Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, United States.
St. Catherine University, Department of Chemistry and Biochemistry, 2004 Randolph Ave., St. Paul, MN 55105, United States.
Biochim Biophys Acta Biomembr. 2018 Jun;1860(6):1335-1341. doi: 10.1016/j.bbamem.2018.02.030. Epub 2018 Mar 2.
Approximately, 70% of the Ca ion transport into the sarcoplasmic reticulum is catalyzed by the sarcoplasmic reticulum Ca-ATPase (SERCA), whose activity is endogenously regulated by phospholamban (PLN). PLN comprises a TM inhibitory region and a cytoplasmic regulatory region that harbors a consensus sequence for cAMP-dependent protein kinase (PKA). The inhibitory region binds the ATPase, reducing its apparent Ca binding affinity. β-adrenergic stimulation activates PKA, which phosphorylates PLN at Ser 16, reversing its inhibitory function. Mutations and post-translational modifications of PLN may lead to dilated cardiomyopathy (DCM) and heart failure. PLN's cytoplasmic region interconverts between a membrane-associated T state and a membrane-detached R state. The importance of these structural transitions on SERCA regulation is emerging, but the effects of natural occurring mutations and their relevance to the progression of heart disease are unclear. Here we use solid-state NMR spectroscopy to investigate the structural dynamics of two lethal PLN mutations, R9C and R25C, which lead to DCM. We found that the R25C mutant enhances the dynamics of PLN and shifts the conformational equilibrium toward the R state confirmation, whereas the R9C mutant drives the amphipathic cytoplasmic domain toward the membrane-associate state, enriching the T state population. The changes in membrane interactions caused by these mutations may explain the aberrant regulation of SERCA.
大约 70%的钙离子进入肌浆网是由肌浆网 Ca-ATP 酶（SERCA）催化的，其活性受肌浆网磷蛋白（PLN）的内源性调节。PLN 由一个 TM 抑制区和一个细胞质调节区组成，该调节区含有一个 cAMP 依赖性蛋白激酶（PKA）的共识序列。抑制区结合 ATP 酶，降低其表观 Ca 结合亲和力。β-肾上腺素能刺激激活 PKA，使 PLN 在 Ser16 磷酸化，从而逆转其抑制功能。PLN 的突变和翻译后修饰可能导致扩张型心肌病（DCM）和心力衰竭。PLN 的细胞质区域在与膜结合的 T 态和与膜分离的 R 态之间相互转换。这些结构转变对 SERCA 调节的重要性正在显现，但天然发生的突变的影响及其与心脏病进展的相关性尚不清楚。在这里，我们使用固态 NMR 光谱研究了导致 DCM 的两种致命 PLN 突变，R9C 和 R25C 的结构动力学。我们发现 R25C 突变增强了 PLN 的动力学，并将构象平衡向 R 态确认转移，而 R9C 突变则将两亲性细胞质结构域推向膜结合状态，增加了 T 态的含量。这些突变引起的膜相互作用的变化可能解释了 SERCA 的异常调节。
