Synthetic Null-Cysteine Phospholamban Analogue and the Corresponding Transmembrane Domain Inhibit the Ca-ATPase

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Phospholamban (PLN) regulates calcium translocation within cardiac myocytes by shifting sarco(endo)plasmic reticulum Ca 2؉ -ATPase (SERCA) affinity for calcium. Although the monomeric form of PLN (6 kDa) is the principal inhibitory species, recent evidence suggests that the PLN pentamer (30 kDa) also is able to bind SERCA. To date, several membrane architectures of the pentamer have been proposed, with different topological orientations for the cytoplasmic domain: (i) extended from the bilayer normal by 50 -60°; (ii) continuous ␣-helix tilted 28°relative to the bilayer normal; (iii) pinwheel geometry, with the cytoplasmic helix perpendicular to the bilayer normal and in contact with the surface of the bilayer; and (iv) bellflower structure, in which the cytoplasmic domain helix makes Ϸ20°angle with respect to the membrane bilayer normal. Using a variety of cell membrane mimicking systems (i.e., lipid vesicles, oriented lipid bilayers, and detergent micelles) and a combination of multidimensional solution/solidstate NMR and EPR spectroscopies, we tested the different structural models. We conclude that the pinwheel topology is the predominant conformation of pentameric PLN, with the cytoplasmic domain interacting with the membrane surface. We propose that the interaction with the bilayer precedes SERCA binding and may mediate the interactions with other proteins such as protein kinase A and protein phosphatase 1.Ca 2ϩ -ATPase ͉ EPR ͉ membrane protein ͉ solid-state NMR ͉ protein dynamics C alcium translocation into the sarcoplasmic reticulum of cardiac myocytes is controlled by the sarco(endo)plasmic reticulum Ca 2ϩ -ATPase (SERCA). Phospholamban (PLN) regulates the activity of SERCA by shifting the apparent calcium affinity for the enzyme. This activity is relieved by phosphorylation of PLN at Ser-16 and/or Thr-17 and high calcium concentration within the cytosol. Wild-type PLN (wt-PLN) forms stable homopentamers in lipid bilayers and in detergent micelles, where each monomer is composed of a helical cytoplasmic domain (residues 1-16), a semiflexible loop (residues 17-21), and a helical transmembrane domain (residues 22-52) (1, 2). Mutagenesis, molecular biology, and in vivo studies revealed that the PLN pentamer depolymerizes into active monomers that bind and inhibit SERCA (3). Similar conclusions were reached by in vitro fluorescence studies (4). Recently, Young and coworkers (5) have reported a cocrystal formed by SERCA and PLN pentamer, suggesting that the pentameric species also is able to bind SERCA. Furthermore, Jones and coworkers (6) hypothesized that the PLN pentamer may act as a chloride ion channel, which is supported by the bellflower structure recently determined by Oxenoid and Chou (2).There are four principal proposed structural models of pentameric wt-PLN, which differ primarily in the topology of the more dynamic cytoplasmic domain. In each of these models (shown in Fig. 1), residues 32-52 are in a coiled helix approximately parallel to the bilayer normal. The first model (extended helix/sheet ...

Section: Methodsmentioning

confidence: 79%

Spectroscopic validation of the pentameric structure of phospholamban

Traaseth¹,

Verardi

Torgersen

et al. 2007

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126

“…Toward these goals, we utilized three different PLN constructs with and without the R9C mutation: (i) synthetic peptides spanning cytoplasmic residues of PLN (PLN wt 1−20 or PLN R9C 1−20 ), (ii) full-length recombinant pentamers (PLN wt and PLN R9C ), and (iii) recombinant monomeric PLN (AFA-PLN), where the three transmembrane cysteines (Cys36, Cys41, Cys46) were mutated into Ala, Phe, Ala, respectively. This triple mutation abolishes PLN oligomerization without altering PLN's inhibitory function (27). We carried out these experiments in the presence of dithiothreitol (DTT ranging from 1 to 20 mM) or hydrogen peroxide (H 2 O 2 ranging from 1-100 μM), chemicals commonly used to mimic physiological redox conditions and oxidative stress (28,29).…”

Section: Resultsmentioning

confidence: 99%

Lethal Arg9Cys phospholamban mutation hinders Ca ²⁺ -ATPase regulation and phosphorylation by protein kinase A

Masterson

Hou

et al. 2011

103

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...

