Secondary Structure and Orientation of Phospholamban Reconstituted in Supported Bilayers from Polarized Attenuated Total Reflection FTIR Spectroscopy

Tatulian, Suren A.; Jones, Larry R.; Reddy, Laxma G.; Stokes, David L.; Tamm, Lukas K.

doi:10.1021/bi00013a038

Cited by 110 publications

(155 citation statements)

References 50 publications

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“…PLB is a small (6000 Da), mostly ␣-helical transmembrane protein that runs as a pentamer by SDS-polyacrylamide gel electrophoresis (11,14). The transmembrane domain is thought to consist of a single ␣-helix (15,16), whereas the cytoplasmic domain contains sites for phosphorylation and for interaction with Ca 2ϩ -ATPase (17,18). Model building suggests that the pentamer could be stabilized by coiled-coil interactions between transmembrane helices (19,20).…”

Section: ؉mentioning

confidence: 99%

Purified, Reconstituted Cardiac Ca2+-ATPase Is Regulated by Phospholamban but Not by Direct Phosphorylation with Ca2+/Calmodulin-dependent Protein Kinase

Reddy

Jones

Pace

et al. 1996

Journal of Biological Chemistry

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Regulation of calcium transport by sarcoplasmic reticulum provides increased cardiac contractility in response to ␤-adrenergic stimulation. This is due to phosphorylation of phospholamban by cAMP-dependent protein kinase or by calcium/calmodulin-dependent protein kinase, which activates the calcium pump (Ca 2؉ -ATPase). Recently, direct phosphorylation of Ca 2؉ -ATPase by calcium/calmodulin-dependent protein kinase has been proposed to provide additional regulation. To investigate these effects in detail, we have purified Ca 2؉ -ATPase from cardiac sarcoplasmic reticulum using affinity chromatography and reconstituted it with purified, recombinant phospholamban. The resulting proteoliposomes had high rates of calcium transport, which was tightly coupled to ATP hydrolysis (ϳ1.7 calcium ions transported per ATP molecule hydrolyzed). Co-reconstitution with phospholamban suppressed both calcium uptake and ATPase activities by ϳ50%, and this suppression was fully relieved by a phospholamban monoclonal antibody or by phosphorylation either with cAMP-dependent protein kinase or with calcium/calmodulin-dependent protein kinase. These effects were consistent with a change in the apparent calcium affinity of Ca -ATPase in longitudinal cardiac sarcoplasmic reticulum vesicles was a substrate for calcium/calmodulin-dependent protein kinase, and accordingly, we found no effect of calcium/calmodulin-dependent protein kinase phosphorylation on V max for calcium transport.

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Section: ؉mentioning

confidence: 99%

Purified, Reconstituted Cardiac Ca2+-ATPase Is Regulated by Phospholamban but Not by Direct Phosphorylation with Ca2+/Calmodulin-dependent Protein Kinase

Reddy

Jones

Pace

et al. 1996

Journal of Biological Chemistry

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“…1), residues 32-52 are in a coiled helix approximately parallel to the bilayer normal. The first model (extended helix/sheet model), based on polarized Fourier transform infrared (FTIR) spectroscopy in a supported lipid bilayer, has residues 22-32 to be in an antiparallel ␤-sheet configuration with the cytoplasmic domain helix oriented Ϸ50-60°relative to the bilayer normal (7). A second model (continuous helix model), based on rotational-echo doubleresonance (REDOR) solid-state NMR and polarized FTIR spectroscopy in lipid bilayers, depicts wt-PLN as a continuous helix (8,9).…”

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confidence: 99%

“…Although the PLN structure is dynamic, extensive data in lipids and micelles show that the predominant Structural models of wt-PLN. The extended helix/sheet and continuous helix models shown were reconstructed from the original papers (7,8) to give the reader a graphical illustration of the models. Graphics were prepared by using Pymol software (www.pymol.org).…”

