Solid-state NMR Reveals Structural Changes in Phospholamban Accompanying the Functional Regulation of Ca2+-ATPase*

Eleri Hughes and David A. Middleton {ddagger}

From the Department of Biomolecular Sciences, University of Manchester Institute of Science and Technology, Manchester M60 1QD, United Kingdom

Received for publication, December 2, 2002 , and in revised form, January 28, 2003.
    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Calcium transport across the sarcoplasmic reticulum of cardiac myocytes is regulated by a reversible inhibitory interaction between the Ca2+-ATPase and the small transmembrane protein phospholamban (PLB). A nullcysteine analogue of PLB, containing isotope labels in the transmembrane domain or cytoplasmic domain, was reconstituted into membranes in the absence and presence of the SERCA1 isoform of Ca2+-ATPase for structural investigation by cross-polarization magic-angle spinning (CP-MAS) NMR. PLB lowered the maximal hydrolytic activity of SERCA1 and its affinity for calcium in membrane preparations suitable for structural analysis by NMR. Novel backbone amide proton-deuterium exchange CP-MAS NMR experiments on the two PLB analogues co-reconstituted with SERCA1 indicated that labeled residues Leu42 and Leu44 were situated well within the membrane interior, whereas Pro21 and Ala24 lie exposed outside the membrane. Internuclear distance measurements on PLB using rotational resonance NMR indicated that the sequences Pro21–Ala24 and Leu42–Leu44 adopt an {alpha}-helical structure in pure lipid bilayers, which is unchanged in the presence of Ca2+-ATPase. By contrast, rotational echo double resonance (REDOR) NMR experiments revealed that the sequence Ala24–Gln26 switches from an {alpha}-helix in pure lipid membranes to a more extended structure in the presence of SERCA1, which may reflect local structural distortions which change the orientations of the transmembrane and cytoplasmic domains. These results suggest that Ca2+-ATPase has a long-range effect on the structure of PLB around residue 25, which promotes the functional association of the two proteins.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Phospholamban (PLB)1 is a 52-amino acid membrane-spanning protein, which is expressed predominantly in the sarcoplasmic reticulum of cardiac myocytes (1). The primary physiological function of PLB is to regulate the active transport of calcium ions into the sarcoplasmic reticulum lumen via an inhibitory association with SERCA2a, the cardiac isoform of Ca2+-ATPase (2, 3). PLB is believed to bind to the calcium-free conformation of SERCA2a (4), and exerts its inhibitory action by reducing the apparent calcium affinity of the enzyme (5). In response to {beta}-adrenergic stimulation, PLB is phosphorylated at Ser16 and Thr17 by cAMP-dependent kinase and calcium/calmodulin-dependent kinase, respectively. PLB phosphorylation relieves SERCA2a inhibition, which increases the rate of calcium uptake into the sarcoplasmic reticulum and results in the accelerated relaxation of cardiac muscle (2). Mechanistic failures in the reversibility of SERCA2a inhibition, or overexpression of PLB relative to SERCA2a, are linked to the disruption of calcium homeostasis in cardiac cells and may contribute to cardiovascular disorders such as congestive heart failure (6).

The exact nature of the interaction between PLB and SERCA enzymes is a subject of debate. PLB readily self-associates to form a homopentamer within the lipid bilayer (7, 8), with a dynamic equilibrium believed to exist between the oligomeric and monomeric state of the protein (9). Most evidence suggests that it is the monomeric form of PLB that binds to and inhibits SERCA, with the pentamer acting as a reservoir from which monomers dissociate (5, 8, 10, 11, 12). In reconstitution studies, the molar stoichiometry of PLB to SERCA required for maximal regulation of calcium transport is between 3:1 and 15:1 (12, 13), whereas a fluorescence energy transfer study has suggested that two PLB monomers interact with two Ca2+ATPase molecules to form a heterodimer (14). In the latter study, it was found that both PLB molecules must be phosphorylated in order to restore the activity of the calcium pump. In mutational studies, Asahi et al. (15) produced functional and physical data to show that PLB interacts with the transmembrane helix M6 of Ca2+-ATPase. By contrast, co-crystallization studies have led to the proposal that a single PLB monomer interacts asymmetrically with two SERCA molecules such that the transmembrane domain of PLB lies close to the M3 helix of SERCA (16). In support of the latter argument, thiol cross-linking analysis demonstrated that PLB residue 30 is in close proximity to Cys318 in helix M4 of Ca2+ATPase (17). Hence, there is still disagreement over the actual interaction sites between PLB and SERCA.

The mechanism for the reversal of SERCA2a inhibition is not fully understood. Although it is clear that phosphorylation of PLB restores SERCA activity, the process does not necessarily involve dissociation of PLB from the enzyme. Rather, the reactivation of the calcium pump may involve a structural rearrangement of PLB at the protein-protein interface (18, 19). Indeed, provided SERCA is in a calcium-free state, the interactions between it and PLB remain stable, and dissociation of the two proteins occurs only under conditions of elevated Ca2+ (19). In addition to contacts with M6, further mutational studies have demonstrated an interaction site between PLB domain IB (residues 17–32) and the L67 loop region of Ca2+ATPase, which links helices M6 and M7 (15). Phosphorylation of PLB may lead to disruption in this region while leaving other interactions intact (15). Consistent with this is the finding that an N27A PLB mutant, although having a normal pentamer to monomer equilibrium, results in super-inhibition of SERCA, which is not relieved by phosphorylation. The conformational alterations generated by the N27A mutation must prevent phosphorylation from overcoming the inhibitory interactions between PLB and SERCA and thus highlights the importance of this region (20).

