Two-Dimensional Solid-State NMR Reveals Two Topologies of Sarcolipin in Oriented Lipid Bilayers

Buffy, Jarrod J.; Traaseth, Nathaniel J.; Mascioni, Alessandro; Gor’kov, Peter L.; Chekmenev, Eduard Y.; Brey, Wallace S.; Veglia, Gianluigi

doi:10.1021/bi060728d

Cited by 48 publications

(67 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The solid-state NMR assignments (Table S1) were carried out by using extensive selective labeling and a combinatorial algorithm for minimizing the resonance positions based on the periodic nature of the helices (22). Fig.…”

Section: Resultsmentioning

confidence: 99%

Structure and topology of monomeric phospholamban in lipid membranes determined by a hybrid solution and solid-state NMR approach

Traaseth

Shi²,

Verardi

et al. 2009

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

Self Cite

158

194

View full text Add to dashboard Cite

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 domains. The N-terminal amphipathic helical domain Ia (residues 1-16) rests on the surface of the lipid membrane with the hydrophobic face of domain Ia embedded in the membrane bilayer interior. The helix comprised of domain Ib (residues 23-30) and transmembrane domain II (residues 31-52) traverses the bilayer with a tilt angle of Ϸ24°. The specific interactions between PLN and lipid membranes may represent an additional regulatory element of its inhibitory function. We propose this hybrid method for the simultaneous determination of structure and topology for membrane proteins with compact folds or proteins whose spatial arrangement is dictated by their specific interactions with lipid bilayers.hybrid method ͉ membrane proteins ͉ oriented solid-state NMR ͉ molecular modeling ͉ PISEMA S tructure and topology are central to membrane protein function (1). Recently determined high-resolution structures reveal the compact folds for several membrane proteins, such as electron and proton-conducting proteins involved in photosynthesis and respiration (http://blanco.biomol.uci.edu/ Membrane Proteins xtal.html). However, a significant population of membrane proteins does not possess a compact tertiary fold, but has its fold space defined through interactions of secondary structure elements (helices, turns, and loops) with the lipid membrane, i.e., the topology (1). This is the case for phospholamban (PLN), a mammalian protein that is essential in the regulation of cardiac muscle contractility (2), and that has recently become a major target for gene therapy to ameliorate cardiomyopathies (3, 4). PLN is located in the sarco(endo)plasmic reticulum (SR) of cardiac myocytes, inhibiting the SR Ca 2ϩ -ATPase (SERCA) by shifting its relative Ca 2ϩ affinity (5). In vitro and in vivo experiments have shown PLN to exist as a homopentamer that deoligomerizes into active monomers that bind SERCA in a 1:1 molar ratio (6). The monomeric form of PLN exists in equilibrium between a dynamically disordered R state and a more restricted T state (7,8) and has 3 ...

show abstract

Section: Resultsmentioning

confidence: 99%

Structure and topology of monomeric phospholamban in lipid membranes determined by a hybrid solution and solid-state NMR approach

Traaseth

Shi²,

Verardi

et al. 2009

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

Self Cite

158

194

View full text Add to dashboard Cite

show abstract

“…Oriented lipid bilayer preparations on glass plate supports have been described previously for a 4/1 molar mixture of DOPC/DOPE (18,53 PISEMA spectra were acquired at a 14.1-T field strength ( 1 H frequency of 600.1 MHz) equipped with a Bruker DMX spectrometer (National High Magnetic Field Laboratory, Tallahassee, FL). The 2D PISEMA experiments (27) were performed at an RF field…”

Section: Methodsmentioning

confidence: 99%

Spectroscopic validation of the pentameric structure of phospholamban

Traaseth¹,

Verardi

Torgersen

et al. 2007

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

Self Cite

100

126

View full text Add to dashboard Cite

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 ...

show abstract

“…SLN comprises a single transmembrane (TM) helix, plus a small cytoplasmic phosphorylation domain and a short lumenal tail (2,7,(13)(14)(15). Dephosphorylated SLN inhibits the apparent calcium affinity of SERCA (1, 2, 16 -19) and decreases the calcium/ATP coupling efficiency (20,21).…”

