Jaud Simon, Tobias Douglas J, Falke Joseph J, White Stephen H
Department of Chemistry, and Department of Physiology and Biophysics, University of California, Irvine, California, USA.
Biophys J. 2007 Jan 15;92(2):517-24. doi: 10.1529/biophysj.106.090704. Epub 2006 Oct 27.
As a first step toward understanding the principles of the targeting of C2 domains to membranes, we have carried out a molecular dynamics simulation of the C2 domain of cytosolic phospholipase A2 (cPLA2-C2) in a 1-palmitoyl-2-oleoyl-phosphatidylcholine bilayer at constant pressure and temperature (NPT, 300 K and 1 atm). Using the high-resolution crystal structure of cPLA2-C2 as a starting point, we embedded two copies of the C2 domain into a pre-equilibrated membrane at the depth and orientation previously defined by electron paramagnetic resonance (EPR). Noting that in the membrane-bound state the three calcium binding loops are complexed to two calcium ions, we initially restrained the calcium ions at the membrane depth determined by EPR. But the depth and orientation of the domains remained within EPR experimental errors when the restraints were later removed. We find that the thermally disordered, chemically heterogeneous interfacial zones of phosphatidylcholine bilayers allow local lipid remodeling to produce a nearly perfect match to the shape and polarity of the C2 domain, thereby enabling the C2 domain to assemble and optimize its own lipid docking site. The result is a cuplike docking site with a hydrophobic bottom and hydrophilic rim. Contrary to expectations, we did not find direct interactions between the protein-bound calcium ions and lipid headgroups, which were sterically excluded from the calcium binding cleft. Rather, the lipid phosphate groups provided outer-sphere calcium coordination through intervening water molecules. These results show that the combined use of high-resolution protein structures, EPR measurements, and molecular dynamics simulations provides a general approach for analyzing the molecular interactions between membrane-docked proteins and lipid bilayers.
作为理解C2结构域靶向细胞膜原理的第一步，我们在恒压和恒温条件下（NPT，300K和1个大气压），对胞质磷脂酶A2的C2结构域（cPLA2-C2）在1-棕榈酰-2-油酰磷脂酰胆碱双层膜中进行了分子动力学模拟。以cPLA2-C2的高分辨率晶体结构为起点，我们将两个C2结构域的拷贝按照先前通过电子顺磁共振（EPR）确定的深度和方向嵌入到预平衡的膜中。注意到在膜结合状态下，三个钙结合环与两个钙离子络合，我们最初将钙离子限制在由EPR确定的膜深度处。但是当后来去除限制时，结构域的深度和方向仍在EPR实验误差范围内。我们发现磷脂酰胆碱双层膜的热无序、化学异质的界面区域允许局部脂质重塑，以产生与C2结构域的形状和极性几乎完美匹配的结构，从而使C2结构域能够组装并优化其自身的脂质对接位点。结果是形成了一个具有疏水底部和亲水边缘的杯状对接位点。与预期相反，我们没有发现蛋白质结合的钙离子与脂质头部基团之间的直接相互作用，脂质头部基团在空间上被排除在钙结合裂隙之外。相反，脂质磷酸基团通过中间水分子提供外层球钙配位。这些结果表明，高分辨率蛋白质结构、EPR测量和分子动力学模拟的联合使用为分析膜结合蛋白与脂质双层之间的分子相互作用提供了一种通用方法。
