Phospholamban Inhibits the Cardiac Calcium Pump Through Reversing the Allosteric Enhancement of Calcium Affinity by ATP

Phospholamban (PLB) is a transmembrane micropeptide that regulates the Ca2+ pump SERCA in cardiac muscle, but the physical mechanism of this regulation remains poorly understood. PLB reduces the Ca2+ sensitivity of active SERCA, increasing the Ca2+ concentration required for pump cycling. However, PLB does not decrease Ca2+ binding to SERCA when ATP is absent, suggesting PLB does not inhibit SERCA Ca2+ affinity. The prevailing explanation for these seemingly conflicting results is that PLB slows the Ca2+-binding step in the SERCA enzymatic cycle, altering the Ca2+ dependence of cycling without affecting the true affinity of the Ca2+ binding sites. Here, we consider another hypothesis, that measurements of Ca2+ binding in the absence of ATP overlook important allosteric effects of nucleotide binding that increase SERCA Ca2+ binding affinity. We speculated that PLB inhibits SERCA by reversing this allostery. To test this, we used a fluorescent SERCA biosensor to quantify the Ca2+ affinity of non-cycling SERCA in the presence and absence of a non-hydrolyzable ATP-analog, AMPPCP. Nucleotide activation increased SERCA Ca2+ affinity, and this effect was reversed by co-expression of PLB. Interestingly, PLB had no effect on Ca2+ affinity in the absence of nucleotide. These results reconcile the previous conflicting observations from ATPase assays versus Ca2+ binding assays. Moreover, structural analysis of SERCA revealed a novel allosteric pathway connecting the ATP- and Ca2+-binding sites. We propose this pathway is interrupted by PLB binding. Thus, PLB reduces the true Ca2+ affinity of SERCA by reversing allosteric activation of the pump by ATP. #### Significance Statement Significance Statement The micropeptide phospholamban (PLB) serves a vital role as the “adrenaline trigger” for the heart. PLB inhibits the calcium pump SERCA at rest, and relief of this inhibition increases cardiac performance during exercise. Still, the physical mechanism for PLB inhibition of SERCA remains poorly understood. Here, our results reveal that PLB regulates SERCA by reversing an allosteric effect of ATP that enhances SERCA’s calcium affinity. We mapped a novel allosteric pathway that structurally couples SERCA’s ATP- and calcium-binding sites. PLB binds residues along this pathway to disrupt communication between the ligand-binding sites. The data reveal an allosteric mechanism that may be conserved in other ATP-dependent ion pumps. This allosteric pathway may provide new opportunities for pharmacological targeting of SERCA. ### Competing Interest Statement The authors have declared no competing interest.