Phosphorylation-dependent Conformational Switch in Spin-labeled Phospholamban Bound to SERCA

Karim, Christine B.; Zhang, Zhiwen; Howard, Edmund C.; Torgersen, Kurt D.; Thomas, David D.

doi:10.1016/j.jmb.2006.02.051

Cited by 104 publications

(242 citation statements)

References 40 publications

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“…The two components correspond to the T and R states of the PLN cytoplasmic domain (15), which are detected in DPC micelles by both EPR and solution NMR (13). Also, a lipid anchor attached to the N terminus of AFA-PLN stabilizes the membrane-bound conformation of the cytoplasmic domain, eliminating the EPR peak attributed to the R state (34), thus confirming that the R state corresponds to the more dynamic and extended conformation (Fig. 7).…”

Section: Resultsmentioning

confidence: 55%

“…and D.D.T. groups using solution NMR (1,(11)(12)(13)(14) and EPR in detergent micelles (13), EPR in lipid vesicles (13,15,34), and solid-state NMR in mechanically oriented lipid bilayers (18). In all of these experiments, the results are unambiguous: the major population of AFA-PLN is L-shaped.…”

Section: Discussionmentioning

confidence: 96%

“…7B, is that the two states (T and R) represent the conformational equilibrium between a more stable, predominantly ␣-helical ensemble and a more dynamic, less populated unfolded state (15). Interestingly, phosphorylation at Ser-16 by protein kinase A and single-site mutations at position 21 show the R state to become more populated, indicating the importance of preexisting equilibria necessary for SERCA recognition (12,14,34). Similar conformational interconversions also have been detected for fd coat protein, in which lipids may induce different topologies consistent with either the arrangement in the virus coat or in the interaction with the lipid membrane (45)(46)(47).…”

Section: Discussionmentioning

confidence: 99%

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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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100

126

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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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Section: Resultsmentioning

confidence: 55%

Section: Discussionmentioning

confidence: 96%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

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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100

126

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“…The bent form inhibits SERCA because of protein-protein interactions between the transmembrane regions of PLB and SERCA (65). Only in the extended form, in which both the cytoplasmic domain and the transmembrane domain of PLB binds to SERCA, the SERCA-PLB complex is capable of reversing SERCA inhibition, because of phosphorylation or mutation (65,66).…”

Section: Introductionmentioning

confidence: 99%

“…Additionally, EPR spectroscopic studies from the Veglia and Thomas groups indicate that a two-state dynamic equilibrium exists for the cytosolic segment of AFA-PLB in a lipid bilayer (58,65,66). Similarly, the Chou group suggested that their structure of PLB in micelles sets the stage for a series of future experiments to characterize the effect of phosphorylation on the orientation and dynamics of its cytoplasmic helix (61).…”

Section: Introductionmentioning

confidence: 99%

Phospholamban and Its Phosphorylated Form Interact Differently with Lipid Bilayers: A³¹P,²H, and¹³C Solid-State NMR Spectroscopic Study

Abu-Baker

Lorigan

2006

Biochemistry

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Phospholamban (PLB) is a 52-amino acid integral membrane protein that helps to regulate the flow of Ca 2+ ions in cardiac muscle cells. Recent structural studies on the PLB pentamer and the functionally active monomer (AFA-PLB) debate whether its cytoplasmic domain, in either the phosphorylated or dephosphorylated states, is α-helical in structure as well as whether it associates with the lipid head groups [Oxenoid, K. (2005 Comparing the secondary structure of the PLB pentamer and its phosphorylated form (P-PLB) as well as their interaction with the lipid bilayer is crucial in order to understand its regulatory function. Therefore, in this study, the full-length wild-type (WT)-PLB and P-PLB were incorporated into 1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine (POPC) phospholipid bilayers and studied utilizing solid-state NMR spectroscopy. The analysis of the 2 H and 31 P solid-state NMR data of PLB and P-PLB in POPC multilamellar vesicles (MLVs) indicates that a direct interaction takes place between both proteins and the phospholipid head groups. However, the interaction of P-PLB with POPC bilayers was less significant when compared to PLB. Moreover, the secondary structure using 13 C=O site-specific isotopically labeled Ala15-PLB and Ala15-P-PLB in POPC bilayers suggests that this residue, located in the cytoplasmic domain, is a part of an α-helical structure for both PLB and P-PLB.

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Spin Labels and Spin Probes

Bardelang

Hardy

Ouari

et al. 2012

Encyclopedia of Radicals in Chemistry, Biology and Materials

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The electron paramagnetic resonance (EPR) parameters of a free radical depend strongly on its microenvironment and stable radicals can be used as reporter molecules for the EPR probing of various chemical and biological systems. Nowadays, nitroxides and trityl radicals are the most important spin labels or spin probes and their use in various applications, spin labeling, spin trapping, oximetry, pH measurement, evaluation of redox status, interactions in supramolecular systems, and so on, are examined.

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Phosphorylation-dependent Conformational Switch in Spin-labeled Phospholamban Bound to SERCA

Cited by 104 publications

References 40 publications

Spectroscopic validation of the pentameric structure of phospholamban

Spectroscopic validation of the pentameric structure of phospholamban

Phospholamban and Its Phosphorylated Form Interact Differently with Lipid Bilayers: A³¹P,²H, and¹³C Solid-State NMR Spectroscopic Study

Spin Labels and Spin Probes

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Phosphorylation-dependent Conformational Switch in Spin-labeled Phospholamban Bound to SERCA

Cited by 104 publications

References 40 publications

Spectroscopic validation of the pentameric structure of phospholamban

Spectroscopic validation of the pentameric structure of phospholamban

Phospholamban and Its Phosphorylated Form Interact Differently with Lipid Bilayers: A31P,2H, and13C Solid-State NMR Spectroscopic Study

Spin Labels and Spin Probes

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Phospholamban and Its Phosphorylated Form Interact Differently with Lipid Bilayers: A³¹P,²H, and¹³C Solid-State NMR Spectroscopic Study