Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN 55455, USA.
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA.
Biochem Biophys Res Commun. 2014 Jun 27;449(2):196-201. doi: 10.1016/j.bbrc.2014.04.166. Epub 2014 May 9.
We have used time-resolved fluorescence resonance energy transfer (TR-FRET) to characterize the interaction between phospholamban (PLB) and the sarcoplasmic reticulum (SR) Ca-ATPase (SERCA) under conditions that relieve SERCA inhibition. Unphosphorylated PLB inhibits SERCA in cardiac SR, but inhibition is relieved by either micromolar Ca(2+) or PLB phosphorylation. In both cases, it has been proposed that inhibition is relieved by dissociation of the complex. To test this hypothesis, we attached fluorophores to the cytoplasmic domains of SERCA and PLB, and reconstituted them functionally in lipid bilayers. TR-FRET, which permitted simultaneous measurement of SERCA-PLB binding and structure, was measured as a function of PLB phosphorylation and [Ca(2+)]. In all cases, two structural states of the SERCA-PLB complex were resolved, probably corresponding to the previously described T and R structural states of the PLB cytoplasmic domain. Phosphorylation of PLB at S16 completely relieved inhibition, partially dissociated the SERCA-PLB complex, and shifted the T/R equilibrium within the bound complex toward the R state. Since the PLB concentration in cardiac SR is at least 10 times that in our FRET measurements, we calculate that most of SERCA contains bound phosphorylated PLB in cardiac SR, even after complete phosphorylation. 4 μM Ca(2+) completely relieved inhibition but did not induce a detectable change in SERCA-PLB binding or cytoplasmic domain structure, suggesting a mechanism involving structural changes in SERCA's transmembrane domain. We conclude that Ca(2+) and PLB phosphorylation relieve SERCA-PLB inhibition by distinct mechanisms, but both are achieved primarily by structural changes within the SERCA-PLB complex, not by dissociation of that complex.
我们使用时间分辨荧光共振能量转移（TR-FRET）技术，在解除 SERCA 抑制的条件下，研究了肌浆网 Ca2+-ATP 酶（SERCA）与磷酸化抑制素（PLB）之间的相互作用。在心脏肌浆网中，未磷酸化的 PLB 抑制 SERCA，但通过微摩尔 Ca2+或 PLB 磷酸化可解除抑制。在这两种情况下，人们提出抑制的解除是通过复合物的解离来实现的。为了验证这一假说，我们将荧光基团连接到 SERCA 和 PLB 的细胞质结构域上，并在脂质双层中重建其功能。TR-FRET 技术可以同时测量 SERCA-PLB 结合和结构，我们检测了 PLB 磷酸化和[Ca2+]对其的影响。在所有情况下，均解析出 SERCA-PLB 复合物的两种结构状态，可能对应于先前描述的 PLB 细胞质结构域的 T 和 R 结构状态。PLB 在 S16 位点的磷酸化完全解除了抑制，部分解离了 SERCA-PLB 复合物，并使结合复合物中的 T/R 平衡向 R 状态移动。由于心脏肌浆网中 PLB 的浓度至少是我们 FRET 测量值的 10 倍，我们计算得出，即使在完全磷酸化后，心脏肌浆网中大多数 SERCA 都含有结合的磷酸化 PLB。4 μM Ca2+完全解除了抑制，但未引起 SERCA-PLB 结合或细胞质结构域结构的可检测变化，这表明其涉及 SERCA 跨膜结构域的结构变化。我们的结论是，Ca2+和 PLB 磷酸化通过不同的机制解除 SERCA-PLB 抑制，但两者主要通过 SERCA-PLB 复合物内的结构变化来实现，而不是通过该复合物的解离来实现。
