Phospholamban inhibits the cardiac calcium pump by interrupting an allosteric activation pathway.

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 versus 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.