The hydrophilic domain of phospholamban inhibits the Ca2+ transport step of the Ca(2+)-ATPase.
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ABSTRACT:
SUBMITTER: Hughes G
PROVIDER: S-EPMC1137357 | biostudies-other | 1994 Oct
REPOSITORIES: biostudies-other
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Project description:The inhibitory interaction of phospholamban (PLN) with the sarco(endo)plasmic reticulum Ca(2+) ATPase isoform 1 (SERCA1a) was modeled on the basis of several constraints which included (i) spontaneous formation of SS-bridges between mutants L321C in transmembrane helix 4 (M4) of SERCA1a and N27C in PLN and between V89C (M4) and V49C (PLN); (ii) definition of the face of the PLN transmembrane helix that interacts with SERCA; (iii) cross-linking between Lys-3 of PLN and Lys-397 and Lys-400 of SERCA2a. The crystal structure of SERCA1a in the absence of Ca(2+), which binds PLN, was used as the structure into which an atomic model of PLN was built. PLN can fit into a transmembrane groove formed by the juxtaposition of M2, the upper part of M4, M6, and M9. In the SERCA1a structure with bound Ca(2+), this groove is closed, accounting for the ability of Ca(2+) to disrupt PLN-SERCA interactions. Near the cytoplasmic surface of the bilayer, the PLN helix is disrupted to prevent its collision with M4. The model can be extended into the cytoplasmic domain so that Lys-3 in PLN can be cross-linked with Lys-397 and Lys-400 in SERCA1a with little unwinding of the N-terminal helix of PLN.
| S-EPMC141018 | biostudies-literature
Project description:Sarcoplasmic reticulum Ca2+-ATPase (SERCA) and phospholamban (PLB) are essential components of the cardiac Ca2+ transport machinery. PLB phosphorylation at residue Ser16 (pSer16) enhances SERCA activity in the heart via an unknown structural mechanism. Here, we report a fully atomistic model of SERCA bound to phosphorylated PLB and study its structural dynamics on the microsecond time scale using all-atom molecular dynamics simulations in an explicit lipid bilayer and water environment. The unstructured N-terminal phosphorylation domain of PLB samples different orientations and covers a broad area of the cytosolic domain of SERCA but forms a stable complex mediated by pSer16 interactions with a binding site formed by SERCA residues Arg324/Lys328. PLB phosphorylation does not affect the interaction between the transmembrane regions of the two proteins; however, pSer16 stabilizes a disordered structure of the N-terminal phosphorylation domain that releases key inhibitory contacts between SERCA and PLB. We found that PLB phosphorylation is sufficient to guide the structural transitions of the cytosolic headpiece that are required to produce a competent structure of SERCA. We conclude that PLB phosphorylation serves as an allosteric molecular switch that releases inhibitory contacts and strings together the catalytic elements required for SERCA activation. This atomistic model represents a vivid atomic-resolution visualization of SERCA bound to phosphorylated PLB and provides previously inaccessible insights into the structural mechanism by which PLB phosphorylation releases SERCA inhibition in the heart.
| S-EPMC7583845 | biostudies-literature
Project description: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.
| S-EPMC2699759 | biostudies-literature
Project description:Sarcoplasmic reticulum Ca2+-ATPase (SERCA) is critical for cardiac Ca2+ transport. Reversal of phospholamban (PLB)-mediated SERCA inhibition by saturating Ca2+ conditions operates as a physiological rheostat to reactivate SERCA function in the absence of PLB phosphorylation. Here, we performed extensive atomistic molecular dynamics simulations to probe the structural mechanism of this process. Simulation of the inhibitory complex at superphysiological Ca2+ concentrations ([Ca2+] = 10 mm) revealed that Ca2+ ions interact primarily with SERCA and the lipid headgroups, but not with PLB's cytosolic domain or the cytosolic side of the SERCA-PLB interface. At this [Ca2+], a single Ca2+ ion was translocated from the cytosol to the transmembrane transport sites. We used this Ca2+-bound complex as an initial structure to simulate the effects of saturating Ca2+ at physiological conditions ([Ca2+]total ? 400 ?m). At these conditions, ?30% of the Ca2+-bound complexes exhibited structural features consistent with an inhibited state. However, in ?70% of the Ca2+-bound complexes, Ca2+ moved to transport site I, recruited Glu771 and Asp800, and disrupted key inhibitory contacts involving the conserved PLB residue Asn34 Structural analysis showed that Ca2+ induces only local changes in interresidue inhibitory interactions, but does not induce repositioning or changes in PLB structural dynamics. Upon relief of SERCA inhibition, Ca2+ binding produced a site I configuration sufficient for subsequent SERCA activation. We propose that at saturating [Ca2+] and in the absence of PLB phosphorylation, binding of a single Ca2+ ion in the transport sites rapidly shifts the equilibrium toward a noninhibited SERCA-PLB complex.
