Lipid-Mediated Folding/Unfolding of Phospholamban as a Regulatory Mechanism for the Sarcoplasmic Reticulum Ca2+-ATPase
Lipid-Mediated Folding/Unfolding of Phospholamban as a Regulatory Mechanism for the Sarcoplasmic Reticulum Ca2+-ATPase
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DOI：
10.1016/j.jmb.2011.03.015
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发表时间：
2011-05-13
影响因子：
5.6
通讯作者：
Veglia, Gianluigi
中科院分区：
文献类型：
--
作者：
Gustavsson, Martin;Traaseth, Nathaniel J.;Veglia, Gianluigi
受磷蛋白（PLN）和肌浆网Ca ~（2+）-ATP酶（SERCA）之间的膜蛋白复合物调节心肌收缩力。在非磷酸化形式中，PLN结合SERCA并抑制Ca 2+通量。当PLN在Ser 16处磷酸化时，抑制作用被逆转。虽然这两种蛋白质的结构细节是新兴的X射线晶体学，冷冻电子显微镜和核磁共振研究，它们的相互作用和调节过程的分子机制仍然缺乏。据推测，SERCA调节取决于PLN结构转变（有序到无序，即，折叠/展开）。在这里，我们研究了PLN的构象变化后，化学解折叠的电子顺磁共振和NMR光谱的组合，揭示了构象转变主要涉及的细胞质区域，与两个伴随的现象：（1）膜结合和折叠的两亲性结构域Ia和（2）折叠/解折叠的PLN的质膜结构域Ib。用PLN的两种磷酸模拟突变体（S16 E和S16 D）分析磷酸化和未磷酸化的PLN显示，结构域Ia和Ib中未折叠状态（T'状态）的群体与通过活性测定测量的SERCA抑制程度线性相关。SERCA的抑制是通过蛋白质（PLN）的折叠基态（T态）进行的，而抑制的缓解涉及促进PLN到激发构象状态（Ser 16磷酸化PLN）。我们建议PLN人口转变（折叠/展开）是SERCA的一个关键调控机制。(C)2011爱思唯尔有限公司保留所有权利。
The integral membrane protein complex between phospholamban (PLN) and sarcoplasmic reticulum Ca2+-ATPase (SERCA) regulates cardiac contractility. In the unphosphorylated form, PLN binds SERCA and inhibits Ca2+ flux. Upon phosphorylation of PLN at Ser16, the inhibitory effect is reversed. Although structural details on both proteins are emerging from X-ray crystallography, cryo-electron microscopy, and NMR studies, the molecular mechanisms of their interactions and regulatory process are still lacking. It has been speculated that SERCA regulation depends on PLN structural transitions (order to disorder, i.e., folding/unfolding). Here, we investigated PLN conformational changes upon chemical unfolding by a combination of electron paramagnetic resonance and NMR spectroscopies, revealing that the conformational transitions involve mostly the cytoplasmic regions, with two concomitant phenomena: (1) membrane binding and folding of the amphipathic domain Ia and (2) folding/unfolding of the juxtamembrane domain lb of PLN. Analysis of phosphorylated and unphosphorylated PLN with two phosphomimetic mutants of PLN (516E and S16D) shows that the population of an unfolded state in domains Ia and lb (T' state) is linearly correlated to the extent of SERCA inhibition measured by activity assays. Inhibition of SERCA is carried out by the folded ground state (T state) of the protein (PLN), while the relief of inhibition involves promotion of PLN to excited conformational states (Ser16 phosphorylated PLN). We propose that PLN population shifts (folding/unfolding) are a key regulatory mechanism for SERCA. (C) 2011 Elsevier Ltd. All rights reserved.