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An investigation of the mechanism of inhibition of the Ca(2+)-ATPase by phospholamban.

ABSTRACT: The Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum has been reconstituted with peptides corresponding to the hydrophobic domain of phospholamban (PLB) with or without the three Cys residues replaced by Ala, and with PLB with the three Cys residues replaced by Ala [PLBcys-(1-52)]. Reconstitution with the hydrophobic domain of PLB[PLB(25-52)] was found to decrease the apparent affinity of the ATPase for Ca2+ with no effect on the maximal rate of ATP hydrolysis observed at saturating concentrations of Ca2+. Reconstitution with PLBCys-(1-52) decreased both the apparent affinity for Ca2+ and the maximal activity; the effect on maximal activity followed from a decrease in the rate of the Ca2+ transport step (E1PCa2-->E2P) as observed with the hydrophilic domain PLB(1-25). The concentration dependences of the effects of the hydrophobic domain and of the whole PLB molecule were very similar, suggesting that the hydrophilic domain made little contribution to the affinity of the ATPase for PLB. The effect of PLB on the ATPase was dependent on the molar ratio of phospholipid to ATPase, suggesting partition of the PLB between its binding site on the ATPase and the bulk lipid phase in the membrane. Neither PLB nor its hydrophobic domain affected the rates of phosphorylation or dephosphorylation of the ATPase. Despite their effects on the apparent affinity of the ATPase for Ca2+, neither PLB nor its hydrophobic domain had any effect on the true affinity of the ATPase for Ca2+, as measured from changes in the tryptophan fluorescence of the ATPase. The effects of PLB on the activity of the ATPase are the sum of the effects of its hydrophilic and hydrophobic domains.

SUBMITTER: Hughes G

PROVIDER: S-EPMC1217713 | biostudies-other | 1996 Sep

REPOSITORIES: biostudies-other

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Structural and dynamic basis of phospholamban and sarcolipin inhibition of Ca(2+)-ATPase.

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Tuning the structural coupling between the transmembrane and cytoplasmic domains of phospholamban to control sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) function.

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Structural basis for relief of phospholamban-mediated inhibition of the sarcoplasmic reticulum Ca2+-ATPase at saturating Ca2+ conditions.

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

Phosphorylated phospholamban stabilizes a compact conformation of the cardiac calcium-ATPase.

Project description:The sarcoendoplasmic reticulum calcium ATPase (SERCA) plays a key role in cardiac calcium handling and is considered a high-value target for the treatment of heart failure. SERCA undergoes conformational changes as it harnesses the chemical energy of ATP for active transport. X-ray crystallography has provided insight into SERCA structural substates, but it is not known how well these static snapshots describe in vivo conformational dynamics. The goals of this work were to quantify the direction and magnitude of SERCA motions as the pump performs work in live cardiac myocytes, and to identify structural determinants of SERCA regulation by phospholamban. We measured intramolecular fluorescence resonance energy transfer (FRET) between fluorescent proteins fused to SERCA cytoplasmic domains. We detected four discrete structural substates for SERCA expressed in cardiac muscle cells. The relative populations of these discrete states oscillated with electrical pacing. Low FRET states were most populated in low Ca (diastole), and were indicative of an open, disordered structure for SERCA in the E2 (Ca-free) enzymatic substate. High FRET states increased with Ca (systole), suggesting rigidly closed conformations for the E1 (Ca-bound) enzymatic substates. Notably, a special compact E1 state was observed after treatment with ?-adrenergic agonist or with coexpression of phosphomimetic mutants of phospholamban. The data suggest that SERCA calcium binding induces the pump to undergo a transition from an open, dynamic conformation to a closed, ordered structure. Phosphorylated phospholamban stabilizes a unique conformation of SERCA that is characterized by a compact architecture.

| S-EPMC3797577 | biostudies-literature

Modeling of the inhibitory interaction of phospholamban with the Ca2+ ATPase.

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

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