| S-EPMC6093236 | biostudies-literature
Project description:Phospholamban (PLB) physically interacts with Ca(2+)-ATPase and regulates contractility of the heart. We have studied this interaction using electron microscopy of large two-dimensional co-crystals of Ca(2+)-ATPase and the I40A mutant of PLB. Crystallization conditions were derived from those previously used for thin, helical crystals, but the addition of a 10-fold higher concentration of magnesium had a dramatic effect on the crystal morphology and packing. Two types of crystals were observed, and were characterized both by standard crystallographic methods and by electron tomography. The two crystal types had the same underlying lattice, which comprised antiparallel dimer ribbons of Ca(2+)-ATPase molecules previously seen in thin, helical crystals, but packed into a novel lattice with p22(1)2(1) symmetry. One crystal type was single-layered, whereas the other was a flattened tube and therefore double-layered. Additional features were observed between the dimer ribbons, which were substantially farther apart than in previous helical crystals. We attributed these additional densities to PLB, and built a three-dimensional model to show potential interactions with Ca(2+)-ATPase. These densities are most consistent with the pentameric form of PLB, despite the use of the presumed monomeric I40A mutant. Furthermore, our results indicate that this pentameric form of PLB is capable of a direct interaction with Ca(2+)-ATPase.
| S-EPMC1459492 | biostudies-literature
Project description:Phospholamban has been suggested to be a key regulator of cardiac sarcoplasmic reticulum (SR) Ca cycling and contractility and a potential therapeutic target in restoring the depressed Ca cycling in failing hearts. Our understanding of the function of phospholamban stems primarily from studies in genetically altered mouse models. To evaluate the significance of this protein in larger mammalian species, which exhibit Ca cycling properties similar to humans, we overexpressed phospholamban in adult rabbit cardiomyocytes. Adenoviral-mediated gene transfer, at high multiplicities of infection, resulted in an insignificant 1.22-fold overexpression of phospholamban. There were no effects on twitch Ca-transient amplitude or decay under basal or isoproterenol-stimulated conditions. Furthermore, the SR Ca load and Na/Ca exchanger function were not altered. These apparent differences between phospholamban overexpression in rabbit compared with previous findings in the mouse may be due to a significantly higher (1.5-fold) endogenous phospholamban-to-sarco(endo)plasmic reticulum Ca-ATPase (SERCA) 2a ratio and potential functional saturation of SERCA2a by phospholamban in rabbit cardiomyocytes. The findings suggest that important species-dependent differences in phospholamban regulation of SERCA2a occur. In larger mammals, a higher fraction of SERCA2a pumps are regulated by phospholamban, and this may influence therapeutic strategies to enhance cardiac contractility and functional cardiac reserve.
| S-EPMC2660221 | biostudies-literature
Project description:The integral membrane protein complex between phospholamban (PLN) and sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) regulates cardiac contractility. In the unphosphorylated form, PLN binds SERCA and inhibits Ca(2+) flux. Upon phosphorylation of PLN at Ser16, the inhibitory effect is reversed. Although structural details on both proteins are emerging from X-ray crystallography, cryo-electron microscopy, and NMR studies, the molecular mechanisms of their interactions and regulatory process are still lacking. It has been speculated that SERCA regulation depends on PLN structural transitions (order to disorder, i.e., folding/unfolding). Here, we investigated PLN conformational changes upon chemical unfolding by a combination of electron paramagnetic resonance and NMR spectroscopies, revealing that the conformational transitions involve mostly the cytoplasmic regions, with two concomitant phenomena: (1) membrane binding and folding of the amphipathic domain Ia and (2) folding/unfolding of the juxtamembrane domain Ib of PLN. Analysis of phosphorylated and unphosphorylated PLN with two phosphomimetic mutants of PLN (S16E and S16D) shows that the population of an unfolded state in domains Ia and Ib (T' state) is linearly correlated to the extent of SERCA inhibition measured by activity assays. Inhibition of SERCA is carried out by the folded ground state (T state) of the protein (PLN), while the relief of inhibition involves promotion of PLN to excited conformational states (Ser16 phosphorylated PLN). We propose that PLN population shifts (folding/unfolding) are a key regulatory mechanism for SERCA.