There remains no clear consensus on the three-dimensional structure of PLB in phospholipid membranes, and the protein is probably structurally sensitive to environmental conditions and to its oligomeric and phosphorylation states. It is generally agreed that the protein structure comprises a largely helical cytoplasmic domain (1a), a helical transmembrane domain (II), and a structurally variable domain (Ib) containing a putative hinge site. NMR studies of PLB in solution have shown that the protein is predominantly {alpha}-helical but is disrupted by a kink in the polypeptide backbone around Pro21, which may act as a hinge between the cytoplasmic and transmembrane domains (21, 22). Solid-state NMR studies have provided local details of the molecular structure of PLB when reconstituted into lipid membranes that mimic the physiological environment of the protein. Selective 13C–13C and 13C–15N interatomic distances along the backbone of wild-type PLB have been measured using the cross-polarization magic-angle spinning (CP-MAS) NMR techniques, rotational echo double resonance (REDOR) and rotational resonance (23). The measurements suggested that the wild-type protein, which is predominantly pentameric, is a continuous {alpha}-helix, even across the region containing the putative proline hinge (23). However, a recent 15N solid-state NMR study of a null-cysteine PLB analogue in uniaxially aligned lipid bilayers has shown that the transmembrane and cytoplasmic domains of the protein are approximately perpendicular to each other (24). Such a structure supports the existence of a hinge region in which the protein backbone deviates from a continuous {alpha}-helical structure. The striking structural differences observed in the two solid-state NMR studies suggest that PLB may undergo conformational transitions as it moves between monomeric and pentameric states. The structure of PLB when associated with Ca2+-ATPase is not known, but a recent fluorescence quenching and time-resolved anisotropy study on a fluorophore-derivatized mutant of PLB has shown that free and ATPase-bound PLB have distinct structures and motional characteristics (25).

In the light of these recent structural insights (21, 23, 24), this work has examined how and where the structure of PLB is affected by its interactions with Ca2+-ATPase, using CP-MAS NMR as a non-perturbing technique to measure interatomic distances within PLB in ATPase-active membranes. Membranes containing null-cysteine, monomeric PLB, and the fasttwitch skeletal muscle (SERCA1) isoform of Ca2+-ATPase were prepared containing the proteins in suitable concentrations for NMR analysis and in which PLB demonstrably regulated ATPase activity. For the NMR analysis two phospholamban samples (PLBI and PLBII) were prepared containing 13C and 15N labels at selective sites in the transmembrane and cytoplasmic domains (Fig. 1). The membrane topology and structure of PLB around the labeled sites was elucidated using a combination of CP-MAS NMR methods to measure linear through-space distances between the labels. New insights into the regulatory mechanism of PLB were gained by comparing the structural details for PLB in Ca2+-ATPase-free membranes with its structure in the presence of Ca2+-ATPase. The accumulation of structural information will be important in the long term for understanding the molecular basis for cardiac disorders such as congestive heart failure.



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FIG. 1.
The primary sequence of human PLB showing the isotope labeling schemes in the two synthetic polypeptides PLB I and PLB II. All 13C and 15N labels were placed in the peptide backbone. A "[1-13C]" prefix denotes labeling at a backbone carbonyl site, and "[2-13C]" denotes labeling at an {alpha}-carbon site. The distances refer to the linear through-space separation of isotope pairs as predicted for an {alpha}-helical structure spanning the region of the labels.

 


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Synthetic PLB was purchased from the Department of Biochemistry, University of Southampton. The three cysteine residues (Cys36, Cys41, Cys46) were replaced by alanine to avoid protein oligomerization (26). Two individual proteins, PLBI and PLBII, were prepared with the labeling schemes shown in Fig. 1.

The detergent n-octyl-{beta}-D-glucopyranoside ({beta}OG) was purchased from Melford Laboratories, and 1,2-bis(2-aminophenoxy)ethaneN,N,N',N'-tetraacetic acid (tetrasodium salt) (BAPTA) was purchased from Molecular Probes. All other chemicals were purchased from Sigma.

Preparation of Ca2+-ATPase—SERCA1 Ca2+-ATPase was prepared from fast-twitch rabbit skeletal muscle according to a method adapted from East and Lee (27). The protein was characterized by SDS-PAGE on a 10% resolving gel. Approximately 150 mg of protein was obtained in one preparation from 100 g of muscle, with concentrations calculated using an adaptation of the Lowry method (28).

Reconstitution of Ca2+-ATPase—SERCA1 was reconstituted into dioleoyl-phosphatidylcholine (DOPC) membranes using an adaptation of methods described previously (8, 29) but with protein concentrations suitable for NMR analysis. Briefly, DOPC in chloroform was dried to a thin film with argon before drying further under vacuum and rehydrating with 1–2 ml of 10 mM Tris, 0.25 M sucrose buffer, pH 7.5, containing 6 mg/ml {beta}OG. C12E8 to 1 mg/ml was added to the required volume of Ca2+-ATPase before adding the protein-detergent mixture to DOPC. The mixture was left for 15 min at room temperature then at 4 °C for 45 min.

Detergent was removed using Amberlite XAD2 resin (~100 mg/ml/h) with stirring for 3 h at room temperature. The resin beads were removed by filtering through muslin and centrifugation at 2000 rpm. Finally a sample of the suspension was centrifuged on a 10–40% density gradient at 60,000 x g for 12 h to confirm successful reconstitution.

