1. Phospholamban inhibits the cardiac calcium pump by interrupting an allosteric activation pathway.
- Author
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Cleary SR, Seflova J, Cho EE, Bisht K, Khandelia H, Espinoza-Fonseca LM, and Robia SL
- Subjects
- Animals, Humans, Adenosine Triphosphate metabolism, Allosteric Regulation, Myocardium metabolism, Dogs, HEK293 Cells, Models, Molecular, Protein Structure, Tertiary, Calcium metabolism, Calcium-Binding Proteins metabolism, Calcium-Binding Proteins chemistry, Sarcoplasmic Reticulum Calcium-Transporting ATPases metabolism, Sarcoplasmic Reticulum Calcium-Transporting ATPases chemistry
- Abstract
Phospholamban (PLB) is a transmembrane micropeptide that regulates the sarcoplasmic reticulum Ca
2+ -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., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)- Published
- 2024
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