Ca2+ Binding to Site I of the Cardiac Ca2+ Pump Is Sufficient to Dissociate Phospholamban

Department of Medicine, Indiana University School of Medicine, Krannert Institute of Cardiology, Indianapolis, Indiana 46202, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 11/2009; 285(5):3253-60. DOI: 10.1074/jbc.M109.080820
Source: PubMed


Phospholamban (PLB) inhibits the activity of SERCA2a, the Ca(2+)-ATPase in cardiac sarcoplasmic reticulum, by decreasing the apparent affinity of the enzyme for Ca(2+). Recent cross-linking studies have suggested that PLB binding and Ca(2+) binding to SERCA2a are mutually exclusive. PLB binds to the E2 conformation of the Ca(2+)-ATPase, preventing formation of E1, the conformation that binds two Ca(2+) (at sites I and II) with high affinity and is required for ATP hydrolysis. Here we determined whether Ca(2+) binding to site I, site II, or both sites is sufficient to dissociate PLB from the Ca(2+) pump. Seven SERCA2a mutants with amino acid substitutions at Ca(2+)-binding site I (E770Q, T798A, and E907Q), site II (E309Q and N795A), or both sites (D799N and E309Q/E770Q) were made, and the effects of Ca(2+) on N30C-PLB cross-linking to Lys(328) of SERCA2a were measured. In agreement with earlier reports with the skeletal muscle Ca(2+)-ATPase, none of the SERCA2a mutants (except E907Q) hydrolyzed ATP in the presence of Ca(2+); however, all were phosphorylatable by P(i) to form E2P. Ca(2+) inhibition of E2P formation was observed only in SERCA2a mutants retaining site I. In cross-linking assays, strong cross-linking between N30C-PLB and each Ca(2+)-ATPase mutant was observed in the absence of Ca(2+). Importantly, however, micromolar Ca(2+) inhibited PLB cross-linking only to mutants retaining a functional Ca(2+)-binding site I. The dynamic equilibrium between Ca(2+) pumps and N30C-PLB was retained by all mutants, demonstrating normal regulation of cross-linking by ATP, thapsigargin, and anti-PLB antibody. From these results we conclude that site I is the key Ca(2+)-binding site regulating the physical association between PLB and SERCA2a.