Reconstitution of PLB—Dimyristoylphosphatidylcholine (DMPC) and PLBI, PLBII, or both were dissolved in 50:50 chloroform:methanol at a molar ratio of 20:1 and dried down to a film as described earlier. Samples were then rehydrated in 10 mM phosphate, pH 7, and centrifuged at 13,000 rpm in a bench-top microcentrifuge. Pellets were stored at –20 °C until required for NMR.

Co-reconstitution of Ca2+-ATPase with PLB—Co-reconstitution of Ca2+ATPase and PLB were carried out by an adaptation of a previous method (8, 29). Initial molar ratios of DOPC/PLB/Ca2+-ATPase were 500:20:1 or 160:10:1. DOPC and PLB were dissolved in 2–5 ml of 50:50 chloroform/methanol and dried as described above. Following rehydration (10 mM Tris, 0.25 M sucrose, pH 7.5, 6 mg/ml {beta}OG) the sample was sonicated. Ca2+-ATPase was then added as described before and incubation carried out. Detergent was removed using XAD2 resin or, for NMR samples, by diluting with 10 mM phosphate, pH 7, and centrifuging at 200,000 x g for 45 min at 4 °C. Finally a sample of the suspension was centrifuged on a 10–40% density gradient at 60,000 x g for 12 h. This provided a single band containing both proteins (as assessed by SDS-PAGE) and exhibiting ATPase and phosphatase activity, which thus confirmed a successful co-reconstitution.

Phosphorylation of PLB—Following co-reconstitution as described above, a 500-µl sample containing 250 µg of PLB was phosphorylated using protein kinase A catalytic subunit (PKA). MgCl2 was added to 10 mM followed by 500 units of PKA. The reaction was initiated by the addition of ATP to 0.6 mM followed by a freeze/thaw cycle and incubation overnight at room temperature. Samples were then taken for ATPase activity measurements. A control co-reconstituted sample was also carried out omitting the PKA but with all other steps identical.

ATPase Activity Measurements—Specific Ca2+-ATPase activity was quantified as the amount of inorganic phosphate (Pi) liberated upon hydrolysis of ATP as measured by formation of a phospho-molybdate complex under acidic conditions. The detergent C12E8 was added to the reaction mixtures to ensure that sealed vesicles were disrupted and that the protein was accessible to reagents on both sides of the membrane.

Samples were made up in a total volume of 0.3 ml of reaction buffer (30 mM Tris, pH 8) with CaCl2 added to give the required free calcium concentration. Following the addition of 0.2 ml of assay medium (4 mM ATP, 4 mM MgCl2, 0.5 mM EGTA, 30 mM imidazole, pH 7.3) samples were incubated for 10 min at 37 °C. The reaction was stopped in 1 ml of quench solution (ice-cold 4.5% ammonium molybdate/perchloric acid, 4:1 v/v) before extraction with 3 ml of n-butylacetate. The mixture was vortexed thoroughly and centrifuged at 2000 rpm for 5 min. The organic layer was then removed and the absorbance read at 320 nm in quartz cuvettes.

The protein concentrations used for calculating specific activity were measured by the Lowry method (28), which detected SERCA1 but was not sensitive to the presence of PLB. Free calcium concentrations were determined essentially as described by Tatulian et al. (30). A buffer solution (1.5 ml) containing 50 µM BAPTA, 20 mM MOPS, 0.1 M KCl, 5 mM MgCl2,5mM ATP, pH 7, and 2 µlofCa2+ATPase stock (160 nM) was prepared. Fluorescence emission scans were carried out on the BAPTA sample at an excitation wavelength of 299 nm, and the emission was measured at a wavelength of 360 nm.

Solid-state NMR Measurements—All solid state NMR experiments were performed on a Bruker Avance 400 spectrometer operating at a magnetic field of 9.3 tesla. CP-MAS experiments were performed by rotating the sample at the magic angle in a 4-mm zirconia rotor. Hartmann-Hahn cross-polarization from 1H to 13C was achieved over a 1.6-ms contact time at a field of 65 kHz, and protons were decoupled during signal acquisition at a field of 85 kHz. REDOR experiments (13C observe, 15N dephase) were conducted at a sample spinning frequency {nu}R of 7000 Hz using a standard spin-echo pulse-sequence with rotorsynchronized {pi} pulses applied at the frequency of 15N (31). Rotational resonance experiments (32) were carried out at sample spinning frequencies of 11000–12500 Hz (n = 1 rotational resonance). Difference intensities, measured from spectra obtained at different mixing times after selective inversion of the carbonyl spins, were simulated numerically to obtain the distance-dependent 13C-13C dipolar coupling constant bIS (32). The sample temperature was maintained at –40 °C (±0.5 °C) in all experiments.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Functional Co-reconstitution—To obtain structural information about PLB when it is functionally associated with Ca2+-ATPase, it was important to develop a reconstitution system in which labeled PLB could be detected by NMR and in which ATPase activity was regulated by PLB. The main requirements of the reconstitution were therefore a high concentration of PLB (1–5 mg in 100 µl of hydrated membranes), a PLB/Ca2+-ATPase ratio giving maximal functional regulation, and a lipid/protein ratio high enough to support ATPase activity. Two reconstitution compositions were examined, one with a lipid/PLB/Ca2+-ATPase ratio of 500:20:1 and one with a lipid/PLB/Ca2+-ATPase ratio of 160:10:1.

