Ball A, Nielsen R, Gelb M H, Robinson B H
Department of Chemistry, University of Washington, Seattle, WA 98195, USA.
Proc Natl Acad Sci U S A. 1999 Jun 8;96(12):6637-42. doi: 10.1073/pnas.96.12.6637.
The C2 domain of cytosolic phospholipase A2 (C2cPLA2) plays an important role in calcium-dependent transfer of the protein from the cytosol to internal cellular membranes as a prelude for arachidonate release from membrane phospholipids. By using a recently developed electron paramagnetic resonance approach together with 13 site-specifically nitroxide spin labeled C2cPLA2s and membrane-permeant and -impermeant spin relaxants, we have determined the orientation of C2cPLA2 with respect to the surface of vesicles of the phospholipid 1,2-dioleoyl-sn-glycero-3-phosphomethanol. The structure reveals that the two calcium-binding regions on C2cPLA2 that display hydrophobic residues, CBR1 and CBR3, are partially inserted into the core of the membrane. CBR2 that contains predominantly hydrophilic residues is close to the membrane but not inserted. The long axis of the cylindrical C2cPLA2 molecule is tilted with respect to the bilayer normal, which brings a cluster of basic protein residues close to the phospholipid headgroups. Such an orientation places the two bound calcium ions close to the membrane surface. All together, the results provide structural support for previous proposals that binding of C2cPLA2 to the membrane interface is driven in part by insertion of hydrophobic surface loops into the membrane core. The results are contrasted with previous studies of the interfacial binding of the first C2 domain of synaptotagmin I, which has shorter surface loops that display basic residues for electrostatic interaction with the bilayer surface.
胞质型磷脂酶A2（C2cPLA2）的C2结构域在该蛋白从胞质溶胶向细胞内膜的钙依赖性转移过程中发挥重要作用，这是膜磷脂释放花生四烯酸的前奏。通过使用最近开发的电子顺磁共振方法，结合13种位点特异性氮氧化物自旋标记的C2cPLA2以及可透过膜和不可透过膜的自旋弛豫剂，我们确定了C2cPLA2相对于磷脂1,2 - 二油酰 - sn - 甘油 - 3 - 磷酸甲醇囊泡表面的取向。该结构表明，C2cPLA2上显示疏水残基的两个钙结合区域CBR1和CBR3部分插入膜的核心。主要包含亲水残基的CBR2靠近膜但未插入。圆柱形C2cPLA2分子的长轴相对于双层法线倾斜，这使得一组碱性蛋白质残基靠近磷脂头部基团。这样的取向使两个结合的钙离子靠近膜表面。总体而言，这些结果为先前的提议提供了结构支持，即C2cPLA2与膜界面的结合部分是由疏水表面环插入膜核心驱动的。这些结果与之前对突触结合蛋白I的第一个C2结构域的界面结合研究形成对比，突触结合蛋白I的表面环较短，显示出用于与双层表面进行静电相互作用的碱性残基。