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    • "e l s e v i e r . c o m / l o c a t e / y b b r c PLB at either micromolar Ca 2+ or after phosphorylation at S16 by PKA [11] [12] [13] [14] [15] [16]. However, recent spectroscopic studies support the subunit model, in which inhibition of SERCA is relieved by structural rearrangements within the SERCA–PLB complex, not by dissociation . "
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    ABSTRACT: We have used time-resolved fluorescence resonance energy transfer (TR-FRET) to characterize the interaction between phospholamban (PLB) and the sarcoplasmic reticulum (SR) Ca-ATPase (SERCA) under conditions that relieve SERCA inhibition. Unphosphorylated PLB inhibits SERCA in cardiac SR, but inhibition is relieved by either micromolar Ca(2+) or PLB phosphorylation. In both cases, it has been proposed that inhibition is relieved by dissociation of the complex. To test this hypothesis, we attached fluorophores to the cytoplasmic domains of SERCA and PLB, and reconstituted them functionally in lipid bilayers. TR-FRET, which permitted simultaneous measurement of SERCA-PLB binding and structure, was measured as a function of PLB phosphorylation and [Ca(2+)]. In all cases, two structural states of the SERCA-PLB complex were resolved, probably corresponding to the previously described T and R structural states of the PLB cytoplasmic domain. Phosphorylation of PLB at S16 completely relieved inhibition, partially dissociated the SERCA-PLB complex, and shifted the T/R equilibrium within the bound complex toward the R state. Since the PLB concentration in cardiac SR is at least 10 times that in our FRET measurements, we calculate that most of SERCA contains bound phosphorylated PLB in cardiac SR, even after complete phosphorylation. 4 μM Ca(2+) completely relieved inhibition but did not induce a detectable change in SERCA-PLB binding or cytoplasmic domain structure, suggesting a mechanism involving structural changes in SERCA's transmembrane domain. We conclude that Ca(2+) and PLB phosphorylation relieve SERCA-PLB inhibition by distinct mechanisms, but both are achieved primarily by structural changes within the SERCA-PLB complex, not by dissociation of that complex.
    Preview · Article · May 2014 · Biochemical and Biophysical Research Communications
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    • "Ca 2+ translocation into the SR induces heart muscle relaxation, while Ca 2+ translocation back to the cytoplasm induces heart muscle contraction [1]. The inhibition of SERCA by PLB can be relieved either upon phosphorylation of PLB at position 16 or 17 by protein kinase A or by elevated Ca 2+ concentrations in the cytoplasm [4] [5]. PLB has been shown to consist of four domains: the cytoplasmic domain Ia (1–16), loop (17–22), domain Ib (23–30) and the hydrophobic transmembrane domain (31–52) [6]. "
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    ABSTRACT: Phospholamban (PLB) is a 52 amino acid integral membrane protein that interacts with the sarcoplasmic reticulum Ca(2+) ATPase (SERCA) and helps to regulate Ca(2+) flow. PLB inhibits SERCA impairing Ca(2+) translocation. The inhibition can be relieved upon phosphorylation of PLB. The Arg9 to Cys (R9C) mutation is a loss of function mutation with reduced inhibitory potency. The effect R9C PLB has on the membrane surface and the hydrophobic region dynamics was investigated by (31)P and (2)H solid-state NMR spectroscopy in multilamellar vesicles (MLVs). The (31)P NMR spectra indicate that, like the phosphorylated PLB (P-PLB), the mutated R9C-PLB protein has significantly less interaction with the lipid bilayer headgroup when compared to wild-type PLB (WT-PLB). Similar to P-PLB, R9C-PLB slightly decreases (31)P T1 values in the lipid headgroup region. (2)H SCD order parameters of (2)H nuclei along the lipid acyl chain decrease less dramatically for R9C-PLB and P-PLB when compared to WT-PLB. The results suggest that R9C-PLB interacts less with the membrane surface and hydrophobic region than WT-PLB. Detachment of the cytoplasmic domain of R9C-PLB from the membrane surface could be related to its loss of function.
    Preview · Article · Jul 2013 · Biochimica et Biophysica Acta
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    • "The mechanism by which PLB regulates SERCA has been the subject of much debate for several decades. Some studies by cross-linking have been interpreted to suggest that relief of SERCA inhibition, by either high [Ca 2? ] or PLB phosphorylation, requires dissociation of PLB from SERCA (Chen et al. 2012; Akin and Jones 2012). However , several lines of evidence, including FRET and co-immunoprecipitation, show clearly that PLB remains bound to SERCA even when phosphorylated by PKA (Asahi et al. 2000; Li et al. 2004a) or when [Ca 2? ] is micromolar (Mueller et al. 2004). "
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    ABSTRACT: We have used site-directed spectroscopic probes to detect structural changes, motions, and interactions due to phosphorylation of proteins involved in the regulation of muscle contraction and relaxation. Protein crystal structures provide static snapshots that provide clues to the conformations that are sampled dynamically by proteins in the cellular environment. Our site-directed spectroscopic experiments, combined with computational simulations, extend these studies into functional assemblies in solution, and reveal details of protein regions that are too dynamic or disordered for crystallographic approaches. Here, we discuss phosphorylation-mediated structural transitions in the smooth muscle myosin regulatory light chain, the striated muscle accessory protein myosin binding protein-C, and the cardiac membrane Ca(2+) pump modulator phospholamban. In each of these systems, phosphorylation near the N terminus of the regulatory protein relieves an inhibitory interaction between the phosphoprotein and its regulatory target. Several additional unifying themes emerge from our studies: (a) The effect of phosphorylation is not to change the affinity of the phosphoprotein for its regulated binding partner, but to change the structure of the bound complex without dissociation. (b) Phosphorylation induces transitions between order and dynamic disorder. (c) Structural states are only loosely coupled to phosphorylation; i.e., complete phosphorylation induces dramatic functional effects with only a partial shift in the equilibrium between ordered and disordered structural states. These studies, which offer atomic-resolution insight into the structural and functional dynamics of these phosphoproteins, were inspired in part by the ground-breaking work in this field by Michael and Kate Barany.
    Full-text · Article · Aug 2012 · Journal of Muscle Research and Cell Motility
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