C2 domain of protein kinase Cα:: Elucidation of the membrane docking surface by site-directed fluorescence and spin labeling
C2 domain of protein kinase Cα:: Elucidation of the membrane docking surface by site-directed fluorescence and spin labeling
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DOI：
10.1021/bi026596f
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发表时间：
2003-02-11
期刊：
影响因子：
2.9
通讯作者：
Falke, JJ
中科院分区：
文献类型：
--
作者：
Kohout, SC;Corbalán-García, S;Falke, JJ
C2结构域是一个保守的信号基序，触发膜对接在Ca 2+依赖的方式，但许多C2结构域的膜对接表面尚未确定。两个极端的模型，可以提出对接的蛋白激酶Calpha（PKCalpha）C2结构域的膜。在平行模型中，膜对接表面包括β链3-4上的Call结合环和阴离子结合位点，使得β链平行于膜取向。在垂直模型中，对接表面定位于Call结合环，并且β链垂直于膜表面取向。本研究利用定点荧光和自旋标记绘制出PKCalpha C2结构域的膜对接表面。单个半胱氨酸残基被设计成分散在蛋白质表面所有区域的18个位置，并用作光谱探针的附着位点。对环境敏感的荧光素探针确定了Ca 2+激活或膜对接触发可测量的荧光变化的位置。Ca 2+结合被发现启动一个全球性的构象变化，而膜对接引发的最大的荧光素环境变化的标记位置上的三个Ca 2+结合环（CBL），从而定位这些环的膜对接表面。使用氮氧自旋探针进行互补EPR功率饱和测量，以确定每个自旋标记的突变体的膜深度参数Phi。阳性膜深度参数指示膜插入被发现的三个位置，都位于Ca 2+结合环：N189上CBL 1，和R249和R252上CBL 3。此外，EPR功率饱和显示，阴离子结合位点附近的5个位置被部分保护免受与水性顺磁探针的碰撞，这表明阴离子结合位点位于或靠近头基层的表面。总之，荧光和EPR结果表明，Ca 2+的第一和第三Ca 2+结合环直接插入到膜的脂质头基区域，并且β链3-4上的阴离子结合位点位于头基附近。该数据支持β-链相对于膜表面朝向平行取向倾斜的模型。
The C2 domain is a conserved signaling motif that triggers membrane docking in a Ca2+- dependent manner, but the membrane docking surfaces of many C2 domains have not yet been identified. Two extreme models can be proposed for the docking of the protein kinase Calpha (PKCalpha) C2 domain to membranes. In the parallel model, the membrane-docking surface includes the Call binding loops and an anion binding site on beta-strands 3-4, such that the beta-strands are oriented parallel to the membrane. In the perpendicular model, the docking surface is localized to the Call binding loops and the beta-strands are oriented perpendicular to the membrane surface. The present study utilizes site-directed fluorescence and spin-labeling to map out the membrane docking surface of the PKCalpha C2 domain. Single cysteine residues were engineered into 18 locations scattered over all regions of the protein surface, and were used as attachment sites for spectroscopic probes. The environmentally sensitive fluorescein probe identified positions where Ca2+ activation or membrane docking trigger measurable fluorescence changes. Ca2+ binding was found to initiate a global conformational change, while membrane docking triggered the largest fluorescein environmental changes at labeling positions on the three Ca2+ binding loops (CBL), thereby localizing these loops to the membrane docking surface. Complementary EPR power saturation measurements were carried out using a nitroxide spin probe to determine a membrane depth parameter, Phi, for each spin-labeled mutant. Positive membrane depth parameters indicative of membrane insertion were found for three positions, all located on the Ca2+ binding loops: N189 on CBL 1, and both R249 and R252 on CBL 3. In addition, EPR power saturation revealed that five positions near the anion binding site are partially protected from collisions with an aqueous paramagnetic probe, indicating that the anion binding site lies at or near the surface of the headgroup layer. Together, the fluorescence and EPR results indicate that the Ca2+ first and third Ca2+ binding loops insert directly into the lipid headgroup region of the membrane, and that the anion binding site on beta-strands 3-4 lies near the headgroups. The data support a model in which the beta-strands are tilted toward the parallel orientation relative to the membrane surface.