Section: Sarcolipin (Sln)mentioning

confidence: 99%

Oligomeric Interactions of Sarcolipin and the Ca-ATPase

Autry

Rubin

Pietrini

et al. 2011

Journal of Biological Chemistry

View full text Add to dashboard Cite

We have detected directly the interactions of sarcolipin (SLN) and the sarcoplasmic reticulum Ca-ATPase (SERCA) by measuring fluorescence resonance energy transfer (FRET) between fusion proteins labeled with cyan fluorescent protein (donor) and yellow fluorescent protein (acceptor). SLN is a membrane protein that helps control contractility by regulating SERCA activity in fast-twitch and atrial muscle. Here we used FRET microscopy and spectroscopy with baculovirus expression in insect cells to provide direct evidence for: 1) oligomerization of SLN and 2) regulatory complex formation between SLN and the fast-twitch muscle Ca-ATPase (SERCA1a isoform). FRET experiments demonstrated that SLN monomers self-associate into dimers and higher order oligomers in the absence of SERCA, and that SLN monomers also bind to SERCA monomers in a 1:1 binary complex when the two proteins are coexpressed. FRET experiments further demonstrated that the binding affinity of SLN for itself is similar to that for SERCA. Mutating SLN residue isoleucine-17 to alanine (I17A) decreased the binding affinity of SLN self-association and converted higher order oligomers into monomers and dimers. The I17A mutation also decreased SLN binding affinity for SERCA but maintained 1:1 stoichiometry in the regulatory complex. Thus, isoleucine-17 plays dual roles in determining the distribution of SLN homooligomers and stabilizing the formation of SERCA-SLN heterodimers. FRET results for SLN self-association were supported by the effects of SLN expression in bacterial cells. We propose that SLN exists as multiple molecular species in muscle, including SERCA-free (monomer, dimer, oligomer) and SERCA-bound (heterodimer), with transmembrane zipper residues of SLN serving to stabilize oligomeric interactions. Sarcolipin (SLN)4 is a 3-kDa membrane protein that reversibly inhibits the activity of the calcium-transporting ATPase (SERCA) in sarcoplasmic reticulum (SR) of fast-twitch, slowtwitch, and atrial muscle (1, 2). SERCA is a 110-kDa membrane protein that relaxes muscle by pumping calcium out of the cytoplasm using energy derived from ATP hydrolysis (3). Transgenic mouse studies have demonstrated that knock-out of SLN enhances atrial contractility (4), cross-expression of SLN inhibits ventricular contractility (2, 5, 6), and ␤-adrenergic stimulation relieves SLN inhibition of contractility (2, 6, 7). Phosphorylation/dephosphorylation of SLN is responsible for SERCA regulation, controlling the rate and amount of calcium loading in SR, which in turn determines the rate of muscle relaxation and the force of subsequent contraction (2, 4 -7). SLN gene expression is up-regulated 2-15-fold in patients with skeletal muscle dysferlinopathy and Takotsubo cardiomyopathy, but down-regulated 2-3-fold in patients with heart failure, atrial fibrillation, and congenital heart defects, indicating that SLN acts as a causative or compensatory factor in human diseases of skeletal and cardiac muscle (8 -12).SLN comprises a single transmembrane (TM) helix, plus a small cytoplasm...

show abstract

Two-Dimensional Solid-State NMR Reveals Two Topologies of Sarcolipin in Oriented Lipid Bilayers

Cited by 48 publications

References 35 publications

Structure and topology of monomeric phospholamban in lipid membranes determined by a hybrid solution and solid-state NMR approach

Structure and topology of monomeric phospholamban in lipid membranes determined by a hybrid solution and solid-state NMR approach

Spectroscopic validation of the pentameric structure of phospholamban

Oligomeric Interactions of Sarcolipin and the Ca-ATPase

Contact Info

Product

Resources

About