Fast-twitch skeletal muscle (SERCA1) Ca2+-ATPase was reconstituted alone or with synthetic null-cysteine PLB into unsaturated lipid (DOPC) membranes using an adaptation of a previously described procedure (29). SERCA1 was examined instead of the cardiac isoform because skeletal muscle readily yields sufficient (milligram) quantities of enzyme for NMR analyses. Previous studies have shown that the functional properties of SERCA1 and its inhibition by PLB are similar to those of SERCA2a (33), and therefore replacement of SERCA2a by SERCA1 is valid here. The null-cysteine analogue of PLB favors the monomeric state, which is believed to regulate Ca2+-ATPase (26) and was therefore studied in preference to wild-type PLB, which forms pentamers. SDS-PAGE of the synthetic PLB reconstituted into pure lipid (DMPC) membranes showed a dominant protein band at 5–6 kDa, which corresponded to monomeric PLB, whereas bands corresponding to higher molecular weight oligomers were insignificant or not observed (data not shown).

The specific hydrolytic activity of reconstituted SERCA1 (lipid/protein ratio of 500:1) was measured at various calcium concentrations (Fig. 2). In both the absence and presence of PLB, maximal ATPase activity was observed at a calcium concentration of 1 µM. The activity was progressively inhibited as the calcium concentration was raised further to 4 µM, an effect that has been reported previously (34). Maximal reconstituted SERCA1 activity in the absence of PLB was 1.1 µmol/mg/min (Fig. 2A), and the Km for calcium dependence was estimated at 0.2 µM (Fig. 2B). In membranes containing PLB and SERCA1 in a molar ratio of 20:1 the maximal activity decreased to 0.4 µmol/mg/min, and Km increased to 0.61 µM (Fig. 2, A and B). Hence, in our hands, PLB both partially inhibited SERCA1 activity and lowered the enzyme affinity for calcium, as has been reported elsewhere for membranes of similar composition (12).



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FIG. 2.
Functional characterization of skeletal muscle (SERCA1) Ca2+-ATPase reconstituted into DOPC membranes. The rate of ATP hydrolysis was measured from SERCA1/DOPC membranes (squares) and from SERCA1/PLB/DOPC membranes at an enzyme/PLB ratio of 1:20 (circles) over the Ca2+ concentration range, 0.04 to 4 µM (A). The lipid/SERCA molar ratio was 500:1 in both cases. The error bars represent the standard deviations of the mean of six independent measurements on the same reconstitutive membrane samples. Normalization of the plots in A to the maximal activities (at 1 µM calcium in both cases) shows that PLB reduces the affinity of SERCA1 for Ca2+ (B). Treatment of the 500:20:1 membranes with PKA raised the specific SERCA1 activity by 275% at 0.4 µM Ca2+ and by 50% at 1 µM Ca2+ (C). The pre- and post-phosphorylation data are shown normalized to the activity of the nontreated membranes at 1 µM Ca2+. Differences in the specific activities ({Delta}A) were obtained by subtracting the activity of SERCA1 co-reconstituted with PLB from the activity of SERCA1 reconstituted alone (D). Mean values of {Delta}A were measured from four individual reconstitutions at calcium concentrations of 0, 0.2, 0.4, and 1 µM. The lipid/SERCA1 ratio in the absence and presence of PLB was 500:1 (squares) or 160:1 (circles) and in co-reconstitutions the SERCA1/PLB ratio was either 20:1 (squares) or 10:1 (circles).

 

Although it is generally accepted that PLB lowers the affinity of Ca2+-ATPase for calcium (5), the effect on Vmax is less well defined. A number of factors appear to be important including lipid to protein ratios and the exact PLB analogue that is used (34, 35, 36, 37, 38). Previous work done on a PLB analogue similar to the one used here demonstrated a similar reduction in Vmaxthe one (35), and in the present work this result has been consistent across a number of separate assays. The important point here is that, in the presence of PLB, the Km is increased as expected, and this is shown more clearly when the data for the 500:20:1 membranes are normalized (Fig. 2B), which is a standard method of activity data presentation (14, 30, 39). In addition, following the phosphorylation of co-reconstituted PLB by incubation with PKA, Ca2+-ATPase activity is re-established (Fig. 2C), demonstrating that the reduction in Vmax is fully reversible and is not due to inactivation or denaturation of the enzyme during reconstitution.

Membranes prepared at a lipid/PLB/SERCA1 ratio of 500: 20:1 were not suitable for structural examination by NMR because of the low protein concentration and the large excess of PLB over SERCA1. The membranes prepared at a lipid/PLB/SERCA1 ratio of 160:10:1 were, in principle, amenable to NMR investigation provided the lower lipid/SERCA and PLB/SERCA ratios supported a functional interaction between the two proteins. Fig. 2D compares the decrease of specific hydrolytic activity ({Delta}A) of DOPC/PLB/SERCA1 membranes in molar ratios of 500:20:1 and 160:10:1 relative to control membranes containing only SERCA1 at the same lipid/protein ratios. The inhibitory effect of PLB was slightly less pronounced in membranes of lower PLB/SERCA1 ratio but nevertheless remained in evidence. Crucially, these experiments confirmed that PLB undergoes a regulatory association with SERCA1 in membranes prepared for NMR analysis.

Topological Analysis of PLB—A novel CP-MAS NMR experiment was developed to ensure that the co-reconstitution procedure incorporated PLB into the lipid bilayer correctly. The experiment invoked proton-deuterium (H/D) exchange to probe the membrane topology of PLB by observing the accessibility of the selectively isotope-labeled sites along the polypeptide backbone in situ and non-invasively in the membrane. When the PLB membranes are suspended in D2O, the rate of H/D exchange at backbone amide positions buried within the lipid bilayer is much slower than H/D exchange at positions in the cytoplasmic domain where exchangeable proton sites are exposed to the aqueous environment.