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confidence: 99%

Spectroscopic validation of the pentameric structure of phospholamban

Traaseth¹,

Verardi

Torgersen

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Phospholamban (PLN) regulates calcium translocation within cardiac myocytes by shifting sarco(endo)plasmic reticulum Ca 2؉ -ATPase (SERCA) affinity for calcium. Although the monomeric form of PLN (6 kDa) is the principal inhibitory species, recent evidence suggests that the PLN pentamer (30 kDa) also is able to bind SERCA. To date, several membrane architectures of the pentamer have been proposed, with different topological orientations for the cytoplasmic domain: (i) extended from the bilayer normal by 50 -60°; (ii) continuous ␣-helix tilted 28°relative to the bilayer normal; (iii) pinwheel geometry, with the cytoplasmic helix perpendicular to the bilayer normal and in contact with the surface of the bilayer; and (iv) bellflower structure, in which the cytoplasmic domain helix makes Ϸ20°angle with respect to the membrane bilayer normal. Using a variety of cell membrane mimicking systems (i.e., lipid vesicles, oriented lipid bilayers, and detergent micelles) and a combination of multidimensional solution/solidstate NMR and EPR spectroscopies, we tested the different structural models. We conclude that the pinwheel topology is the predominant conformation of pentameric PLN, with the cytoplasmic domain interacting with the membrane surface. We propose that the interaction with the bilayer precedes SERCA binding and may mediate the interactions with other proteins such as protein kinase A and protein phosphatase 1.Ca 2ϩ -ATPase ͉ EPR ͉ membrane protein ͉ solid-state NMR ͉ protein dynamics C alcium translocation into the sarcoplasmic reticulum of cardiac myocytes is controlled by the sarco(endo)plasmic reticulum Ca 2ϩ -ATPase (SERCA). Phospholamban (PLN) regulates the activity of SERCA by shifting the apparent calcium affinity for the enzyme. This activity is relieved by phosphorylation of PLN at Ser-16 and/or Thr-17 and high calcium concentration within the cytosol. Wild-type PLN (wt-PLN) forms stable homopentamers in lipid bilayers and in detergent micelles, where each monomer is composed of a helical cytoplasmic domain (residues 1-16), a semiflexible loop (residues 17-21), and a helical transmembrane domain (residues 22-52) (1, 2). Mutagenesis, molecular biology, and in vivo studies revealed that the PLN pentamer depolymerizes into active monomers that bind and inhibit SERCA (3). Similar conclusions were reached by in vitro fluorescence studies (4). Recently, Young and coworkers (5) have reported a cocrystal formed by SERCA and PLN pentamer, suggesting that the pentameric species also is able to bind SERCA. Furthermore, Jones and coworkers (6) hypothesized that the PLN pentamer may act as a chloride ion channel, which is supported by the bellflower structure recently determined by Oxenoid and Chou (2).There are four principal proposed structural models of pentameric wt-PLN, which differ primarily in the topology of the more dynamic cytoplasmic domain. In each of these models (shown in Fig. 1), residues 32-52 are in a coiled helix approximately parallel to the bilayer normal. The first model (extended helix/sheet ...

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“…In contrast, the anti-PLB monoclonal antibody, 2D12, which recognizes residues 9 -13 of PLB and disrupts the functional interaction between PLB and SERCA2a by mimicking PLB phosphorylation (8), prevented the cross-linking of residues 27 and 30 of PLB to residues 318 (7) and 328 of SERCA2a. Conformational changes in the vicinity of this epitope are likely to result from phosphorylation of Ser 16 during ␤-adrenergic stimulation (43), and it will therefore be important in future experiments to establish the sites of interaction between this important region of PLB and the Ca 2ϩ pump to understand the mechanism of regulation in a physiological context.…”

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confidence: 99%

Spatial and Dynamic Interactions between Phospholamban and the Canine Cardiac Ca2+ Pump Revealed with Use of Heterobifunctional Cross-linking Agents

