Structural Characterization of Ca 2+ -ATPase-Bound Phospholamban in Lipid Bilayers by Solid-State Nuclear Magnetic Resonance (NMR) Spectroscopy † , ‡ (original) (raw)
Related papers
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2010
Phospholamban (PLB) is an integral membrane protein regulating Ca 2+ transport through inhibitory interaction with sarco(endo)plasmic reticulum calcium ATPase (SERCA). The Asn27 to Ala (N27A) mutation of PLB has been shown to function as a superinhibitor of the affinity of SERCA for Ca 2+ and of cardiac contractility in vivo. The effects of this N27A mutation on the side-chain and backbone dynamics of PLB were investigated with 2 H and 15 N solid-state NMR spectroscopy in phospholipid multilamellar vesicles (MLVs). 2 H and 15 N NMR spectra indicate that the N27A mutation does not significantly change the side-chain or backbone dynamics of the transmembrane and cytoplasmic domains when compared to wild-type PLB. However, dynamic changes are observed for the hinge region, in which greater mobility is observed for the CD 3 -labeled Ala24 N27A-PLB. The increased dynamics in the hinge region of PLB upon N27A mutation may allow the cytoplasmic helix to more easily interact with the Ca 2+ -ATPase; thus, showing increased inhibition of Ca 2+ -ATPase.
Proceedings of the National Academy of Sciences, 2005
We have used magnetic resonance to map the interaction surface of an integral membrane protein for its regulatory target, an integral membrane enzyme. Phospholamban (PLN) regulates cardiac contractility via its modulation of sarco(endo)plasmic reticulum calcium ATPase (SERCA) activity. Impairment of this regulatory process causes heart failure. To map the molecular details of the PLN/SERCA interaction, we have functionally reconstituted SERCA with labeled PLN in dodecylphosphocholine micelles for high-resolution NMR spectroscopy and in both micelles and lipid bilayers for EPR spectroscopy. Differential perturbations in NMR linewidths and chemical shifts, measured as a function of position in the PLN sequence, provide a vivid picture of extensive SERCA contacts in both cytoplasmic and transmembrane domains of PLN and provide structural insight into previously reported functional mutagenesis data. NMR and EPR data show clear and complementary evidence for a dynamic (μs-to-ms) equilibr...
2007
Phospholamban (PLN) and sarcolipin (SLN) are two single-pass membrane proteins that regulate Ca 2+ -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.
Activating and Deactivating Roles of Lipid Bilayers on the Ca2+-ATPase/Phospholamban Complex
Biochemistry, 2011
The physicochemical properties of the lipid bilayer shape the structure and topology of membrane proteins and regulate their biological function. Here, we investigated the functional effects of various lipid bilayer compositions on the sarcoplasmic reticulum (SR) Ca 2+-ATPase (SERCA) in the presence and absence of its endogenous regulator, phospholamban (PLN). In the cardiac muscle, SERCA hydrolyzes one ATP molecule to translocate two Ca 2+ ions into the SR membrane per enzymatic cycle. Unphosphorylated PLN reduces SERCA's affinity for Ca 2+ and affects the enzymatic turnover. We varied bilayer thickness, head group, fluidity, and found that both the maximal velocity (V max) of the enzyme and its apparent affinity for Ca 2+ (K Ca) are strongly affected. Our results show that a) SERCA V max has a biphasic dependence on bilayer thickness, reaching its maximum with 22-carbon lipid chain length, b) phosphatidylethanolamine (PE) and phosphatidylserine (PS) decrease K Ca , and c) monounsaturated lipids afford higher SERCA V max and Ca 2+-affinity than diunsaturated lipids. The presence of PLN removes the activating effect of PE and shifts SERCA's activity profile, with a maximal activity reached in bilayers with 20-carbon lipid chain length. Our results in synthetic lipid systems compare well with those carried out in native SR lipids. Importantly, we found that specific membrane compositions closely reproduce PLN effects (V max and K Ca) found in living cells, reconciling an ongoing controversy regarding the regulatory role of PLN on SERCA function. Taken with the physiological changes occurring in the SR membrane composition, these studies underscore a possible allosteric role of the lipid bilayers on the SERCA/PLN complex. Sarcoplasmic reticulum (SR) Ca 2+-ATPase (SERCA) is a multi-domain membrane-spanning enzyme that regulates muscle relaxation by Ca 2+ translocation from the cytosol into the SR lumen (1, 2). SERCA is a P-type ATPase and transports two Ca 2+ ions per hydrolyzed ATP (3). Phospholamban (PLN) is the main endogenous inhibitor of SERCA activity in cardiomyocytes and its inhibition is relieved upon phosphorylation at Ser 16 or Thr 17 of PLN (4, 5, 6, 7). Since SERCA and PLN are the main regulators of the cardiac output, they are targets for treatment of cardiac disease (8, 9).