The H/D exchange procedure exploits the modified REDOR experiment described by Gullion (40), which reintroduces the dipolar interaction between a spin half-nucleus such as 13C and a spin 1 nucleus such as 2H. The recoupled dipolar interaction is observed as a reduction of the peak intensity, S, from the observed nucleus relative to the peak intensity, S0, measured in a control experiment. The drop in the peak intensity, which can be normalized as S/S0, is related to the through-space internuclear distance rIS between the two nuclei and to the experimental echo period te during which dipolar recoupling is allowed to occur.

H/D exchange was monitored indirectly from the 13C observed, 2H dephased REDOR NMR spectrum of PLB with labels in the transmembrane (PLB II) and cytoplasmic (PLB I) domains at selective backbone carbonyl and {alpha}-carbon positions (Fig. 1). Following H/D exchange on the amide (i+1) adjacent to 13C-labeled carbonyl, i, the 2H nucleus dephases the carbonyl 13C signal by an amount dependent on te (set in the REDOR experiment) and rIS, which is structurally fixed at 2.0 Å (Fig. 3A). Similarly, dephasing of a labeled {alpha}-carbon signal will occur following H/D exchange on the adjacent amide in the same residue. The experiment was applied to DOPC membranes containing SERCA1 and an equimolar mixture of PLBI and PLBII (Fig. 1) in a lipid/PLB/SERCA ratio of 160:10:1. The membranes were suspended in solution in 90% D2O, incubated at 4 °C for 15 min, centrifuged to a pellet, flash-frozen, and maintained at –40 °C during 13C,2H-REDOR NMR analysis to prevent further H/D exchange. The echo time, te, was set to 3.5 ms, which is predicted to produce the maximum possible dephasing for an internuclear distance of 2.0 Å (40). By examining mixed membranes of PLBI-labeled [1-13C]Pro21–[2-13C]Ala24 in the cytoplasmic domain and PLBII-labeled [1-13C]Leu42–[2-13C]Leu44 in the transmembrane domain, the extent of H/D exchange at the different sites could be compared directly.



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FIG. 3.
The results of a backbone amide proton-deuterium exchange CP-MAS NMR experiment on mixed membranes of PLBI and PLBII co-reconstituted with SERCA1 (PLB/SERCA1/lipid ratio = 10:1:160). After suspension of the protein in D2O, exposed amide protons are exchanged for deuterons (A). The 13C,2H-REDOR experiment dephases 13C signals from labeled carbon sites (denoted by an asterisk) adjacent to amide groups that have undergone H/D exchange. A REDOR echo time, te, of 3.5 ms gives maximum dephasing for the 13C–2H distances shown. The control, full-echo 13C,2H-REDOR spectrum (te = 3.5 ms; {nu}R = 7000 Hz) shows a broad peak from the carbonyl at 175 ppm and two peaks from the {alpha}-carbons at 53 and 54.5 ppm (B). The dephased echo REDOR spectrum shows diminished intensities relative to the control of the carbonyl peak and C{alpha} peak at 53 ppm (C). The standard 13C CP-MAS spectrum of PLBII in DOPC membranes with SERCA1 confirms that the peak at 54.5 ppm is from the {alpha}-carbon of Leu44 (D). By difference, the peak at 53 ppm in the REDOR spectra is from the {alpha}-carbon of Ala24.

 

The results of the H/D exchange experiment are shown in Fig. 3, B–D. The control REDOR NMR spectrum showed superimposed contributions from PLB in the carbonyl region (170 ppm) and two partially overlapping peaks from the C{alpha} labels at 53 and 54.5 ppm (Fig. 3B). The 2H dephased REDOR spectrum of the co-reconstituted proteins showed a pronounced loss of signal intensity in the carbonyl region relative to the control spectrum (Fig. 3C) indicating that H/D exchange had occurred. In the C{alpha} region of the spectrum, the intensity of the 53 ppm peak diminished relative to the control, but the intensity of the 54.5 ppm peak changed little. A comparison of the control REDOR spectrum with the spectrum of pure PLBII in DOPC membranes (Fig. 3D) assigned the 54.5 ppm peak to the C{alpha} of Leu44 in the transmembrane domain and, by difference, the 53 ppm peak to Ala24 in the cytoplasmic domain. Hence, REDOR dephasing indicates that the extent of amide H/D exchange at Leu44 (and Leu42) is considerably lower than at Ala24 (and Pro21). This experiment therefore confirms that Leu44 is buried within the membrane and that Ala24 lies outside the membrane where it is exposed to the aqueous medium.

Structural Analysis of PLB—The goal of this work was to examine the structure of PLB in a membrane environment in which the protein participates in a functional association with Ca2+-ATPase. Functional studies (Fig. 2) confirmed that the inhibitory interaction between SERCA1 and PLB was reproduced in lipid membranes suitable for NMR analysis. CP-MAS NMR measurements of the co-reconstituted proteins would therefore reflect the structure of PLB when associated with SERCA1. Identical experiments on PLB in the absence of SERCA1 would help to elucidate any structural changes in PLB in the presence of Ca2+-ATPase, which have been detected in fluorescence quenching and anisotropy studies (25).

