Sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA) and phospholamban (PLB) are essential for intracellular Ca2+ transport in myocytes. Ca2+-dependent activation of SERCA-PLB provides a control function that regulates cytosolic and SR Ca2+ levels. Although experimental and computational studies alone have led to a greater insight into SERCA-PLB regulation, the structural mechanisms for Ca2+ binding reversing inhibition of the complex remain poorly understood. Therefore, we have performed atomistic simulations totaling 32.7 μs and cell-based intramolecular fluorescence resonance energy transfer (FRET) experiments to determine structural changes of PLB-bound SERCA in response to binding of a single Ca2+ ion. Complementary MD simulations and FRET experiments showed that open-to-closed transitions in the structure of the headpiece underlie PLB inhibition of SERCA, and binding of a single Ca2+ ion is sufficient to shift the protein population toward a structurally closed structure of the complex. Closure is accompanied by functional interactions between the N-domain β5-β6 loop and the A-domain and the displacement of the catalytic phosphorylation domain toward a competent structure. We propose that reversal of SERCA-PLB inhibition is achieved by stringing together its controlling modules (A-domain and loop Nβ5-β6) with catalytic elements (P-domain) to regulate function during intracellular Ca2+ signaling. We conclude that binding of a single Ca2+ is a critical mediator of allosteric signaling that dictates structural changes and motions that relieve SERCA inhibition by PLB. Understanding allosteric regulation is of paramount importance to guide therapeutic modulation of SERCA and other evolutionarily related ion-motive ATPases.

中文翻译：

---

动力学驱动的变构是 Ca2+ 介导的受磷蛋白 SERCA 抑制释放的基础

肌浆网 (SR) Ca2+-ATP 酶 (SERCA) 和受磷蛋白 (PLB) 对于肌细胞内 Ca2+ 转运至关重要。SERCA-PLB 的 Ca2+ 依赖性激活提供了调节胞质和 SR Ca2+ 水平的控制功能。尽管仅凭实验和计算研究就可以更深入地了解 SERCA-PLB 调节，但对 Ca2+ 结合逆转复合物抑制的结构机制仍知之甚少。因此，我们进行了总计 32.7 μs 的原子模拟和基于细胞的分子内荧光共振能量转移 (FRET) 实验，以确定 PLB 结合的 SERCA 响应单个 Ca2+ 离子结合的结构变化。互补的 MD 模拟和 FRET 实验表明，头件结构中的开放到封闭转变是 PLB 对 SERCA 抑制的基础，单个 Ca2+ 离子的结合足以将蛋白质群转变为复合物的结构封闭结构。闭合伴随着 N 结构域 β5-β6 环和 A 结构域之间的功能相互作用以及催化磷酸化结构域向主管结构的移位。我们提出，SERCA-PLB 抑制的逆转是通过将其控制模块（A 结构域和环 Nβ5-β6）与催化元件（P 结构域）串在一起来调节细胞内 Ca2+ 信号传导过程中的功能来实现的。我们得出的结论是，单个 Ca2+ 的结合是变构信号传导的关键介质，它决定了减轻 PLB 对 SERCA 抑制的结构变化和运动。了解变构调节对于指导 SERCA 和其他进化相关的离子动力 ATP 酶的治疗调节至关重要。