Allosteric regulation of SERCA by phosphorylation-mediated conformational shift of phospholamban

Abstract

The membrane protein complex between the sarcoplasmic reticulum Ca 2+ -ATPase (SERCA) and phospholamban (PLN) controls Ca 2+ transport in cardiomyocytes, thereby modulating cardiac contractility. β-Adrenergic-stimulated phosphorylation of PLN at Ser-16 enhances SERCA activity via an unknown mechanism. Using solid-state nuclear magnetic resonance spectroscopy, we mapped the physical interactions between SERCA and both unphosphorylated and phosphorylated PLN in membrane bilayers. We found that the allosteric regulation of SERCA depends on the conformational equilibrium of PLN, whose cytoplasmic regulatory domain interconverts between three different states: a ground T state (helical and membrane associated), an excited R state (unfolded and membrane detached), and a B state (extended and enzymebound), which is noninhibitory. Phosphorylation at Ser-16 of PLN shifts the populations toward the B state, increasing SERCA activity. We conclude that PLN's conformational equilibrium is central to maintain SERCA's apparent Ca 2+ affinity within a physiological window. This model represents a paradigm shift in our understanding of SERCA regulation by posttranslational phosphorylation and suggests strategies for designing innovative therapeutic approaches to enhance cardiac muscle contractility. -ATPase (SERCA)/phospholamban (PLN) complex regulates Ca 2+ translocation into the sarcoplasmic reticulum (SR) of cardiomyocytes and constitutes the main mechanism of cardiac relaxation (diastole) (1-3). SERCA is a P-type ATPase that translocates two Ca 2+ ions per ATP molecule hydrolyzed in exchange for three H + ( Fig. 1) (4, 5). PLN binds and allosterically inhibits SERCA function, decreasing its apparent affinity for Ca 2+ ions (3, 6). On β-adrenergic stimulation, cAMP-dependent protein kinase A phosphorylates PLN at Ser-16, reversing the inhibition and augmenting cardiac output (3). Disruptions in this regulatory mechanism degenerate into Ca 2+ mishandling and heart failure (3). Several X-ray structures of SERCA have been determined along its enzymatic coordinates, providing atomic details on the structural transitions in the absence of PLN (4, 5). The first image of the SERCA/PLN complex resulted from cryo-EM studies (7), but the low-resolution data prevent an atomic view of PLN structure and architecture within the complex. In addition, mutagenesis and cross-linking data were used to model the complex, suggesting that the inhibitory transmembrane (TM) region of PLN is positioned into a binding groove far from the putative Ca 2+ entry, as well as the ATP binding site, and located between TM helices M2, M4, M6, and M9 of SERCA. The location of PLN's TM domain agrees with a recent crystal structure of the SERCA/PLN complex (8) and is remarkably similar to the one recently identified for a PLN homolog, sarcolipin, in complex with SERCA (9, 10) (Fig. 1).In the SERCA/PLN model, which was further refined using NMR constraints (11), the loop bridging the TM and cytoplasmic domain of PLN adopts an unfolded configuration, stretching tow...

Mitogen‐Activated Protein Kinase and Intracellular Polyamine Signaling Is Involved in TRPV1 Activation–Induced Cardiac Hypertrophy

“…It has been reported that PLN inhibits Serca2a activity by decreasing the apparent affinity of the Serca2a for Ca 2+ , and this inhibition is relieved by phosphorylation of PLN at serine 16 or threonine 17 41. This suggests that the activity of Serca2a in TAC‐induced cardiac hypertrophy is inhibited because of decreased phosphorylation of PLN, resulting in a decrease in the rate of relaxation of cardiac muscle and a positive inotropic effect.…”

Tead1 is required for maintaining adult cardiomyocyte function, and its loss results in lethal dilated cardiomyopathy

“…SERCA2a activity is allosterically inhibited by Pln. Phosphorylation of Pln at Ser16/Thr17 sites -by PKA and CaMK, respectively -relieves this inhibition and enhances SERCA2a activity (31,32). Hence, we next measured the phosphorylation status of Pln in Tead1-deleted hearts.…”

Acute Inotropic and Lusitropic Effects of Cardiomyopathic R9C Mutation of Phospholamban

“…The R9C-dependent structural transition and the phosphorylation-dependent conformational change are similar in direction and magnitude (ϩ1.2 and ϩ4 Å, respectively). A regulatory complex structural change is the primary mechanism for relief of inhibition of SERCA by PLB phosphorylation (50,51). How the R9C mutation may imitate phosphorylation of PLB is not clear, but we speculate that there may be modification of the Cys at position 9 to a negatively charged species that resembles phosphorylated residues of neighboring PKA/CaMKII sites on the PLB.…”