Kim N. Ha

Controlling the inhibition of the sarcoplasmic Ca2+-ATPase by tuning phospholamban structural dynamics.

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 Ca2+-ATPase (SERCA) and phospholamban (PLN), which in humans is responsible for ∼70% 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-of-function (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.

Lethal Arg9Cys phospholamban mutation hinders Ca2+-ATPase regulation and phosphorylation by protein kinase A

The regulatory interaction of phospholamban (PLN) with Ca2+-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 Ca2+-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 PLNR9C 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.

Expression and purification of isotopically labeled peptide inhibitors and substrates of cAMP-dependant protein kinase A for NMR analysis.

Extensive X-ray crystallographic studies carried out on the catalytic-subunit of protein kinase A (PKA-C) enabled the atomic characterization of inhibitor and/or substrate peptide analogues 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 the enzymatic cycle. NMR spectroscopy 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 solid-phase 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 correspond to the cytoplasmic regions of the wild-type and lethal mutants of the membrane protein phospholamban, while the fourth peptide correspond to the binding epitope of the heat-stable protein kinase inhibitor (PKI(5-24)). 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.

What can we learn from a small regulatory membrane protein

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 SERCAs 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.

Effects of naturally occurring arginine 14 deletion on phospholamban conformational dynamics and membrane interactions

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 adsorbed on the membrane surface, and an R state, where the cytoplasmic domain is unfolded and membrane detached. Previous studies have shown that the PLN conformational equilibrium is crucial to SERCA regulation. Here, we used a combination of solution and solid-state NMR to compare the structural topology and conformational dynamics of monomeric PLN (PLN(AFA)) with that of the PLN(R14del), 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 PLN(R14del) is similar to that of the native sequence. Conversely, 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 SERCAs activity assays. These findings 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.

Effects of the Arg9Cys and Arg25Cys mutations on phospholamban's conformational equilibrium in membrane bilayers

Approximately, 70% of the Ca2+ ion transport into the sarcoplasmic reticulum is catalyzed by the sarcoplasmic reticulum Ca2+-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 Ca2+ 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. PLNs 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.

Tuning the Structural Coupling Between the Transmembrane and Cytoplasmic Domains of Phospholamban to Control SERCA Inhibition

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. Based on the success of the Chien Group with S16E PLN, a pseudo-phosphorylated PLN species which halted the progression of heart failure in animal models, we wish to further develop PLN species to improve SERCA function by rationally designing mutations based on the structural and biophysical data available on the system. The present study seeks to introduce single and multiple glycine mutations in the loop domain of PLN in order to tune the structural coupling between the cytoplasmic and transmembrane helix, and ascertain the effects on SERCA function. These studies further develop the model by which the control of enzyme function is performed by altering the structural dynamics of a small inhibitor can then be translated to other membrane enzymes, such as the Na/K-ATPase.