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Mechanisms for cardiac calcium pump activation by its substrate and a synthetic allosteric modulator using fluorescence lifetime imaging.

Jaroslava ŠeflováCarlos Cruz-CortésGuadalupe Guerrero-SernaSeth L RobiaL Michel Espinoza-Fonseca
Published in: PNAS nexus (2023)
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 Ca 2+ -ATPase, a pharmacologically important pump that transports Ca 2+ at the expense of adenosine triphosphate (ATP) hydrolysis. We first tested whether the dissociation of sarcoplasmic reticulum Ca 2+ -ATPase from its regulatory protein phospholamban is required for small-molecule activation. We found that CDN1163, a validated sarcoplasmic reticulum Ca 2+ -ATPase activator, does not have significant effects on the stability of the sarcoplasmic reticulum Ca 2+ -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 Ca 2+ -ATPase, increasing the fraction of catalytically competent structures at physiologically relevant Ca 2+ concentrations. Unlike ATP, CDN1163 alone has no significant effects on the Ca 2+ -dependent shifts in the structural populations of sarcoplasmic reticulum Ca 2+ -ATPase, and it does not increase the pump's affinity for Ca 2+ ions. However, we found that CDN1163 enhances the ATP-mediated modulatory effects to increase the population of catalytically competent sarcoplasmic reticulum Ca 2+ -ATPase structures. Importantly, this structural shift occurs within the physiological window of Ca 2+ concentrations at which sarcoplasmic reticulum Ca 2+ -ATPase operates. We demonstrated that ATP is both a substrate and modulator of sarcoplasmic reticulum Ca 2+ -ATPase and showed that CDN1163 and ATP act synergistically to populate sarcoplasmic reticulum Ca 2+ -ATPase structures that are primed for phosphorylation. This study provides novel insights into the structural mechanisms for sarcoplasmic reticulum Ca 2+ -ATPase activation by its substrate and a synthetic allosteric modulator.
Keyphrases
  • small molecule
  • protein kinase
  • high resolution
  • heart failure
  • protein protein
  • quantum dots
  • inflammatory response
  • atrial fibrillation
  • nuclear factor
  • fluorescence imaging