Analysis of Cytochrome P450 CYP119 Ligand-dependent Conformational Dynamics by Two-dimensional NMR and X-ray Crystallography
Analysis of Cytochrome P450 CYP119 Ligand-dependent Conformational Dynamics by Two-dimensional NMR and X-ray Crystallography
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DOI：
10.1074/jbc.m114.627935
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发表时间：
2015-04-17
影响因子：
4.8
通讯作者：
de Montellano, Paul R. Ortiz
中科院分区：
文献类型：
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作者：
Basudhar, Debashree;Madrona, Yarrow;de Montellano, Paul R. Ortiz
确定细胞色素P450活性位点的构象状态对于设计最小化药物-药物相互作用的药物、开发异构体特异性P450抑制剂以及设计新型氧化催化剂至关重要。我们使用二维H-1，N-15 HSQC化学位移微扰映射的N-15-标记的苯丙氨酸残基和X-射线晶体学检查CYP 119的配体依赖的构象动力学。活性位点苯丙氨酸残基的影响最大的唑类抑制剂和脂肪酸底物的结合，在协议的构象变化的活性位点定位。这得到了结晶学的支持，结晶学揭示了F-G环与各种唑的运动。然而，由唑类和底物引起的NMR化学位移扰动是可区分的。与几个唑类显着的化学位移扰动的情况下，揭示了配体的结合到一个开放的构象类似的配体的自由状态。与此相反，4-苯基咪唑引起明显的NMR变化，涉及Phe-87，Phe-144，和Phe-153，支持在晶体结构中发现的闭合构象。通过NMR和晶体学观察到4-苯基咪唑上具有对氟取代基的相同闭合构象，但对氯或溴取代基产生第二闭合构象。因此，在含有许多唑类配体的溶液中，开放构象是有利的，但对位取代的苯基咪唑产生两种闭合构象，这取决于对位取代基的大小。结果表明，配体选择性地稳定离散的细胞色素P450构象状态。
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. We used two-dimensional H-1,N-15 HSQC chemical shift perturbation mapping of N-15-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. The results suggest that ligands selectively stabilize discrete cytochrome P450 conformational states.