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首页> 外文期刊>Biochemistry >Membrane Interactions, Ligand-Dependent Dynamics, and Stability of Cytochrome P4503A4 in Lipid Nanodiscs
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Membrane Interactions, Ligand-Dependent Dynamics, and Stability of Cytochrome P4503A4 in Lipid Nanodiscs

机译：膜相互作用，配体依赖动力学和脂质纳米盘中细胞色素P4503A4的稳定性。

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Membrane-bound cytochrome P4503A4 (CYP3A4) is the major source of enzymatic drug metabolism. Although several structural models of CYP3A4 in various ligand complexes are available, none includes a lipid bilayer. Details of the effects of the membrane on protein dynamics and solvation, and access channels for ligands, remain uncertain. H/D exchange mass spectrometry (H/DXMS) with ligand free CYP3A4 containing a deletion of residues 3-12, compared to that of the full length wild type, in lipid nanodiscs afforded 91% sequence coverage. Deuterium exchange was fast in the F- and G-helices, HI loop, and C-terminal loop. In contrast, there is very low exchange in the F'- and G'-helices. The results are consistent with the overall membrane orientation of CYP3A4 suggested by published MD simulations and spectroscopic results, and the solvent accessibility of the F/G loop suggests that it is not deeply membrane embedded. Addition of ketoconazole results in only modest, but global, changes in solvent accessibility. Interestingly, with ketoconazole bound some peptides become less solvent accessible or dynamic, including the F- and G-helices, but several peptides demonstrate modestly increased accessibility. Differential scanning calorimetry (DSC) of CYP3A4-nanodiscs suggests membrane-induced stabilization compared to that of aggregated CYP3A4 in buffer, and this stabilization is enhanced upon addition of the ligand ketoconazole. This ligand-induced stabilization is accompanied by a very large increase in Delta H for CYP3A4 denaturation in nanodiscs, possibly due to increased CYP3A4-membrane interactions. Together, the results suggest a distinct orientation of CYP3A4 on the lipid membrane, and they highlight likely solvent access channels, which are consistent with several MD simulations.
机译：膜结合细胞色素P4503A4（CYP3A4）是酶促药物代谢的主要来源。尽管在各种配体复合物中有几种CYP3A4的结构模型可用，但没有一个模型包括脂质双层。膜对蛋白质动力学和溶剂化作用以及配体通道的影响的细节仍然不确定。在脂质纳米光盘中，与全长野生型相比，含有不含残基3-12的残基缺失的不含配体的CYP3A4的H / D交换质谱法（H / DXMS）提供了91％的序列覆盖率。氘交换在F和G螺旋，HI环和C端环中快速进行。相反，F'和G'螺旋的交换非常低。结果与已发表的MD模拟和光谱结果表明的CYP3A4的整体膜取向一致，F / G环的溶剂可及性表明该分子未深层嵌入膜中。酮康唑的加入只会导致溶剂可及性发生适度但整体的变化。有趣的是，与酮康唑结合后，某些肽的溶剂可及性或动态性降低，包括F螺旋和G螺旋，但一些肽显示出适度的可及性。 CYP3A4纳米糊的差示扫描量热法（DSC）表明，与缓冲液中的CYP3A4聚集相比，膜诱导的稳定，并且在加入配体酮康唑后这种稳定作用得以增强。这种配体诱导的稳定作用伴随着纳米光盘中CYP3A4变性的Delta H极大增加，这可能是由于CYP3A4膜相互作用增加所致。总之，这些结果表明CYP3A4在脂质膜上的定向不同，并且它们突出了可能的溶剂通道，这与几种MD模拟是一致的。

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