Institute of Enzymology, Hungarian Academy of Sciences, Karolina út 29, Budapest, Hungary.
FASEB J. 2010 Oct;24(10):3829-39. doi: 10.1096/fj.10-155614. Epub 2010 Jun 3.
Lipid-protein interactions are rarely characterized at a structural molecular level due to technical difficulties; however, the biological significance of understanding the mechanism of these interactions is outstanding. In this report, we provide mechanistic insight into the inhibitory complex formation of the lipid mediator sphingosylphosphorylcholine with calmodulin, the most central and ubiquitous regulator protein in calcium signaling. We applied crystallographic, thermodynamic, kinetic, and spectroscopic approaches using purified bovine calmodulin and bovine cerebral microsomal fraction to arrive at our conclusions. Here we present 1) a 1.6-Å resolution crystal structure of their complex, in which the sphingolipid occupies the conventional hydrophobic binding site on calmodulin; 2) a peculiar stoichiometry-dependent binding process: at low or high protein-to-lipid ratio calmodulin binds lipid micelles or a few lipid molecules in a compact globular conformation, respectively, and 3) evidence that the sphingolipid displaces calmodulin from its targets on cerebral microsomes. We have ascertained the specificity of the interaction using structurally related lipids as controls. Our observations reveal the structural basis of selective calmodulin inhibition by the sphingolipid. On the basis of the crystallographic and biophysical characterization of the calmodulin-sphingosylphosphorylcholine interaction, we propose a novel lipid-protein binding model, which might be applicable to other interactions as well.
由于技术上的困难，脂质-蛋白质相互作用很少在结构分子水平上得到描述；然而，理解这些相互作用机制的生物学意义是非常突出的。在本报告中，我们提供了关于脂质介体鞘磷脂磷酸胆碱与钙调蛋白相互作用抑制复合物形成的机制见解，钙调蛋白是钙信号转导中最核心和最普遍的调节蛋白。我们应用了结晶学、热力学、动力学和光谱学方法，使用纯化的牛钙调蛋白和牛脑微粒体部分来得出我们的结论。在这里，我们提出了 1）它们复合物的 1.6Å 分辨率晶体结构，其中鞘磷脂占据钙调蛋白的常规疏水性结合位点；2）一种特殊的、依赖于计量比的结合过程：在低或高蛋白-脂质比下，钙调蛋白分别结合脂质胶束或少量脂质分子，形成紧凑的球状构象；3）证据表明，鞘磷脂将钙调蛋白从脑微粒体上的靶蛋白中置换出来。我们使用结构上相关的脂质作为对照来确定相互作用的特异性。我们的观察结果揭示了鞘磷脂对钙调蛋白选择性抑制的结构基础。基于钙调蛋白-鞘磷脂磷酸胆碱相互作用的晶体学和生物物理特性，我们提出了一种新的脂质-蛋白质结合模型，该模型可能也适用于其他相互作用。
