Phosphorylation induces a conformational transition near the lipid-water interface of phospholamban reconstituted with the Ca-ATPase.
Phosphorylation induces a conformational transition near the lipid-water interface of phospholamban reconstituted with the Ca-ATPase.
复制标题
磷酸化会在用 Ca-ATP 酶重构的受磷蛋白的脂质-水界面附近诱导构象转变。
DOI：
10.1021/bi0266030
复制
发表时间：
2002
期刊：
影响因子：
2.9
通讯作者：
Bigelow,DianaJ
中科院分区：
文献类型：
--
作者：
Chen,Baowei;Bigelow,DianaJ
我们用含一个半胱氨酸(Cys-24)的工程化磷蛋白与肌浆网钙-三磷酸腺苷酶(SR Ca-ATPase)相互作用和cAMP依赖的蛋白激酶(PKA)对丝氨酸-16(Ser-16)的磷酸化，检测了磷蛋白的构象变化。这种修饰的突变体PLB与脂质体中亲和纯化的Ca-ATPase共重组后具有完整的功能。ANSmal发射特性和其溶剂可及性表明，Cys-24处于膜外的水环境中。ANSmal-PLB的荧光猝灭和时间分辨各向异性测量表明，在自由态和Ca-ATPase结合态下，PLB具有不同的结构。当PLB与Ca-ATPase相互作用时，ANSmal的溶剂暴露和探针运动都得到了增强。这种构象转变需要将游离的PLB转化为对PLB的磷酸化状态敏感的构象。当钙-−酶结合的原磷脂被磷酸化后，ANSMALL的溶剂暴露水平降低，这表明原磷脂在脂质ATPase水界面附近的氨基酸序列起着构象开关的作用，对原磷脂的磷酸化做出反应。更长的关联时间，由各向异性测量解决，对应于多肽链的波动，实质上受到PLB与Ca-ATPase相互作用的限制。这种限制不能被pLB的磷酸化逆转，这表明靠近脂质−水界面的Cys-24附近的区域不会从Ca-ATPase解离。这些结果表明，蛋白激酶A的磷酸化导致原磷脂−钙-三磷酸腺苷酶蛋白接触的重新分布，从而减轻原蛋白B对钙转运激活的抑制作用。
We have measured conformational changes of phospholamban (PLB) induced both by its interaction with the SR Ca-ATPase and by phosphorylation of Ser-16 by cAMP-dependent protein kinase (PKA) using an engineered PLB having a single cysteine (Cys-24) derivatized with the fluorophore 2-(4‘-maleimidylanilino)naphthalene-6-sulfonic acid (ANSmal). This modified mutant PLB is fully functional when co-reconstituted with the affinity-purified Ca-ATPase in liposomes. ANSmal emission properties and its solvent accessibility indicate that Cys-24 is in an aqueous environment outside the membrane. Fluorescence quenching and time-resolved anisotropy measurements of ANSmal-PLB demonstrate distinct structures for PLB in the free and Ca-ATPase-bound state. Both solvent exposure and probe motions of ANSmal are enhanced upon interaction of PLB with the Ca-ATPase. This conformational transition entails conversion of free PLB in a conformation which is insensitive to one which is sensitive to the phosphorylation state of PLB. Upon phosphorylation of Ca-ATPase-bound PLB, a decreased level of solvent exposure of ANSmal is observed, suggesting that the amino acid sequence of PLB near the lipid−water interface acts as a conformational switch in response to the phosphorylation of PLB. A longer correlation time, resolved by anisotropy measurements, corresponding to polypeptide chain fluctuations, is substantially restricted by interaction of PLB with the Ca-ATPase. This restriction is not reversed by phosphorylation of PLB, indicating that the region around Cys-24 near the lipid−water interface does not undergo dissociation from the Ca-ATPase. These results suggest that the phosphorylation by PKA induces a redistribution of PLB−Ca-ATPase protein contacts to relieve the inhibitory effect of PLB for the activation of calcium transport.