The high resolution 13C CP-MAS experiments REDOR and rotational resonance were used to probe the local structure of PLB by measuring distances between selectively 13C- and 15N-labeled sites (Fig. 1). In PLBI the labels were placed at structurally informative positions in the cytoplasmic domain close to the putative Pro21 hinge site where conformational changes have been observed following phosphorylation at Ser16/Thr17 (3). The sites were selected such that 13C–13C and 13C–15N distances are related to the backbone torsional angles that define secondary structure (Fig. 1 and Table I). PLBII contains a pair of labels in the transmembrane domain. The transmembrane domain is highly unlikely to deviate from an {alpha}-helical structure (41, 42), and therefore distance measurements in this region would provide a convenient reference against which to test the reliability of measurements of other, potentially more flexible, regions of the protein.


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TABLE I
A summary of internuclear distance measurements for PLB in lipid membranes

 

Rotational resonance experiments were carried out to measure 13C–13C distances between the labeled carbonyl and C{alpha} sites. The sample spinning frequency {nu}R, was adjusted to the first order rotational resonance condition for each pair of 13C labels ({nu}R = {Delta}{nu}, where {Delta}{nu} is the difference in resonance frequencies of the labels) and the carbonyl spin polarization was selectively inverted (32). Sample spinning at rotational resonance selectively reintroduces 13C–13C dipolar interactions, which are measured as magnetization exchange curves of difference intensities at different mixing times. The exchange curve is a function of several variables including chemical shift tensor orientations, which were taken from previous measurements (41), a zero quantum relaxation term, which was estimated with error limits from the spectral line widths and the dipolar coupling constant, bIS, which is to be determined. These terms were included in numerical simulations to find the value of bIS giving the best fit to the experimental curves. Calculation of the internuclear distance rIS from bIS is trivial. Errors in the calculation of rIS were based on uncertainties in the experimental data and on the value of the relaxation term.

In Fig. 4 experimental magnetization exchange curves are shown from PLBI and PLBII reconstituted with SERCA1 into DOPC membranes and from PLBI alone in DMPC membranes. Simulations of magnetization exchange in Fig. 4, which intersect the upper and lower limits of the experimental errors, were used to calculate the range of the internuclear distances consistent with the experimental data (Table I). Also shown in Table I are the predicted internuclear distances between [1-13C]Pro21–[2-13C]Ala24 and [1-13C]Leu42–[2-13C]Leu44 for different secondary structures. The internuclear distance measured for [1-13C]Leu42-[2-13C]Leu44 in the presence of SERCA1 agrees closely with the distance predicted for a helix. Indeed, previous work suggests that the transmembrane region containing Leu42 and Leu44 is {alpha}-helical (42), and therefore the accuracy of the distance measurement has been confirmed. The internuclear distance between [1-13C]Pro21 and [2-13C]Ala24 measured from PLB in the absence of SERCA1 indicates that the sequence Pro21-Gln22-Gln23-Ala24 lies in a helical region of the cytoplasmic domain (Table I). The distance remains the same in membranes containing SERCA1, which suggests that the functional interaction of PLB with Ca2+-ATPase does not involve major structural changes in this region of the protein.



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FIG. 4.
Plots of rotational resonance magnetization exchange for PLBII (A) and PLBI (B and C) reconstituted into DOPC membranes with and without SERCA1 (PLB/SERCA1/lipid ratio = 10:1:160). A series of 13C spectra was obtained at the first order rotational resonance condition ({nu}R = 11263–12800 Hz) with respect to the carbonyl and C{alpha} resonance frequencies. Data points were calculated from the difference in the intensities of the positive and inverted peaks from the labeled sites. Simulations of the experimental data were performed by calculating a series of curves for a range of dipolar coupling constants bIS and zero quantum relaxation times. The curves shown, and the corresponding distances, represent fits to the upper and lower error limits of the experimental data. Error bars were calculated from the noise.

 

Distances between the 15N and 13C labels in PLBI were measured using 13C observe, 15N dephase REDOR NMR (31). Dephasing curves were plotted from measurements of S/S0 at different echo times, and simulations of the dephasing provided the internuclear distance (Fig. 5 and Table I). In DMPC membranes containing only PLBI, curves of S/S0 exhibited no measurable dephasing even at long echo times, indicating that the Ala25–Gln26 distance was beyond the limits of detection and greater than 5 Å (Fig. 5A). The distance expected for an {alpha}-helix is 4.4 Å, which would give rise to a dephasing curve close to the simulation shown in Fig. 5A (solid line). Hence, the sequence Ala24-Arg25-Gln26 is non-helical when PLB is unable to associate with Ca2+-ATPase. A striking change in the dephasing was observed when the experiment was repeated on membranes containing SERCA1 (Fig. 5B). The measured dephasing followed closely the curve calculated for a distance of 4.4 Å, which is consistent with an {alpha}-helix (Fig. 5).



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FIG. 5.
Plots of magnetization dephasing (S/S0) from 13C,15N-REDOR experiments on PLBI. A, values of S/S0 were obtained for PLBI reconstituted into DMPC bilayers at protein/lipid ratio of 1:10. The solid line shows the dephasing calculated for a 13C–15N distance of 4.4 Å, which is the predicted internuclear separation if [2-13C]Ala24 and [15N]Gln lie within an {alpha}-helix. The dashed line shows the dephasing calculated for a 13C–15N distance of 5.0 Å. Values of S/S0 were also obtained for PLBI reconstituted with SERCA1 into DOPC membranes (PLB/SERCA1/lipid ratio = 10:1:160). B, the solid and dashed lines are as described in A. The dotted line represents an internuclear distance of 4.7 Å and is the best fit to the upper error limits of the experimental data. REDOR spectra were recorded at a spinning frequency of 7000 Hz.

