Activation mechanism of the cardiac calcium pump by a small-molecule allosteric modulator

ABSTRACT The discovery of small-molecule 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 (TCSPC) imaging approach to investigate the activation mechanism of a druggable protein by a small-molecule allosteric modulator. We tested this approach using the cardiac sarcoplasmic reticulum Ca 2+ -ATPase (SERCA2a), an important pharmacological target that transports Ca 2+ at the expense of ATP hydrolysis in the heart. We found that CDN1163, a validated SERCA2a activator, does not dissociate the endogenous complex between SERCA2a and its regulator phospholamban (PLN) in the presence of either Ca 2+ or AMP-PCP, a non-hydrolyzable ATP analog. CDN1163 does not influence SERCA2a’s affinity for Ca 2+ ions at functionally relevant conditions. Global analysis of the fluorescence lifetimes showed that ATP is both a substrate and a modulator that populates competent SERCA2a conformations. Interestingly, CDN1163 alone does not significantly induce changes in the structural populations of SERCA2a. Instead, CDN1163 potentiates the effects of ATP to further shift the equilibrium toward a competent SERCA2a conformation. Importantly, this population shift occurs at sub-physiological conditions, and within physiological Ca 2+ concentrations at which SERCA2a operates. We propose an activation mechanism whereby a small-molecule modulator synergizes with ATP to stabilize a conformation of SERCA2a primed for activation. This study demonstrates the power of TCSPC to reveal novel insights into how structural and biochemical states are coupled to allosterically activate a pharmacological target in the heart.