Structural and Dynamic Basis of Phospholamban and Sarcolipin Inhibition of Ca<sup>2+</sup>-ATPase

Traaseth, Nathaniel J.; Ha, Kim N.; Verardi, Raffaello; Shi, Lei; Buffy, Jarrod J.; Masterson, Larry R.; Veglia, Gianluigi

doi:10.1021/bi701668v

Cited by 117 publications

(122 citation statements)

References 62 publications

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“…4C). This topology agrees with rotational dynamics measurements carried out by EPR (51) and solid-state NMR (4). MD calculations show that the pinwheel structure does not deviate significantly from the initial topology, with the domains Ia maintaining a tight association with the lipid bilayer (Fig.…”

Section: Discussionsupporting

confidence: 88%

“…PLN is a 52-residue transmembrane (TM) protein highly conserved across mammals (2). Its helix-loop-helix secondary structure is further subdivided into four dynamic domains: domain Ia (1-16), loop (17)(18)(19)(20)(21)(22), domain Ib (23)(24)(25)(26)(27)(28)(29)(30), and domain II (3,4). The hydrophobic TM domain II is the most conserved and responsible for SERCA inhibition, whereas the cytoplasmic domain harbors two phosphorylation sites that reverse PLN inhibitory function (2).…”

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confidence: 99%

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Structural topology of phospholamban pentamer in lipid bilayers by a hybrid solution and solid-state NMR method

Verardi

Shi

Traaseth

et al. 2011

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

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155

210

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Phospholamban (PLN) is a type II membrane protein that inhibits the sarcoplasmic reticulum Ca 2þ -ATPase (SERCA), thereby regulating calcium homeostasis in cardiac muscle. In membranes, PLN forms pentamers that have been proposed to function either as a storage for active monomers or as ion channels. Here, we report the T-state structure of pentameric PLN solved by a hybrid solution and solid-state NMR method. In lipid bilayers, PLN adopts a pinwheel topology with a narrow hydrophobic pore, which excludes ion transport. In the T state, the cytoplasmic amphipathic helices (domains Ia) are absorbed into the lipid bilayer with the transmembrane domains arranged in a left-handed coiled-coil configuration, crossing the bilayer with a tilt angle of approximately 11°with respect to the membrane normal. The tilt angle difference between the monomer and pentamer is approximately 13°, showing that intramembrane helix-helix association forces dominate over the hydrophobic mismatch, driving the overall topology of the transmembrane assembly. Our data reveal that both topology and function of PLN are shaped by the interactions with lipids, which fine-tune the regulation of SERCA.hybrid NMR method | PISEMA | calcium regulation | oligomeric protein | dipolar assisted rotational resonance recoupling T he membrane protein complex formed by Ca 2þ -ATPase (SERCA) and phospholamban (PLN) regulates Ca 2þ concentration within the sarcoplasmic reticulum (SR), thereby controlling muscle excitation-contraction coupling (1, 2). PLN is a 52-residue transmembrane (TM) protein highly conserved across mammals (2). Its helix-loop-helix secondary structure is further subdivided into four dynamic domains: domain Ia (1-16), loop (17)(18)(19)(20)(21)(22), and domain II (31-52) (3, 4). The hydrophobic TM domain II is the most conserved and responsible for SERCA inhibition, whereas the cytoplasmic domain harbors two phosphorylation sites that reverse PLN inhibitory function (2). PLN has a direct role in the pathophysiology of the heart muscle, with three lethal mutations linked to dilated cardiomyopathy in humans (R9C-PLN, R14del, and L39-truncated-PLN) (5). In both synthetic and cell membranes, PLN forms pentamers that dissociate into monomers upon interacting with SERCA (1, 6). Although the stoichiometry of the SERCA/PLN complex has been assessed (1, 6), both the role and the structure of the PLN pentamer remain a matter of active debate. Because PLN expression in both atria and ventricles is higher than SERCA, it is likely that oligomerization may participate in SERCA regulation (7). Insights into PLN organization in the membrane have come from biochemical and biophysical data (2,6,8). Initial electrophysiological measurements indicated that PLN formed Ca 2þ channels (9). However, more recent electrochemical studies concluded that PLN does not conduct Cl − or Ca 2þ ions (10).Divergent structural models for the PLN pentamer have been proposed in the literature (8). Although very similar in the secondary structure content, these models differ in t...