 

In summary, the sequence Ala24-Arg25-Gln6 in the cytoplasmic domain of PLB undergoes a conformational transition from a non-helical structure in the absence of SERCA1 to a helix in the presence of the enzyme. The neighboring region Pro21-Gln22-Gln23-Ala24 in the cytoplasmic domain appears to be conformationally stable and remains helical.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Functional Association of PLB and Ca2+-ATPase—A physiologically important interaction between PLB and SERCA2a has been identified in vivo and in vitro in both functional and physical studies. Physical interactions between SERCA2a and PLB co-expressed in HEK-293 cells were detected by co-immunoprecipitation of the two proteins by an anti-PLB monoclonal antibody (43). The elevation of calcium from micromolar to millimolar concentration reduced the amount of co-immunoprecipitation by 80%, indicating that SERCA and PLB dissociate at high calcium concentrations. By contrast, the co-immunoprecipitation experiments showed that phosphorylation of PLB at Ser16 by protein kinase A did not result in the dissociation of the two proteins (43). Further studies on the interaction have been carried out using Cys-scanning mutagenesis of PLB and the cross-linking agent, 1,6-bismaleimidohexane (17). At low calcium concentrations the N30C mutant of PLB could be cross-linked with SERCA2a in insect cell microsomes. Sequence analysis of a digested peptide fragment of the cross-linked proteins indicated that the site of protein-protein interaction might include residues around Cys318 in transmembrane helix 4 of SERCA2a. Cross-linking was abolished at millimolar concentrations of calcium in parallel with the restoration of ATPase activity (17), which was consistent with the dissociation of the two proteins at high calcium concentrations. Recent structural insights into the conformational changes of Ca2+-ATPase during its catalytic cycle have shown that the association and dissociation of Ca2+ from the enzyme involve large rearrangements of the transmembrane helices as well as in the cytoplasmic domain (44). If in the presence of PLB the affinity for calcium were reduced, higher concentrations of calcium would be required to activate the catalytic cycle of the enzyme. Once activated, the large-scale conformational changes of Ca2+-ATPase may provide the mechanism that triggers the dissociation of PLB. However, kinetic measurements have shown that PLB alters the activation barrier for the conformational change accompanying calcium activation of Ca2+-ATPase (45).

Here, a synthetic null-cysteine analogue of PLB was functionally reconstituted with the skeletal muscle isoform of Ca2+-ATPase into DOPC membranes. The initial molar ratio of PLB to SERCA1 in the reconstitution mixture prior to detergent removal and centrifugation was either 20:1 or 10:1. The membranes were similar in composition to those examined by Reddy et al. (12), but the initial PLB/SERCA ratio was lower than that required for ATPase inhibition in much earlier work (46). The molar ratio in the final membrane preparation was not determined, but previous studies using the same reconstitution procedure have shown that the final molar ratio is ~60% of the initial value at the beginning of the reconstitution procedure (12). Hence, more realistic estimates of the PLB/SERCA ratios in the reconstituted membranes are ~12:1 and 6:1.

The functional measurements confirmed that PLB interacts with SERCA1 and regulates its activity in a membrane composition that is suitable for structural analysis by solid-state NMR, with maximal inhibition occurring at calcium concentrations between 0.4 and 1 µM (Fig. 2).

Structural Changes in PLB—Despite the publication of several NMR studies of the molecular details of PLB (21, 22, 23, 24), there remains little agreement on the structure of the protein when embedded in lipid membranes. Indeed, PLB probably exists in multiple structural forms, which are dependent on environment, oligomeric, and phosphorylation states and on interactions with Ca2+-ATPase. There is agreement that PLB is predominantly {alpha}-helical in the membrane, but a major point of conflict is in the angle between the transmembrane and cytoplasmic domains. Selective distance measurements of wild-type PLB by rotational resonance and REDOR NMR indicate that the protein is a continuous helix; somewhat surprisingly, the helix is unbroken around Pro21 (23). Solid-state 15N NMR studies on a null-cysteine analogue of PLB in uniformly aligned lipid bilayers showed that the interhelical angle between the transmembrane and cytoplasmic domain Ia is ~90°, that is, the two domains are close to perpendicular (24). Although these two solid-state NMR studies (23, 24) are apparently contradictory, they may reflect structural differences between the pentameric form of wild-type PLB and the monomeric state of the null-cysteine analogue.

Recent fluorescent quenching and anisotropy studies on fluorophore-labeled PLB have revealed that its interactions with Ca2+-ATPase provoke structural changes in the cytoplasmic domain of PLB (25). To investigate the structural changes in more detail, the conformation of the cytoplasmic sequence from Pro21 to Gln26 was compared in the absence and presence of SERCA1. Distance measurements using rotational resonance NMR confirmed that the region Pro21-Gln22-Gln23-Ala24 is helical in SERCA1-free membranes and remains helical in membranes containing SERCA1 (Fig. 4 and Table I). However, the sequence Ala24-Arg25-Gln26 appears to be structurally sensitive to the presence of Ca2+-ATPase. The 13C–15N distance between labels in Ala24 and Gln26 was greater than 5 Å in the absence of SERCA1 (Fig. 5A). In the presence of SERCA1, the distance shortened significantly to 4.4 Å (Fig. 5 and Table I), which indicates that interactions of PLB with the ATPase cause the sequence Ala24-Arg25-Gln26 to adopt a helical structure.