Chen

Stokes

Rice

et al. 2003

Journal of Biological Chemistry

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Heterobifunctional thiol to amine cross-linking agents were used to gain new insights on the dynamics and conformational factors governing the interaction between the cardiac Ca 2؉ pump (SERCA2a) and phospholamban (PLB). PLB is a small protein inhibitor of SERCA2a that reduces enzyme affinity for Ca 2؉ and thereby regulates cardiac contractility. We found that the PLB monomer with Asn 27 or Asn 30 changed to Cys (N27C-PLB or N30C-PLB) cross-linked to lysine of SERCA2a within seconds with >80% efficiency. Optimal cross-linking occurred at spacer chain lengths of 10 and 15 Å for N27C and N30C, respectively. The rapid time course of cross-linking indicated that neither dissociation of PLB pentamers nor binding of PLB monomers to SERCA2a was rate-limiting. Cross-linking occurred only to the E2 (Ca 2؉ -free) conformation of SERCA2a, was strongly favored by nucleotide binding to this state, and was completely inhibited by thapsigargin. Protein sequencing in combination with mutagenesis identified of Lys 328 of SERCA2a as the target of cross-linking. A three-dimensional map of interacting residues indicated that the cross-linking distances were entirely compatible with the 10-Å distance recently determined between N30C of PLB and Cys 318 of SERCA2a. In contrast, Lys 3 of PLB did not cross-link to any Lys (or Cys) of SERCA2a, suggesting that previous three-dimensional models that constrain Lys 3 near residues 397-400 of thapsigargin-inhibited SERCA2a should be viewed with caution. Furthermore, although earlier models of PLB⅐SERCA2a are based on thapsigargin-bound SERCA, our results suggest that the nucleotide-bound, E2 conformation is substantially different and represents the key conformational state for interacting with PLB. PLB1 is a small phosphoprotein modulator of the Ca 2ϩ -pump (SERCA2a) in cardiac SR, which is critically involved in regulating the strength and duration of the heartbeat (1, 2). In the dephosphorylated state, PLB inhibits the Ca 2ϩ -ATPase by decreasing its apparent affinity for Ca 2ϩ (3). Phosphorylation of PLB at Ser 16 and Thr 17 during ␤-adrenergic stimulation of the heart reverses PLB inhibition and augments Ca 2ϩ loading of the SR (4), thus producing positive inotropic and lusitropic effects (2, 5). PLB is a single-span membrane protein composed of 52 amino acids, which forms a homopentamer within the SR membrane (1, 4). Recent work suggests that there is a dynamic equilibrium between PLB monomers and pentamers (6), and that the PLB monomer is responsible for binding to SERCA2a (7) and inhibiting it (8, 9). In intact myocardium, ␤-adrenergic receptor stimulation disrupts the inhibitory interaction between PLB and SERCA2a rapidly; PLB phosphorylation, Ca 2ϩ transport, and contractility all increase within seconds after ␤-receptor activation (4, 10). This suggests that PLB monomers must associate and dissociate quickly, over a time course of seconds or faster, to allow for dynamic regulation of SERCA2a and the strength of contractility.The emerging picture for the mechanism of SERCA2a inhibit...

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Secondary Structure and Orientation of Phospholamban Reconstituted in Supported Bilayers from Polarized Attenuated Total Reflection FTIR Spectroscopy

Cited by 110 publications

References 50 publications

Purified, Reconstituted Cardiac Ca2+-ATPase Is Regulated by Phospholamban but Not by Direct Phosphorylation with Ca2+/Calmodulin-dependent Protein Kinase

Purified, Reconstituted Cardiac Ca2+-ATPase Is Regulated by Phospholamban but Not by Direct Phosphorylation with Ca2+/Calmodulin-dependent Protein Kinase

Spectroscopic validation of the pentameric structure of phospholamban

Spatial and Dynamic Interactions between Phospholamban and the Canine Cardiac Ca2+ Pump Revealed with Use of Heterobifunctional Cross-linking Agents

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