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Section: Discussionsupporting

confidence: 88%

mentioning

confidence: 99%

Structural topology of phospholamban pentamer in lipid bilayers by a hybrid solution and solid-state NMR method

Verardi

Shi

Traaseth

et al. 2011

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

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155

210

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“…dues [23][24][25][26][27][28][29][30], and II (residues 31-52) (9). As measured by NMR (10,11) and EPR spectroscopies (7,8,12,13), the T state is predominant (Ϸ84%) in both micelles and lipid bilayers (14).…”

mentioning

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.

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194

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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 ...

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“…The superior performance of the HE-PELF over the conventional PELF experiment is demonstrated for a U-15 N single-pass membrane protein sarcolipin 42,46,47 reconstituted in magnetically aligned DMPC/D6PC bicelles. Top panel of Figure 5 shows the PELF, H1-PELF, and H-PELF spectra plotted at the same noise level.…”

Section: Resultsmentioning

confidence: 99%

Proton evolved local field solid-state nuclear magnetic resonance using Hadamard encoding: Theory and application to membrane proteins

Gopinath¹,

Mote²,

Veglia

2011

The Journal of Chemical Physics

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NMR anisotropic parameters such as dipolar couplings and chemical shifts are central to structure and orientation determination of aligned membrane proteins and liquid crystals. Among the separated local field experiments, the proton evolved local field (PELF) scheme is particularly suitable to measure dynamically averaged dipolar couplings and give information on local molecular motions. However, the PELF experiment requires the acquisition of several 2D datasets at different mixing times to optimize the sensitivity for the complete range of dipolar couplings of the resonances in the spectrum. Here, we propose a new PELF experiment that takes the advantage of the Hadamard encoding (HE) to obtain higher sensitivity for a broad range of dipolar couplings using a single 2D experiment. The HE scheme is obtained by selecting the spin operators with phase switching of hard pulses. This approach enables one to detect four spin operators, simultaneously, which can be processed into two 2D spectra covering a broader range of dipolar couplings. The advantages of the new approach are illustrated for a U-15 N NAL single crystal and the U-15 N labeled single-pass membrane protein sarcolipin reconstituted in oriented lipid bicelles. The HE-PELF scheme can be implemented in other multidimensional experiments to speed up the characterization of the structure and dynamics of oriented membrane proteins and liquid crystalline samples.

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Structural and Dynamic Basis of Phospholamban and Sarcolipin Inhibition of Ca²⁺-ATPase

Cited by 117 publications

References 62 publications

Structural topology of phospholamban pentamer in lipid bilayers by a hybrid solution and solid-state NMR method

Structural topology of phospholamban pentamer in lipid bilayers by a hybrid solution and solid-state NMR method

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

Proton evolved local field solid-state nuclear magnetic resonance using Hadamard encoding: Theory and application to membrane proteins

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Structural and Dynamic Basis of Phospholamban and Sarcolipin Inhibition of Ca2+-ATPase

Cited by 117 publications

References 62 publications

Structural topology of phospholamban pentamer in lipid bilayers by a hybrid solution and solid-state NMR method

Structural topology of phospholamban pentamer in lipid bilayers by a hybrid solution and solid-state NMR method

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

Proton evolved local field solid-state nuclear magnetic resonance using Hadamard encoding: Theory and application to membrane proteins

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Structural and Dynamic Basis of Phospholamban and Sarcolipin Inhibition of Ca²⁺-ATPase