Landgraf Kyle E, Malmberg Nathan J, Falke Joseph J
Department of Chemistry and Biochemistry and the Molecular Biophysics Program, University of Colorado, Boulder, Colorado 80309-0215, USA.
Biochemistry. 2008 Aug 12;47(32):8301-16. doi: 10.1021/bi800711t. Epub 2008 Jul 9.
Protein kinase C isoform alpha (PKCalpha) is a ubiquitous, conventional PKC enzyme that possesses a conserved C2 domain. Upon activation by cytoplasmic Ca (2+) ions, the C2 domain specifically binds to the plasma membrane inner leaflet where it recognizes the target lipids phosphatidylserine (PS) and phosphatidylinositol-4,5-bisphosphate (PIP 2). The membrane penetration depth and docking angle of the membrane-associated C2 domain is not well understood. The present study employs EPR site-directed spin labeling and relaxation methods to generate a medium-resolution model of the PKCalpha C2 domain docked to a membrane of lipid composition similar to the plasma membrane inner leaflet. The approach measures EPR depth parameters for 10 function-retaining spin labels coupled to the C2 domain, and for spin labels coupled to depth calibration molecules. The resulting depth parameters, together with the known structure of the free C2 domain, provide a sufficient number of constraints to define two membrane docking geometries for C2 domain bound to physiological membranes lacking or containing PIP 2, respectively. In both the absence and presence of PIP 2, the two bound Ca (2+) ions of the C2 domain lie near the anionic phosphate plane in the headgroup region, consistent with the known ability of the Ca (2+) and membrane-binding loops (CMBLs) to bind the headgroup of the PS target lipid. In the absence of PIP 2, the polybasic lipid binding site on the beta3-beta4 hairpin is occupied with PS, but in the presence of PIP 2 this larger, higher affinity target lipid competitively displaces PS and causes the long axis of the domain to tilt 40 +/- 10 degrees toward the bilayer normal. The ability of the beta3-beta4 hairpin site to bind PS as well as PIP 2 extends the lifetime of the membrane-docked state and is predicted to enhance the kinase turnover number of PKCalpha during a single membrane docking event. In principle, PIP 2-induced tilting of the C2 domain could modulate the activity of membrane-docked PKCalpha as it diffuses between membrane regions with different local PS and PIP 2 concentrations. Finally, the results demonstrate that EPR relaxation methods are sufficiently sensitive to detect signaling-induced changes in the membrane docking geometries of peripheral membrane proteins.
蛋白激酶Cα亚型（PKCα）是一种普遍存在的传统蛋白激酶C酶，具有保守的C2结构域。在被细胞质钙离子（Ca²⁺）激活后，C2结构域特异性结合到质膜内小叶，在那里它识别靶脂质磷脂酰丝氨酸（PS）和磷脂酰肌醇-4,5-二磷酸（PIP₂）。膜相关C2结构域的膜穿透深度和对接角度尚不清楚。本研究采用电子顺磁共振（EPR）定点自旋标记和弛豫方法，生成了一个与质膜内小叶脂质组成相似的膜上对接PKCα C2结构域的中等分辨率模型。该方法测量了与C2结构域偶联的10个保留功能的自旋标记以及与深度校准分子偶联的自旋标记的EPR深度参数。所得深度参数与游离C2结构域的已知结构一起，提供了足够数量的约束条件，以分别定义C2结构域与缺乏或含有PIP₂的生理膜结合的两种膜对接几何结构。在不存在和存在PIP₂的情况下，C2结构域的两个结合钙离子（Ca²⁺）都位于头基区域的阴离子磷酸平面附近，这与Ca²⁺和膜结合环（CMBLs）结合PS靶脂质头基的已知能力一致。在不存在PIP₂的情况下，β3-β4发夹上的多碱性脂质结合位点被PS占据，但在存在PIP₂的情况下，这种更大、亲和力更高的靶脂质竞争性取代PS，并导致结构域的长轴朝双层法线方向倾斜40±10度。β3-β4发夹位点结合PS以及PIP₂的能力延长了膜对接状态的寿命，并预计在单次膜对接事件中提高PKCα的激酶转换数。原则上，PIP₂诱导的C2结构域倾斜可在膜对接的PKCα在具有不同局部PS和PIP₂浓度的膜区域之间扩散时调节其活性。最后，结果表明EPR弛豫方法足够灵敏，能够检测外周膜蛋白膜对接几何结构中信号诱导的变化。