Proceedings of the National Academy of Sciences, 2009
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...
Structural and dynamic basis of phospholamban and sarcolipin inhibition of Ca2+-ATPase
Towards the full …, 2008
Phospholamban (PLN) and sarcolipin (SLN) are two single-pass membrane proteins that regulate Ca 2+ -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.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2010
Wild-type phospholamban (WT-PLB) is a pentameric transmembrane protein that regulates the cardiac cycle (contraction and relaxation). From a physiological prospective, unphosphorylated WT-PLB inhibits sarcoplasmic reticulum ATPase activity; whereas, its phosphorylated form relieves the inhibition in a mechanism that is not completely understood. In this study, site-specifically 15 N-Ala-11-and 15 N-Leu-7-labeled WT-PLB and the corresponding phosphorylated forms (P-PLB) were incorporated into 1,2-dioleoyl-sn-glycero-3-phosphocholine/2-dioleoyl-sn-glycero-3phosphoethanolamine (DOPC/DOPE) mechanically oriented lipid bilayers. The aligned 15 N-labeled Ala-11 and Leu-7 WT-PLB samples show 15 N resonance peaks at approximately 71 ppm and 75 ppm, respectively, while the corresponding phosphorylated forms P-PLB show 15 N peaks at 92 ppm and 99 ppm, respectively. These 15 N chemical shift changes upon phosphorylation are significant and in agreement with previous reports, which indicate that phosphorylation of WT-PLB at Ser-16 alters the structural properties of the cytoplasmic domain with respect to the lipid bilayers.
Biochemistry, 2009
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 PKAdependent 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-iodoacetamido-fluorescein (IAF) bound to Cys 674 within the Nor P-domains respectively, to detect structural coupling within oligomeric complexes. Accompanying the phosphorylation of PLB, neighboring Ca-ATPase polypeptide chains exhibit a 4 ± 2 Å decrease in the proximity between FITC sites within the Ndomain and a 9 ± 3 Å 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. Ca-ATPases function to maintain low cytoplasmic calcium levels, acting to resequester calcium into the sarcoplasmic reticulum (SR) lumen after each contractile event, thus controlling relaxation rates in the heart and other muscle cells. Co-expression of the regulatory protein phospholamban (PLB) in slow-twitch skeletal muscle and the heart permits the modulation of Ca-ATPase transport function and thus the force and rate of muscle contraction. Specifically, in response to β-adrenergic stimulation, the activation of cAMP-dependent protein kinase (PKA) results in the phosphorylation of Ser 16 in PLB, which releases inhibitory interactions with the Ca-ATPase that enhance transport activity at submicromolar
Journal of Biological Chemistry, 2007
Cardiac contraction and relaxation are regulated by conformational transitions of protein complexes that are responsible for calcium trafficking through cell membranes. Central to the muscle relaxation phase is a dynamic membrane protein complex formed by Ca2+-ATPase (SERCA) and phospholamban (PLN), which in humans is responsible for approximately 70% of the calcium re-uptake in the sarcoplasmic reticulum. Dysfunction in this regulatory mechanism causes severe pathophysiologies. In this report, we used a combination of nuclear magnetic resonance, electron paramagnetic resonance, and coupled enzyme assays to investigate how single mutations at position 21 of PLN affects its structural dynamics and, in turn, its interaction with SERCA. We found that it is possible to control the activity of SERCA by tuning PLN structural dynamics. Both increased rigidity and mobility of the PLN backbone cause a reduction of SERCA inhibition, affecting calcium transport. Although the more rigid, loss-of-function (LOF) mutants have lower binding affinities for SERCA, the more dynamic LOF mutants have binding affinities similar to that of PLN. Here, we demonstrate that it is possible to harness this knowledge to design new LOF mutants with activity similar to S16E (a mutant already used in gene therapy) for possible application in recombinant gene therapy. As proof of concept, we show a new mutant of PLN, P21G, with improved LOF characteristics in vitro.