Solid-state NMR Reveals Structural Changes in Phospholamban Accompanying the Functional Regulation of Ca2+-ATPase

Abstract

Calcium transport across the sarcoplasmic reticulum of cardiac myocytes is regulated by a reversible inhibitory interaction between the Ca 2؉ -ATPase and the small transmembrane protein phospholamban (PLB). A nullcysteine analogue of PLB, containing isotope labels in the transmembrane domain or cytoplasmic domain, was reconstituted into membranes in the absence and presence of the SERCA1 isoform of Ca 2؉ -ATPase for structural investigation by cross-polarization magic-angle spinning (CP-MAS) NMR. PLB lowered the maximal hydrolytic activity of SERCA1 and its affinity for calcium in membrane preparations suitable for structural analysis by NMR. switches from an ␣-helix in pure lipid membranes to a more extended structure in the presence of SERCA1, which may reflect local structural distortions which change the orientations of the transmembrane and cytoplasmic domains. These results suggest that Ca 2؉ -ATPase has a long-range effect on the structure of PLB around residue 25, which promotes the functional association of the two proteins. Phospholamban (PLB)1 is a 52-amino acid membrane-spanning protein, which is expressed predominantly in the sarcoplasmic reticulum of cardiac myocytes (1). The primary physiological function of PLB is to regulate the active transport of calcium ions into the sarcoplasmic reticulum lumen via an inhibitory association with SERCA2a, the cardiac isoform of Ca 2ϩ -ATPase (2, 3). PLB is believed to bind to the calcium-free conformation of SERCA2a (4), and exerts its inhibitory action by reducing the apparent calcium affinity of the enzyme (5). In response to ␤-adrenergic stimulation, PLB is phosphorylated at Ser 16 and Thr 17 by cAMP-dependent kinase and calcium/calmodulin-dependent kinase, respectively. PLB phosphorylation relieves SERCA2a inhibition, which increases the rate of calcium uptake into the sarcoplasmic reticulum and results in the accelerated relaxation of cardiac muscle (2). Mechanistic failures in the reversibility of SERCA2a inhibition, or overexpression of PLB relative to SERCA2a, are linked to the disruption of calcium homeostasis in cardiac cells and may contribute to cardiovascular disorders such as congestive heart failure (6).The exact nature of the interaction between PLB and SERCA enzymes is a subject of debate. PLB readily self-associates to form a homopentamer within the lipid bilayer (7,8), with a dynamic equilibrium believed to exist between the oligomeric and monomeric state of the protein (9). Most evidence suggests that it is the monomeric form of PLB that binds to and inhibits SERCA, with the pentamer acting as a reservoir from which monomers dissociate (5, 8, 10 -12). In reconstitution studies, the molar stoichiometry of PLB to SERCA required for maximal regulation of calcium transport is between 3:1 and 15:1 (12, 13), whereas a fluorescence energy transfer study has suggested that two PLB monomers interact with two Ca 2ϩ ATPase molecules to form a heterodimer (14). In the latter study, it was found that both PLB molecules must be phosphorylated in order t...

Electron Paramagnetic Resonance Reveals a Large-Scale Conformational Change in the Cytoplasmic Domain of Phospholamban upon Binding to the Sarcoplasmic Reticulum Ca-ATPase

“…Experiments with a monomeric PLB mutant fluorescently labeled at A24C showed that addition of SERCA increased the solvent exposure of the label, as well as the rate of local probe motion (50). A contradictory result, obtained by solidstate NMR of monomeric PLB, suggested not only that residues 21-24 remain helical upon addition of SERCA but also that association with SERCA induces helical structure between residues 24 and 26 (51). Clearly, investigating the relationship between the structural dynamics of PLB's cytoplasmic domain secondary structure will be an important factor in understanding PLB-SERCA binding.…”

Solid-state NMR and Functional Measurements Indicate That the Conserved Tyrosine Residues of Sarcolipin Are Involved Directly in the Inhibition of SERCA1

“…SLN was co-reconstituted with SERCA1a into DOPC membranes at a lipid/SLN/SERCA1a molar ratio of 160:10:1 or 160:3:1 (21). For the reconstitution of SLN alone, DOPC and SLN were dissolved in 50:50 chloroform/methanol at an l/p molar ratio of 53:1 and dried to a thin film as described above.…”

“…Activity Measurements-Specific Ca 2ϩ -ATPase activity was quantified as the amount of inorganic phosphate (P i ) liberated upon ATP hydrolysis at 37°C, as described previously (21). Ca 2ϩ uptake by SR microsomes was measured on a Carey Eclipse fluorimeter by monitoring the change in fluorescence of the calcium indicator BAPTA in a method adapted from Refs.…”

The Cytoplasmic Domains of Phospholamban and Phospholemman Associate with Phospholipid Membrane Surfaces

“…Structural analysis of PLB in organic solvents has revealed that the N-terminally situated cytoplasmic domain is predominantly helical and is linked to the transmembrane helix via a short disordered region (9). Analysis of PLB in lipid bilayers using solid-state nuclear magnetic resonance (NMR) methods showed that the sequence Ala24-Gln26, which connects the cytoplasmic and transmembrane domains, is helical when the protein is alone in the membrane but undergoes a transition to a more extended structure in the presence of SERCA (10). It was argued that the conformational adjustment might reflect changes in the orientations of the transmembrane and cytoplasmic domains as PLB encounters, and moves into contact with, SERCA (10).…”