Zhang Meng, Huang Rui, Im Sang-Choul, Waskell Lucy, Ramamoorthy Ayyalusamy
From the Department of Chemistry and Biophysics, University of Michigan, Ann Arbor, Michigan 48109-1055 and.
the Department of Anesthesiology, University of Michigan and Veterans Affairs Medical Center, Ann Arbor, Michigan 48105.
J Biol Chem. 2015 May 15;290(20):12705-18. doi: 10.1074/jbc.M114.597096. Epub 2015 Mar 20.
Mammalian cytochrome P450 (P450) is a membrane-bound monooxygenase whose catalytic activities require two electrons to be sequentially delivered from its redox partners: cytochrome b5 (cytb5) and cytochrome P450 reductase, both of which are membrane proteins. Although P450 functional activities are known to be affected by lipids, experimental evidence to reveal the effect of membrane on P450-cytb5 interactions is still lacking. Here, we present evidence for the influence of phospholipid bilayers on complex formation between rabbit P450 2B4 (CYP2B4) and rabbit cytb5 at the atomic level, utilizing NMR techniques. General line broadening and modest chemical shift perturbations of cytb5 resonances characterize CYP2B4-cytb5 interactions on the intermediate time scale. More significant intensity attenuation and a more specific protein-protein binding interface are observed in bicelles as compared with lipid-free solution, highlighting the importance of the lipid bilayer in stabilizing stronger and more specific interactions between CYP2B4 and cytb5, which may lead to a more efficient electron transfer. Similar results observed for the interactions between CYP2B4 lacking the transmembrane domain (tr-CYP2B4) and cytb5 imply interactions between tr-CYP2B4 and the membrane surface, which might assist in CYP2B4-cytb5 complex formation by orienting tr-CYP2B4 for efficient contact with cytb5. Furthermore, the observation of weak and nonspecific interactions between CYP2B4 and cytb5 in micelles suggests that lipid bilayer structures and low curvature membrane surface are preferable for CYP2B4-cytb5 complex formation. Results presented in this study provide structural insights into the mechanism behind the important role that the lipid bilayer plays in the interactions between P450s and their redox partners.
哺乳动物细胞色素P450（P450）是一种膜结合单加氧酶，其催化活性需要两个电子从其氧化还原伴侣依次传递：细胞色素b5（cytb5）和细胞色素P450还原酶，这两者都是膜蛋白。尽管已知P450的功能活性会受到脂质的影响，但仍缺乏揭示膜对P450 - cytb5相互作用影响的实验证据。在此，我们利用核磁共振技术，在原子水平上展示了磷脂双层对兔P450 2B4（CYP2B4）和兔cytb5之间复合物形成的影响。cytb5共振的一般谱线展宽和适度化学位移扰动表征了中间时间尺度上CYP2B4 - cytb5的相互作用。与无脂质溶液相比，在双分子层中观察到更显著的强度衰减和更特异的蛋白质 - 蛋白质结合界面，突出了脂质双层在稳定CYP2B4和cytb5之间更强且更特异的相互作用中的重要性，这可能导致更有效的电子转移。对于缺乏跨膜结构域的CYP2B4（tr - CYP2B4）和cytb5之间的相互作用观察到的类似结果表明，tr - CYP2B4与膜表面之间存在相互作用，这可能通过使tr - CYP2B4定向以与cytb5有效接触来辅助CYP2B4 - cytb5复合物的形成。此外，在胶束中观察到CYP2B4和cytb5之间存在弱且非特异性的相互作用，这表明脂质双层结构和低曲率膜表面更有利于CYP2B4 - cytb5复合物的形成。本研究呈现的结果为脂质双层在P450与其氧化还原伴侣相互作用中所起重要作用背后的机制提供了结构见解。