| S-EPMC3573877 | biostudies-literature
Project description:Coefficients for active transport of ions and heat in vesicles with Ca(2+)-ATPase from sarcoplasmic reticulum are defined in terms of a newly proposed thermodynamic theory and calculated using experiments reported in the literature. The coefficients characterize in a quantitative manner different performances of the enzyme isoforms. Four enzyme isoforms are examined, namely from white and red muscle tissue, from blood platelets, and from brown adipose mitochondria. The results indicate that the isoforms have a somewhat specialized function. White muscle tissue and brown adipose tissue have the same active transport coefficient ratio, but the activity level of the enzyme in white muscle is higher than in brown adipose tissue. The thermogenesis ratio is high in both white muscle and brown adipose tissue, in agreement with a specific role in nonshivering thermogenesis. Other isoforms do not have this ability to generate heat. A calcium-dependence of the coefficients is found, which can be understood as being in accordance with the role of this ion as a messenger in muscle contraction as well as in thermogenesis. The investigation points to new experiments related to structure as well as to function of the isoforms.
| S-EPMC2711487 | biostudies-literature
Project description:Oligomeric interactions between Ca-ATPase polypeptide chains and their modulation by phospholamban (PLB) were measured in native cardiac sarcoplasmic reticulum (SR) microsomes. Progressive modification of Lys(514) with fluorescein 5-isothiocyanate (FITC), which physically blocks access to the nucleotide binding site by ATP, demonstrates that Ca-ATPase active sites function independently of one another prior to the phosphorylation of PLB. However, upon cAMP-dependent protein kinase (PKA) phosphorylation of PLB, a second-order dependence between residual enzyme activity and the fraction of active sites is observed, consistent with a dimeric functional complex. Complementary distance measurements were made using FITC or 5-iodoacetamidofluorescein (IAF) bound to Cys(674) within the N- or P-domains, respectively, to detect structural coupling within oligomeric complexes. Accompanying the phosphorylation of PLB, neighboring Ca-ATPase polypeptide chains exhibit a 4 +/- 2 A decrease in the proximity between FITC sites within the N-domain and a 9 +/- 3 A increase in the proximity between IAF sites within P-domains. Thus, the phosphorylation of PLB induces spatial rearrangements between the N- and P-domain elements of proximal Ca-ATPase polypeptide chains which restore functional interactions between neighboring polypeptide chains and, in turn, result in increased rates of catalytic turnover. These results are interpreted in terms of a structural model, calculated through optimization of shape complementarity, desolvation, and electrostatic energies, which suggests a dimeric arrangement of Ca-ATPase polypeptide chains through the proximal association of N-domains that accommodates interaction with PLB. We suggest that the phosphorylation of PLB acts to release constraints involving interdomain subunit interactions that enhance catalytically important N-domain motions.
| S-EPMC2765579 | biostudies-literature
Project description:Intracellular Ca2+ mobilization is closely linked with the initiation of salivary secretion in parotid acinar cells. Reactive oxygen species (ROS) are known to be related to a variety of oxidative stress-induced cellular disorders and believed to be involved in salivary impairments. In this study, we investigated the underlying mechanism of hydrogen peroxide (H2O2) on cytosolic Ca2+ accumulation in mouse parotid acinar cells. Intracellular Ca2+ levels were slowly elevated when 1 mM H2O2 was perfused in the presence of normal extracellular Ca2+. In a Ca2+-free medium, 1 mM H2O2 still enhanced the intracellular Ca2+ level. Ca2+ entry tested using manganese quenching technique was not affected by perfusion of 1 mM H2O2. On the other hand, 10 mM H2O2 induced more rapid Ca2+ accumulation and facilitated Ca2+ entry from extracellular fluid. Ca2+ refill into intracellular Ca2+ store and inositol 1,4,5-trisphosphate (1 µM)-induced Ca2+ release from Ca2+ store was not affected by 1 mM H2O2 in permeabilized cells. Ca2+ efflux through plasma membrane Ca2+-ATPase (PMCA) was markedly blocked by 1 mM H2O2 in thapsigargin-treated intact acinar cells. Antioxidants, either catalase or dithiothreitol, completely protected H2O2-induced Ca2+ accumulation through PMCA inactivation. From the above results, we suggest that excessive production of H2O2 under pathological conditions may lead to cytosolic Ca2+ accumulation and that the primary mechanism of H2O2-induced Ca2+ accumulation is likely to inhibit Ca2+ efflux through PMCA rather than mobilize Ca2+ ions from extracellular medium or intracellular stores in mouse parotid acinar cells.
| S-EPMC5840080 | biostudies-literature