Phospholamban (PLN) and sarcolipin (SLN) are two single-pass membrane proteins that regulate Ca2+-ATPase (SERCA), an ATP-driven pump that translocates calcium ions into the lumen of the sarcoplasmic reticulum, initiating muscle relaxation. Both proteins bind SERCA through intramembrane interactions, impeding calcium translocation. While phosphorylation of PLN at Ser-16 and/or Thr-17 reestablishes calcium flux, the regulatory mechanism of SLN remains elusive. SERCA has been crystallized in several different states along the enzymatic reaction coordinates, providing remarkable mechanistic information; however, the lack of high-resolution crystals in the presence of PLN and SLN limits the current understanding of the regulatory mechanism. This brief review offers a survey of our hybrid structural approach using solution and solid-state NMR methodologies to understand SERCA regulation from the point of view of PLN and SLN. These results have improved our understanding of the calcium translocation process and are the basis for designing new therapeutic approaches to ameliorate muscle malfunctions.

The regulatory interaction of phospholamban (PLN) with Ca 2þ -ATPase controls the uptake of calcium into the sarcoplasmic reticulum, modulating heart muscle contractility. A missense mutation in PLN cytoplasmic domain (R9C) triggers dilated cardiomyopathy in humans, leading to premature death. Using a combination of biochemical and biophysical techniques both in vitro and in live cells, we show that the R9C mutation increases the stability of the PLN pentameric assembly via disulfide bridge formation, preventing its binding to Ca 2þ -ATPase as well as phosphorylation by protein kinase A. These effects are enhanced under oxidizing conditions, suggesting that oxidative stress may exacerbate the cardiotoxic effects of the PLN R9C mutant. These results reveal a regulatory role of the PLN pentamer in calcium homeostasis, going beyond the previously hypothesized role of passive storage for active monomers.SERCA | ventricular dilatation | calcium regulation | heart failure | membrane proteins H eart failure (HF) is the leading cause of morbidity and mortality worldwide (1, 2). The most prominent disorder leading to HF is dilated cardiomyopathy (DCM), a disease characterized by left ventricular dilatation and impaired systolic function (1, 2). DCM has both acquired and genetic etiologies (1, 2). Recent genome sequencing has revealed a high incidence of DCM-associated mutations in cytoskeletal, nuclear, as well as sarcomeric proteins (3). A number of mutations have been indentified in calcium handling proteins, which play a central role in the mechanics of heart muscle contractility (3-6).Cardiac muscle contraction (systole) begins when an action potential causes membrane depolarization, activating the sarcolemmal L-type calcium (Ca 2þ ) channels. Ca 2þ flows through the L-type Ca 2þ -channels into the cytosol. This increase in Ca 2þ concentration induces a large-scale release of Ca 2þ into the cytosol from intracellular stores by the sarcoplasmic reticulum (SR) Ca 2þ -release channels (or ryanodine receptors). Ca 2þ then moves toward the contractile apparatus, where it binds the troponin complex and initiates contraction. Muscle relaxation (diastole) occurs when Ca 2þ is sequestered into the SR by the SR Ca 2þ -ATPase (SERCA) (7) a membrane-embedded Ca 2þ pump (8). SERCA is regulated by phospholamban (PLN), which reduces its apparent Ca 2þ affinity (9, 10). PLN's inhibition is reversed by cAMP-dependent protein kinase A (PKA), which phosphorylates PLN at Ser16, enhancing cardiac contractility and reestablishing Ca 2þ flux (11).PLN is a single-pass membrane protein, which comprises three structural domains (12-14), further subdivided into four dynamic domains [cytoplasm: domain Ia (residues 1-16), loop (residues 17-22), domain Ib (residues 23-30); transmembrane: domain II (residues 31-52)] (15) (Fig. S1). In membranes, PLN forms homopentamers arranged in a pinwheel topology that are in equilibrium with monomers (16, 17) that bind SERCA with 1∶1 stoichiometry (6, 18-21). Also, it has been proposed that the PLN monomer-pen...

Cardiac contraction and relaxation are regulated by conformational transitions of protein complexes that are responsible for calcium trafficking through cell membranes. Central to the muscle relaxation phase is a dynamic membrane protein complex formed by Ca 2؉ -ATPase (SERCA) and phospholamban (PLN), which in humans is responsible for ϳ70% of the calcium re-uptake in the sarcoplasmic reticulum. Dysfunction in this regulatory mechanism causes severe pathophysiologies. In this report, we used a combination of nuclear magnetic resonance, electron paramagnetic resonance, and coupled enzyme assays to investigate how single mutations at position 21 of PLN affects its structural dynamics and, in turn, its interaction with SERCA. We found that it is possible to control the activity of SERCA by tuning PLN structural dynamics. Both increased rigidity and mobility of the PLN backbone cause a reduction of SERCA inhibition, affecting calcium transport. Although the more rigid, loss-offunction (LOF) mutants have lower binding affinities for SERCA, the more dynamic LOF mutants have binding affinities similar to that of PLN. Here, we demonstrate that it is possible to harness this knowledge to design new LOF mutants with activity similar to S16E (a mutant already used in gene therapy) for possible application in recombinant gene therapy. As proof of concept, we show a new mutant of PLN, P21G, with improved LOF characteristics in vitro.

