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

Basudhar, Debashree; Madrona, Yarrow; Kandel, Sylvie; Lampe, Jed N.; Nishida, Clinton R.; de Montellano, Paul R.  Ortiz

doi:10.1074/jbc.M114.627935

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

Journal Article · Mon Feb 09 23:00:00 EST 2015 · Journal of Biological Chemistry

DOI:https://doi.org/10.1074/jbc.M114.627935· OSTI ID:1349037

Basudhar, Debashree ^[1]; Madrona, Yarrow ^[2]; Kandel, Sylvie ^[3]; Lampe, Jed N. ^[4]; Nishida, Clinton R. ^[2]; de Montellano, Paul R. Ortiz ^[2]

Univ. of California, San Francisco, CA (United States); Office of Scientific and Technical Information (OSTI)
Univ. of California, San Francisco, CA (United States)
XenoTech, LLC, Lenexa, KA (United States)
Univ. of Kansas Medical Center, Kansas City, KA (United States)

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.

View Accepted Manuscript (DOE)

Research Organization:: Univ. of California, San Francisco, CA (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)

Grant/Contract Number:: AC02-05CH11231; AC02-76SF00515

OSTI ID:: 1349037

Journal Information:: Journal of Biological Chemistry, Journal Name: Journal of Biological Chemistry Journal Issue: 16 Vol. 290; ISSN 0021-9258

Publisher:: American Society for Biochemistry and Molecular BiologyCopyright Statement

Country of Publication:: United States

Language:: English

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