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

Phospholamban (PLB) is a transmembrane micropeptide that regulates the sarcoplasmic reticulum Ca²⁺-ATPase (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 transitions in the SERCA enzymatic cycle associated with Ca²⁺ binding, altering transport Ca²⁺ dependence without actually affecting the equilibrium binding affinity of the Ca²⁺-coordinating 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 disrupted by PLB binding. Thus, PLB reduces the equilibrium Ca²⁺ affinity of SERCA by interrupting allosteric activation of the pump by ATP.