Phospholamban (PLB) is a transmembrane micropeptide that regulates the sarcoplasmic reticulum Ca-ATPase (SERCA) in cardiac muscle, but the physical mechanism of this regulation remains poorly understood. PLB reduces the Ca sensitivity of active SERCA, increasing the Ca concentration required for pump cycling. However, PLB does not decrease Ca binding to SERCA when ATP is absent, suggesting PLB does not inhibit SERCA Ca affinity. The prevailing explanation for these seemingly conflicting results is that PLB slows transitions in the SERCA enzymatic cycle associated with Ca binding, altering transport Ca dependence without actually affecting the equilibrium binding affinity of the Ca-coordinating sites. Here, we consider another hypothesis, that measurements of Ca binding in the absence of ATP overlook important allosteric effects of nucleotide binding that increase SERCA Ca binding affinity. We speculated that PLB inhibits SERCA by reversing this allostery. To test this, we used a fluorescent SERCA biosensor to quantify the Ca affinity of non-cycling SERCA in the presence and absence of a non-hydrolyzable ATP-analog, AMPPCP. Nucleotide activation increased SERCA Ca affinity, and this effect was reversed by co-expression of PLB. Interestingly, PLB had no effect on Ca affinity in the absence of nucleotide. These results reconcile the previous conflicting observations from ATPase assays Ca binding assays. Moreover, structural analysis of SERCA revealed a novel allosteric pathway connecting the ATP- and Ca-binding sites. We propose this pathway is disrupted by PLB binding. Thus, PLB reduces the equilibrium Ca affinity of SERCA by interrupting allosteric activation of the pump by ATP.

中文翻译：

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Phospholamban 通过中断变构激活途径来抑制心脏钙泵


受磷蛋白 (PLB) 是一种跨膜微肽，可调节心肌中的肌浆网 Ca-ATP 酶 (SERCA)，但这种调节的物理机制仍知之甚少。 PLB 降低了活性 SERCA 的 Ca 敏感性，增加了泵循环所需的 Ca 浓度。然而，当 ATP 不存在时，PLB 不会减少 Ca 与 SERCA 的结合，表明 PLB 不会抑制 SERCA Ca 亲和力。对这些看似矛盾的结果的普遍解释是，PLB 减缓了与 Ca2+ 结合相关的 SERCA 酶循环的转变，改变了 Ca2+ 转运依赖性，但实际上并不影响 Ca2+ 配位位点的平衡结合亲和力。在这里，我们考虑另一个假设，即在没有 ATP 的情况下测量 Ca2+ 结合忽略了核苷酸结合的重要变构效应，这种效应增加了 SERCA Ca2+ 结合亲和力。我们推测 PLB 通过逆转这种变构来抑制 SERCA。为了测试这一点，我们使用荧光 SERCA 生物传感器来量化在存在和不存在不可水解 ATP 类似物 AMPPCP 的情况下非循环 SERCA 的 Ca 亲和力。核苷酸激活增加了 SERCA Ca 亲和力，并且这种效应通过 PLB 的共表达而逆转。有趣的是，在没有核苷酸的情况下，PLB 对 Ca 亲和力没有影响。这些结果与之前 ATP 酶测定 Ca 结合测定的相互矛盾的观察结果相一致。此外，SERCA 的结构分析揭示了连接 ATP 和 Ca 结合位点的新变构途径。我们认为该途径被 PLB 结合破坏。因此，PLB 通过中断 ATP 对泵的变构激活来降低 SERCA 的平衡 Ca 亲和力。