“…Most of the selectively labeled samples were also expressed in E. coli (Table S3). PLN samples that showed isotopic scrambling, [ 15 N-Ser] and [ 15 N-Gln], were synthesized by using Fmoc solid-phase peptide synthesis as reported (62,63). For solution NMR experiments, AFA-PLN was solubilized in 300 mM DPC, 120 mM NaCl, and 20 mM NaH2PO4 (pH 6.0).…”

Section: Methodsmentioning

confidence: 99%

Structure and topology of monomeric phospholamban in lipid membranes determined by a hybrid solution and solid-state NMR approach

Traaseth

Shi²,

Verardi

et al. 2009

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194

Phospholamban (PLN) is an essential regulator of cardiac muscle contractility. The homopentameric assembly of PLN is the reservoir for active monomers that, upon deoligomerization form 1:1 complexes with the sarco(endo)plasmic reticulum Ca 2؉ -ATPase (SERCA), thus modulating the rate of calcium uptake. In lipid bilayers and micelles, monomeric PLN exists in equilibrium between a bent (or resting) T state and a more dynamic (or active) R state. Here, we report the high-resolution structure and topology of the T state of a monomeric PLN mutant in lipid bilayers, using a hybrid of solution and solid-state NMR restraints together with molecular dynamics simulations in explicit lipid environments. Unlike the previous structural ensemble determined in micelles, this approach gives a complete picture of the PLN monomer structure in a lipid bilayer. This hybrid ensemble exemplifies the tilt, rotation, and depth of membrane insertion, revealing the interaction with the lipids for all protein domains. The N-terminal amphipathic helical domain Ia (residues 1-16) rests on the surface of the lipid membrane with the hydrophobic face of domain Ia embedded in the membrane bilayer interior. The helix comprised of domain Ib (residues 23-30) and transmembrane domain II (residues 31-52) traverses the bilayer with a tilt angle of Ϸ24°. The specific interactions between PLN and lipid membranes may represent an additional regulatory element of its inhibitory function. We propose this hybrid method for the simultaneous determination of structure and topology for membrane proteins with compact folds or proteins whose spatial arrangement is dictated by their specific interactions with lipid bilayers.hybrid method ͉ membrane proteins ͉ oriented solid-state NMR ͉ molecular modeling ͉ PISEMA S tructure and topology are central to membrane protein function (1). Recently determined high-resolution structures reveal the compact folds for several membrane proteins, such as electron and proton-conducting proteins involved in photosynthesis and respiration (http://blanco.biomol.uci.edu/ Membrane Proteins xtal.html). However, a significant population of membrane proteins does not possess a compact tertiary fold, but has its fold space defined through interactions of secondary structure elements (helices, turns, and loops) with the lipid membrane, i.e., the topology (1). This is the case for phospholamban (PLN), a mammalian protein that is essential in the regulation of cardiac muscle contractility (2), and that has recently become a major target for gene therapy to ameliorate cardiomyopathies (3, 4). PLN is located in the sarco(endo)plasmic reticulum (SR) of cardiac myocytes, inhibiting the SR Ca 2ϩ -ATPase (SERCA) by shifting its relative Ca 2ϩ affinity (5). In vitro and in vivo experiments have shown PLN to exist as a homopentamer that deoligomerizes into active monomers that bind SERCA in a 1:1 molar ratio (6). The monomeric form of PLN exists in equilibrium between a dynamically disordered R state and a more restricted T state (7,8) and has 3 ...