The 13C–15N distance of more than 5 Å in the absence of SERCA1 could not be translated unambiguously into a backbone conformation of the Ala24-Arg25-Gln26 region because the labeled sites were separated by too many degrees of rotational freedom. A computational grid search, in which the tripeptide sequence was scanned through conformational space, found several structures that were consistent with the measured distance. From these, two structures emerged that represented the two extremes of the structure space (Fig. 6). In one structure the Ala24-Arg25-Gln26 region is distorted but maintains the transmembrane and cytoplasmic domains in an essentially parallel orientation (Fig. 6A). The other structure also contains a distortion in the Ala24-Arg25-Gln26 region, but the backbone {varphi} and {Psi} angles are such that the cytoplasmic and transmembrane domains are presented at an angle of ~90° to each other. This model is consistent with previous results from 15N-labeled null-cysteine PLB in oriented lipid bilayers (24), but our data are not sufficient to provide independent confirmation.



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FIG. 6.
Models of PLB in lipid membranes in the absence (SERCA) and presence (SERCA +) of Ca2+-ATPase. The polypeptide sequences (inset) highlight the measured interatomic distances between Pro21–Ala24 and Leu42–Leu44, which corresponded to a helical structure in both regions (shown in yellow in the full model) in SERCA1 + and SERCA – membranes. The 13C–15N distance across the region Ala24–Gln26 is shown in green in the full models. In SERCA1 + membranes, the distance was consistent with a helix across Ala24–Gln26 (left). In SERCA – membranes, the measured distance was consistent with an elongated, non-helical structure across Ala24–Gln26 (right). The two models on the right-hand side represent extreme structures that are consistent with the measured distance from Ala24–Gln26, which differ in the relative orientations of the cytoplasmic and transmembrane domains. In all the models of PLB, the protein is assumed to be fully {alpha}-helical outside of the regions examined by NMR.

 

The NMR data presented clearly report that the presence of Ca2+-ATPase in the membrane induces a marked conformational change in the protein backbone around Arg25. A model for the conformational change can be proposed that fits these data and also reconciles the two apparently conflicting structures previously determined by solid-state NMR (23, 24). In the absence of SERCA1, the cytoplasmic domain lies parallel to the membrane surface where charge interactions occur between residues in the cytoplasmic domain and the phospholipid head groups. When PLB encounters SERCA1, PLB undergoes a conformational change around Pro21–Gln26, and the two domains switch from a perpendicular arrangement to a continuous {alpha}-helix. As a continuous helix PLB would be able to interact with residues in the cytoplasmic domain of SERCA1 (15).

Long-range Effects of Ca2+-ATPase—If it is assumed that PLB interacts with SERCA1 in a 1:1 stoichiometry, and protein losses during reconstitution are taken into account, ~17% of the total PLB is associated with SERCA1 and 83% is free in the bulk lipid. The experimental magnetization curves in Figs. 4 and 5 therefore represent a superposition of curves from free and SERCA1-associated PLB, and the calculated internuclear distances are population-weighted average values. It is not possible to obtain precise molecular details for PLB when bound to SERCA1 from these data; nevertheless, general conclusions can be drawn about the overall influence of Ca2+-ATPase on PLB structure.

The NMR results raise the question of why the region Ala24-Arg25-Gln26 in the free protein undergoes a conformational change to a helix in membranes containing SERCA1, although over 80% of the PLB molecules are not associated with the enzyme. The answer to this question may by sought by examining the proportions and dimensions of the individual membrane components. Studies of protein-lipid interactions have estimated that 30 lipid molecules form the annulus around the transmembrane section of Ca2+-ATPase (47). If it is assumed that the mean surface area of a phosphatidylcholine molecule is 60 Å2 (48), from simple model building it is estimated that the mean distance between the transmembrane sections of individual Ca2+-ATPase molecules is 50 Å at a SERCA1/lipid ratio of 1:160. From the crystal structure of the Ca2+-ATPase (44), the separation of the large cytoplasmic domains of individual enzyme molecules is expected to be less than 20 Å.

It has been proposed that the cytoplasmic domain of PLB lies parallel to the membrane surface in the absence of SERCA1 (Fig. 6). The rotational diffusion of PLB in fluid membranes would allow the cytoplasmic domain to sweep over the membrane surface in a circular path. A helical cytoplasmic domain would be ~30 Å in length, and the diameter of its circular trajectory is therefore 60 Å. In the presence of SERCA1, the mean separation of enzyme molecules is much less than the diameter of the rotational path of PLB, which means that a membrane-parallel cytoplasmic domain would encounter one or more enzyme molecules even when PLB is not bound to SERCA1. Such collisions might provide the energy for PLB to adopt a continuous helix with parallel transmembrane and cytoplasmic domains. Hence, Ca2+-ATPase appears to have a long-range effect on the conformation of PLB, which may trigger PLB into adopting its regulatory structure.


    FOOTNOTES
 
* This work was supported by the British Heart Foundation (Project Grant PG/2000043). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed. Tel.: 44-161-2004217; Fax: 44-161-2360409; E-mail: david.a.middleton{at}umist.ac.uk.

1 The abbreviations used are: PLB, phospholamban; SERCA, sarco-(endo)plasmic reticulum CaATPase; CP-MAS, cross-polarization magic-angle spinning; {beta}OG, n-octyl-{beta}-D-glucopyranoside; PKA, protein kinase A catalytic subunit; DOPC, dioleoylphosphatidylcholine; DMPC, dimyristoylphosphatidylcholine; REDOR, rotational-echo double resonance; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (tetrasodium salt); MOPS, 4-morpholinepropanesulfonic acid. Back


    ACKNOWLEDGMENTS
 
We thank Dr. Ram Sharma (Southampton University) for protein synthesis and purification.



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 ABSTRACT
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 DISCUSSION
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