.
@article{osti_1349037,
title = {Analysis of Cytochrome P450 CYP119 Ligand-dependent Conformational Dynamics by Two-dimensional NMR and X-ray Crystallography},
author = {Basudhar, Debashree and Madrona, Yarrow and Kandel, Sylvie and Lampe, Jed N. and Nishida, Clinton R. and de Montellano, Paul R. Ortiz},
abstractNote = {Defining the conformational states of cytochrome P450 active sites is critical for the design of agents that minimize drug-drug interactions, the development of isoform-specific P450 inhibitors, and the engineering of novel oxidative catalysts. In this paper, we used two-dimensional 1H,15N HSQC chemical shift perturbation mapping of 15N-labeled Phe residues and x-ray crystallography to examine the ligand-dependent conformational dynamics of CYP119. Active site Phe residues were most affected by the binding of azole inhibitors and fatty acid substrates, in agreement with active site localization of the conformational changes. This was supported by crystallography, which revealed movement of the F-G loop with various azoles. Nevertheless, the NMR chemical shift perturbations caused by azoles and substrates were distinguishable. The absence of significant chemical shift perturbations with several azoles revealed binding of ligands to an open conformation similar to that of the ligand-free state. In contrast, 4-phenylimidazole caused pronounced NMR changes involving Phe-87, Phe-144, and Phe-153 that support the closed conformation found in the crystal structure. The same closed conformation is observed by NMR and crystallography with a para-fluoro substituent on the 4-phenylimidazole, but a para-chloro or bromo substituent engendered a second closed conformation. An open conformation is thus favored in solution with many azole ligands, but para-substituted phenylimidazoles give rise to two closed conformations that depend on the size of the para-substituent. Finally, the results suggest that ligands selectively stabilize discrete cytochrome P450 conformational states.},
doi = {10.1074/jbc.M114.627935},
journal = {Journal of Biological Chemistry},
number = 16,
volume = 290,
place = {United States},
year = {Tue Feb 10 00:00:00 EST 2015},
month = {Tue Feb 10 00:00:00 EST 2015}
}

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