Configuration of PKCα-C2 Domain Bound to Mixed SOPC/SOPS Lipid Monolayers
Configuration of PKCα-C2 Domain Bound to Mixed SOPC/SOPS Lipid Monolayers
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DOI：
10.1016/j.bpj.2009.08.037
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发表时间：
2009-11-18
影响因子：
3.4
通讯作者：
Schlossman, Mark L.
中科院分区：
文献类型：
--
作者：
Chen, Chiu-Hao;Malkova, Sarka;Schlossman, Mark L.
X射线反射率测量用于确定蛋白激酶C α（PKC α-C2）的C2结构域的构型，所述蛋白激酶C α（PKC α-C2）结合于负载在缓冲水溶液上的1-硬脂酰基-2-油酰基-sn-甘油基-3-磷酸胆碱和1-硬脂酰基-2-油酰基-sn-甘油基-3-磷酸丝氨酸的7：3混合物的脂质单层。根据PKC alpha-C2的已知晶体结构和脂质层的平板模型表示来分析反射率。脂质结合的PKC α-C2的构型由定义其取向的两个角度（θ = 35度+/-10度和phi = 210度+/-30度）和进入脂质层的穿透深度（=7.5 +/-2埃）来描述。在这种结构中，PKC α-C2的β-折叠几乎垂直于脂质层，并且结构域渗透到脂质层的头基区域，但不渗透到尾基区域。通过我们的X射线反射率确定的PKC α-C2的这种构型与许多先前的发现，特别是突变研究一致，并且还提供了我们认为是对PKC α酶激活机制的新分子见解。我们的分析方法，这使我们能够测试所有可能的蛋白质取向，表明我们的数据不能被解释为蛋白质的取向平行于膜，如早期的工作所建议的。
X-ray reflectivity measurements are used to determine the configuration of the C2 domain of protein kinase C alpha (PKC alpha-C2) bound to a lipid monolayer of a 7:3 mixture of 1-stearoyl-2-oleoyi-sn-glycero-3-phosphocholine and 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoserine supported on a buffered aqueous solution. The reflectivity is analyzed in terms of the known crystallographic structure of PKC alpha-C2 and a slab model representation of the lipid layer. The configuration of lipid-bound PKC alpha-C2 is described by two angles that define its orientation, theta = 35 degrees +/- 10 degrees and phi = 210 degrees +/- 30 degrees, and a penetration depth (=7.5 +/- 2 angstrom) into the lipid layer. In this structure, the beta-sheets of PKC alpha-C2 are nearly perpendicular to the lipid layer and the domain penetrates into the headgroup region of the lipid layer, but not into the tailgroup region. This configuration of PKC alpha-C2 determined by our x-ray reflectivity is consistent with many previous findings, particularly mutational studies, and also provides what we believe is new molecular insight into the mechanism of PKC alpha enzyme activation. Our analysis method, which allows us to test all possible protein orientations, shows that our data cannot be explained by a protein that is orientated parallel to the membrane, as suggested by earlier work.