Phospholamban (PLN) is the endogenous inhibitor of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), the integral membrane enzyme responsible for 70% of the Ca2+ shuttling into the SR, inducing cardiac muscle relaxation in humans. Dysfunctions in SERCA:PLN interactions have been implicated as having a critical role in cardiac disease, and targeting Ca2+ transport has been demonstrated to be a promising avenue in treating conditions of heart failure. Here, we designed a series of new mutants able to tune SERCA function, targeting the loop sequence that connects the transmembrane and cytoplasmic helices of PLN. We found that a variable degree of loss of inhibition mutants is attainable by engineering glycine mutations along PLN’s loop domain. Remarkably, a double glycine mutation results in a complete loss-of-function mutant, fully mimicking the phosphorylated state of PLN. Using nuclear magnetic resonance (NMR) spectroscopy, we rationalized the effects of these mutations in terms of entropic control on PLN function, whose inhibitory function can be modulated by increasing its conformational dynamics. However, if PLN mutations go past a threshold set by the phosphorylated state, they break the structural coupling between the transmembrane and cytoplasmic domains, resulting in a species that behaves as the inhibitory transmembrane domain alone. These studies provide new potential candidates for adenovirus gene therapy to reverse the effects of heart failure.

Approximately, 70% of the Ca ion transport into the sarcoplasmic reticulum is catalyzed by the sarcoplasmic reticulum Ca-ATPase (SERCA), whose activity is endogenously regulated by phospholamban (PLN). PLN comprises a TM inhibitory region and a cytoplasmic regulatory region that harbors a consensus sequence for cAMP-dependent protein kinase (PKA). The inhibitory region binds the ATPase, reducing its apparent Ca binding affinity. β-adrenergic stimulation activates PKA, which phosphorylates PLN at Ser 16, reversing its inhibitory function. Mutations and post-translational modifications of PLN may lead to dilated cardiomyopathy (DCM) and heart failure. PLN's cytoplasmic region interconverts between a membrane-associated T state and a membrane-detached R state. The importance of these structural transitions on SERCA regulation is emerging, but the effects of natural occurring mutations and their relevance to the progression of heart disease are unclear. Here we use solid-state NMR spectroscopy to investigate the structural dynamics of two lethal PLN mutations, R9C and R25C, which lead to DCM. We found that the R25C mutant enhances the dynamics of PLN and shifts the conformational equilibrium toward the R state confirmation, whereas the R9C mutant drives the amphipathic cytoplasmic domain toward the membrane-associate state, enriching the T state population. The changes in membrane interactions caused by these mutations may explain the aberrant regulation of SERCA.

Phospholamban (PLN) is a single-pass membrane protein that regulates the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA). Phosphorylation of PLN at Ser16 reverses its inhibitory function under β-adrenergic stimulation, augmenting Ca2+ uptake in the sarcoplasmic reticulum and muscle contractility. PLN exists in two conformations; a T state, where the cytoplasmic domain is helical and absorbed on the membrane surface, and an R state, where the cytoplasmic domain is unfolded and membrane detached. Previous studies from our group have shown that the PLN conformational equilibrium is crucial to SERCA regulation. Here, we used a combination of solution and solid-state NMR techniques to compare the structural topology and conformational dynamics of monomeric PLN (PLNAFA) with that of the PLNR14del, a naturally occurring deletion mutant that is linked to the progression of dilated cardiomyopathy. We found that the behavior of the inhibitory transmembrane domain of PLNR14del is similar to that of the native sequence. In contrast, the conformational dynamics of R14del both in micelles and lipid membranes are enhanced. We conclude that the deletion of Arg14 in the cytoplasmic region weakens the interactions with the membrane and shifts the conformational equilibrium of PLN toward the disordered R state. This conformational transition is correlated with the loss-of-function character of this mutant and is corroborated by SERCA’s activity assays. These findings further support our hypothesis that SERCA function is fine-tuned by PLN conformational dynamics and begin to explain the aberrant regulation of SERCA by the R14del mutant.

Extensive x-ray crystallographic studies carried out on the catalytic-subunit of protein kinase A (PKAc) enabled the atomic characterization of inhibitor and/or substrate peptide analogs trapped at its active site. Yet, the structural and dynamic transitions of these peptides from the free to the bound state are missing. These conformational transitions are central to understanding molecular recognition and enzymatic cycle. NMR allows one to study these phenomena under functionally relevant conditions. However, the amounts of isotopically labeled peptides required for this technique present prohibitive costs for peptide synthesis. To enable NMR studies, we have optimized both expression and purification of isotopically enriched substrate/inhibitor peptides using a recombinant fusion protein system. Three of these peptides corresponded to the cytoplasmic regions of the wildtype and lethal mutants of the membrane protein phospholamban, while the fourth peptide corresponded to the binding epitope of the heat-stable protein kinase inhibitor ). The target peptides were fused to the maltose binding protein (MBP), which is further purified using a His 6 tag approach. This convenient protocol allows for the purification of milligram amounts of peptides necessary for NMR analysis.

This chapter reviews the molecular biology, biochemical, and NMR methods that we used to study the structural dynamics, membrane topology and interaction of phospholamban (PLN), a small regulatory membrane protein involved in the regulation of the sarcoplasmic reticulum Ca-ATPase (SERCA). In particular, we show the progression of our research from the initial hypotheses toward understanding the molecular mechanisms of SERCA's regulation, including the effects of PLN oligomerization and posttranslational phosphorylation. Finally, we show how the knowledge of the molecular mechanism of the structural dynamics and topology of free and bound proteins can lead to the rational design of PLN analogs for possible use in gene therapy.