Magic-angle spinning solid-state NMR Spectroscopy of nanodisc-embedded human CYP3A4
Magic-angle spinning solid-state NMR Spectroscopy of nanodisc-embedded human CYP3A4
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DOI：
10.1021/bi701411g
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发表时间：
2007-12-04
期刊：
影响因子：
2.9
通讯作者：
Rienstra, Chad M.
中科院分区：
文献类型：
--
作者：
Kijac, Aleksandra Z.;Li, Ying;Rienstra, Chad M.
细胞色素P450（CYP 3A 4）通过氧化大量结构不同的底物，促进约50%的商业药物的代谢。与其他内质网定位的P450一样，CYP 3A 4含有膜锚定N-末端螺旋和大量疏水结构域，这对CYP 3A 4与膜之间的相互作用很重要。虽然膜影响CYP 3A 4配体结合的特异性，但迄今为止尚未揭示相互作用的结构细节，因为X射线晶体学研究仅适用于CYP 3A 4的可溶性结构域。在这里，我们报告样品制备和初始魔角旋转（MAS）固态NMR（SSNMR）的CYP 3A 4（三角洲3-12）嵌入在纳米级膜双层，或Nanodisc。生长方案从IL生长培养基中产生类似于2.5mg酶活性的、均匀富集C-13、N-15的CYP 3A 4。Nanodisks中聚乙二醇3350沉淀的CYP 3A 4产生高分辨率和灵敏度的光谱，与折叠的均质蛋白一致。通过溴隐亭结合监测，Nanodisces中的CYP 3A 4在整个沉淀方案中保持酶活性。从C-13-C-13 2D化学位移相关光谱测量的C-13线宽类似于0.5ppm。从C-13化学位移确定的几种氨基酸类型内的二级结构分布与无配体的X射线结构一致。这些结果表明，MAS SSNMR可以在折叠的活性状态下对纳米盘嵌入的膜蛋白进行。SSNMR和Nanodisc方法的结合为获得CYP 3A 4和其他完整膜蛋白的结构信息提供了新的可能性，并完全保留了功能。
Cytochrome P450 (CYP) 3A4 contributes to the metabolism of approximately 50% of commercial drugs by oxidizing a large number of structurally diverse substrates. Like other endoplasmic reticulum-localized P450s, CYP3A4 contains a membrane-anchoring N-terminal helix and a significant number of hydrophobic domains, important for the interaction between CYP3A4 and the membrane. Although the membrane affects specificity of CYP3A4 ligand binding, the structural details of the interaction have not been revealed so far because X-ray crystallography studies are available only for the soluble domain of CYP3A4. Here we report sample preparation and initial magic-angle spinning (MAS) solidstate NMR (SSNMR) of CYP3A4 (Delta 3-12) embedded in a nanoscale membrane bilayer, or Nanodisc. The growth protocol yields similar to 2.5 mg of the enzymatically active, uniformly C-13,N-15-enriched CYP3A4 from I L of growth medium. Polyethylene glycol 3350-precipitated CYP3A4 in Nanodiscs yields spectra of high resolution and sensitivity, consistent with a folded, homogeneous protein. CYP3A4 in Nanodiscs remains enzymatically active throughout the precipitation protocol as monitored by bromocriptine binding. The C-13 line widths measured from C-13-C-13 2D chemical shift correlation spectra are similar to 0.5 ppm. The secondary structure distribution within several amino acid types determined from C-13 chemical shifts is consistent with the ligand-free X-ray structures. These results demonstrate that MAS SSNMR can be performed on Nanodisc-embedded membrane proteins in a folded, active state. The combination of SSNMR and Nanodisc methodologies opens up new possibilities for obtaining structural information on CYP3A4 and other integral membrane proteins with full retention of functionality.