Phosphorylation-dependent conformational switch in spin-labeled phospholamban bound to SERCA
Phosphorylation-dependent conformational switch in spin-labeled phospholamban bound to SERCA
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DOI：
10.1016/j.jmb.2006.02.051
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发表时间：
2006-05-12
影响因子：
5.6
通讯作者：
Thomas, DD
中科院分区：
文献类型：
--
作者：
Karim, CB;Zhang, ZW;Thomas, DD
我们已经使用化学合成，功能重建，和电子顺磁共振（EPR）探测受磷蛋白（PLB）的功能动力学，调节心肌肌浆网的钙ATP酶（SERCA）。PLB的跨膜结构域在低[Ca 2 +]下抑制SERCA，但胞质结构域在Ser16磷酸化后解除这种抑制。用2，2，6，6-四甲基哌啶-1-氧基-4-氨基-4-羧酸（TOAC）自旋标记物代替Ala 11合成了单体PLB，其直接报告肽骨架动力学。PLB在存在或不存在SERCA的情况下重构成膜。TOAC-PLB显示正常的抑制功能，其被磷酸化逆转。Ser16或微摩尔[Ca2 +]。EPR表明，PLB胞质结构域具有两个解决的构象，紧张的T状态，是有序的和放松的R状态，是动态无序和扩展。PLB磷酸化将这种平衡向R状态转移，使其更具动态性（超扩展）。磷酸化强烈扰乱了与SERCA结合的PLB的动力学，而不会解离复合物，而微摩尔[Ca 2 +]对PLB动力学没有影响。合成连接到PLB N末端的脂质锚允许Ca依赖性SERCA抑制，但防止磷酸化诱导的无序和抑制逆转。我们的结论是，PLB磷酸化的SERCA抑制的救济是由于PLB的胞质结构域中的有序到无序的过渡，这使得该结构域延伸到膜表面以上，并诱导SERCA的胞质结构域的结构变化。该机制不同于在加入微摩尔[Ca2 +]后缓解PLB依赖性SERCA抑制的机制。(c)2006爱思唯尔有限公司保留所有权利。
We have used chemical synthesis, functional reconstitution, and electron paramagnetic resonance (EPR) to probe the functional dynamics of phospholamban (PLB), which regulates the Ca-ATPase (SERCA) in cardiac sarcoplasmic reticulum. The transmembrane domain of PLB inhibits SERCA at low [Ca2+], but the cytoplasmic domain relieves this inhibition upon Ser16 phosphorylation. Monomeric PLB was synthesized with Ala11 replaced by the 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) spin label, which reports peptide backbone dynamics directly. PLB was reconstituted into membranes in the presence or absence of SERCA. TOAC-PLB showed normal inhibitory function, which was reversed by phosphorylation. at Ser16 or by micromolar [Ca2+]. EPR showed that the PLB cytoplasmic domain exhibits two resolved conformations, a tense T state that is ordered and a relaxed R state that is dynamically disordered and extended. PLB phosphorylation shifts this equilibrium toward the R state and makes it more dynamic (hyperextended). Phosphorylation strongly perturbs the dynamics of SERCA-bound PLB without dissociating the complex, while micromolar [Ca2+] has no effect on PLB dynamics. A lipid anchor synthetically attached to the N terminus of PLB permits Ca-dependent SERCA inhibition but prevents the phosphorylation-induced disordering and reversal of inhibition. We conclude that the relief of SERCA inhibition by PLB phosphorylation is due to an order-to-disorder transition in the cytoplasmic domain of PLB, which allows this domain to extend above the membrane surface and induce a structural change in the cytoplasmic domain of SERCA. This mechanism is distinct from the one that relieves PLB-dependent SERCA inhibition upon the addition of micromolar [Ca2+]. (c) 2006 Elsevier Ltd. All rights reserved.