Mechanisms for cardiac calcium pump activation by its substrate and a synthetic allosteric modulator using fluorescence lifetime imaging.
Mechanisms for cardiac calcium pump activation by its substrate and a synthetic allosteric modulator using fluorescence lifetime imaging.
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DOI：
10.1093/pnasnexus/pgad453
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发表时间：
2024-01
期刊：
影响因子：
--
通讯作者：
Espinoza-Fonseca, L. Michel
中科院分区：
文献类型：
--
作者：
Seflova, Jaroslava;Cruz-Cortes, Carlos;Guerrero-Serna, Guadalupe;Robia, Seth L.;Espinoza-Fonseca, L. Michel
变构调节剂的发现是药物发现中的新兴范例，并且信号转导是具有挑战性的微妙和动态过程。我们开发了一种时间相关的单光子计数成像方法来研究心肌肌浆网Ca 2 +-ATPase的小分子激活的结构机制，Ca 2 +-ATPase是一种非常重要的泵，以三磷酸腺苷（ATP）水解为代价转运Ca 2+。我们首先测试了肌浆网Ca 2 +-ATP酶从其调节蛋白受磷蛋白的解离是否是小分子激活所必需的。我们发现，CDN 1163，一种有效的肌浆网Ca 2 +-ATP酶激活剂，对肌浆网Ca 2 +-ATP酶-受磷蛋白复合物的稳定性没有显著影响。使用不可水解的ATP类似物β，γ-亚甲基腺苷5′-三磷酸（AMP-PCP）的时间相关单光子计数成像实验表明，ATP是肌浆网Ca 2 +-ATP酶的变构调节剂，在生理相关的Ca 2+浓度下增加催化活性结构的比例。与ATP不同，单独的CDN 1163对肌浆网Ca 2 +-ATP酶结构群体中的Ca 2+依赖性变化没有显著影响，并且它不增加泵对Ca 2+离子的亲和力。然而，我们发现，CDN 1163增强ATP介导的调节作用，以增加具有催化活性的肌浆网Ca 2 +-ATP酶结构的数量。重要的是，这种结构转变发生在肌浆网Ca 2 +-ATP酶工作的Ca 2+浓度的生理窗口内。我们证明了ATP既是肌浆网Ca 2 +-ATP酶的底物又是其调节剂，并且表明CDN 1163和ATP协同作用以填充为磷酸化准备的肌浆网Ca 2 +-ATP酶结构。本研究提供了新的见解的结构机制，肌浆网Ca 2 +-ATP酶激活其底物和合成的变构调节剂。
The discovery of allosteric modulators is an emerging paradigm in drug discovery, and signal transduction is a subtle and dynamic process that is challenging to characterize. We developed a time-correlated single photon-counting imaging approach to investigate the structural mechanisms for small-molecule activation of the cardiac sarcoplasmic reticulum Ca2+-ATPase, a pharmacologically important pump that transports Ca2+ at the expense of adenosine triphosphate (ATP) hydrolysis. We first tested whether the dissociation of sarcoplasmic reticulum Ca2+-ATPase from its regulatory protein phospholamban is required for small-molecule activation. We found that CDN1163, a validated sarcoplasmic reticulum Ca2+-ATPase activator, does not have significant effects on the stability of the sarcoplasmic reticulum Ca2+-ATPase–phospholamban complex. Time-correlated single photon-counting imaging experiments using the nonhydrolyzable ATP analog β,γ-Methyleneadenosine 5′-triphosphate (AMP-PCP) showed ATP is an allosteric modulator of sarcoplasmic reticulum Ca2+-ATPase, increasing the fraction of catalytically competent structures at physiologically relevant Ca2+ concentrations. Unlike ATP, CDN1163 alone has no significant effects on the Ca2+-dependent shifts in the structural populations of sarcoplasmic reticulum Ca2+-ATPase, and it does not increase the pump's affinity for Ca2+ ions. However, we found that CDN1163 enhances the ATP-mediated modulatory effects to increase the population of catalytically competent sarcoplasmic reticulum Ca2+-ATPase structures. Importantly, this structural shift occurs within the physiological window of Ca2+ concentrations at which sarcoplasmic reticulum Ca2+-ATPase operates. We demonstrated that ATP is both a substrate and modulator of sarcoplasmic reticulum Ca2+-ATPase and showed that CDN1163 and ATP act synergistically to populate sarcoplasmic reticulum Ca2+-ATPase structures that are primed for phosphorylation. This study provides novel insights into the structural mechanisms for sarcoplasmic reticulum Ca2+-ATPase activation by its substrate and a synthetic allosteric modulator.
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