Biology subjects

Cleary, S. R.

Publications and source records attributed to Cleary, S. R..

4 recordsLinked to original sources

bioRxiv2023

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. O_TEXTBOXSignificance StatementThe 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 SERCAs calcium affinity. We mapped a novel allosteric pathway that structurally couples SERCAs 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. C_TEXTBOX

biochemistry↗

bioRxiv2023

Dilated cardiomyopathy variant R14del increases phospholamban pentamer stability, blunting dynamic regulation of cardiac calcium handling

The sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) is a membrane transporter that creates and maintains intracellular Ca2+ stores. In the heart, SERCA is regulated by an inhibitory interaction with the monomeric form of the transmembrane micropeptide phospholamban (PLB). PLB also forms avid homo-pentamers, and dynamic exchange of PLB between pentamers and the regulatory complex with SERCA is an important determinant of cardiac responsiveness to exercise. Here, we investigated two naturally occurring pathogenic mutations of PLB, a cysteine substitution of arginine 9 (R9C) and an in-frame deletion of arginine 14 (R14del). Both mutations are associated with dilated cardiomyopathy. We previously showed that the R9C mutation causes disulfide crosslinking and hyperstabilization of pentamers. While the pathogenic mechanism of R14del is unclear, we hypothesized that this mutation may also alter PLB homo-oligomerization and disrupt the PLB-SERCA regulatory interaction. SDS-PAGE revealed a significantly increased pentamer:monomer ratio for R14del-PLB when compared to WT-PLB. In addition, we quantified homo-oligomerization and SERCA-binding in live cells using fluorescence resonance energy transfer (FRET) microscopy. R14del-PLB showed an increased affinity for homo-oligomerization and decreased binding affinity for SERCA compared to WT, suggesting that, like R9C, the R14del mutation stabilizes PLB in its pentameric form, decreasing its ability to regulate SERCA. Moreover, the R14del mutation reduces the rate of PLB unbinding from the pentamer after a transient Ca2+ elevation, limiting the rate of re-binding to SERCA. A computational model predicted that hyperstabilization of PLB pentamers by R14del impairs the ability of cardiac Ca2+ handling to respond to changing heart rates between rest and exercise. We postulate that impaired responsiveness to physiological stress contributes to arrhythmogenesis in human carriers of the R14del mutation.

physiology↗

bioRxiv2021

STOICHIOMETRY OF THE SODIUM PUMP-PHOSPHOLEMMAN REGULATORY COMPLEX

The sodium-potassium ATPase (NKA) establishes ion gradients that facilitate many physiological processes. In the heart, NKA activity is regulated by its interaction with phospholemman (PLM, FXYD1). Here we used a novel fluorescence lifetime-based assay to investigate the structure, stoichiometry, and affinity of the NKA-PLM regulatory complex. We observed concentration dependent association of the subunits of NKA-PLM regulatory complex, with avid association of the alpha subunit with the essential beta subunit followed by lower affinity alpha-alpha and alpha-PLM interactions. The data provide the first evidence that the regulatory complex is composed of two alpha subunits associated with two beta subunits, decorated with two PLM regulatory subunits in intact cells. Docking and molecular dynamics simulations generated a structural model of the complex that is consistent with our experimental observations. We propose that alpha-alpha subunit interactions support conformational coupling of the catalytic subunits, which may enhance NKA turnover rate. These observations provide insight into the pathophysiology of heart failure, wherein low NKA expression may be insufficient to support formation of the complete regulatory complex with stoichiometry (alpha-beta-PLM)2.

biophysics↗

bioRxiv2021

Inhibitory and Stimulatory Micropeptides Preferentially Bind to Different Conformations of the Cardiac Calcium Pump

The ATP-dependent ion pump SERCA sequesters Ca2+ in the endoplasmic reticulum to establish a reservoir for cell signaling. Because of its central importance in physiology, this transporter is tightly controlled by physical interactions with tissue-specific regulatory micropeptides that tune SERCA function to match changing physiological conditions. In the heart, phospholamban (PLB) inhibits SERCA, while dwarf open reading frame (DWORF) stimulates SERCA. These competing interactions determine cardiac performance by modulating the amplitude of Ca2+ signals that drive the contraction/relaxation cycle. The distinct functions of these peptides may relate to their reciprocal preferences for SERCA binding. While SERCA binds PLB more avidly at low cytoplasmic Ca2+, it binds DWORF better at high Ca2+. In the present study, we determined that this opposing Ca2+ sensitivity is due to preferential binding of DWORF and PLB to different intermediate conformations that the pump samples during the Ca2+ transport cycle. The results suggest a mechanistic basis for inhibitory and stimulatory micropeptide function. In addition, fluorescence resonance energy transfer (FRET) measurements revealed dynamic shifts in SERCA-micropeptide binding equilibria during cellular Ca2+ elevations. The data suggest Ca2+-dependent dynamic exchange of inhibitory and stimulatory micropeptides from SERCA during the cardiac cycle. Together, these mechanisms provide beat-to-beat modulation of cardiac Ca2+ handling and contribute to the hearts adaptation to the increased physiological demands of exercise. O_TEXTBOXSignificance Interactions between SERCA and its regulatory micropeptides modulate cardiac performance. A previous study demonstrated that while the SERCA inhibitor, phospholamban, loses affinity during intracellular Ca2+ elevations, its competitor DWORF stimulates SERCA and binds better at elevated Ca2+. Here, we found this Ca2+-dependent difference in affinity is driven by preferential micropeptide binding to different SERCA conformations sampled during its enzymatic cycle. Tight binding to different conformations of the pump may underly the distinct mechanistic functions of these regulators. Lower affinity during alternating phases of the cardiac cycle may drive dynamic exchange of inhibitory and stimulatory micropeptides during contraction and relaxation. Our results reveal mechanisms that modulate cardiac Ca2+ handling on a beat-to-beat basis and help the heart adapt to exercise. C_TEXTBOX

biophysics↗