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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 1732) 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
-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 13C13C and
13C15N 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
-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
-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.
|
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The detergent n-octyl--D-glucopyranoside (
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+-ATPaseSERCA1 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+-ATPaseSERCA1 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 12 ml of 10 mM Tris, 0.25 M
sucrose buffer, pH 7.5, containing 6 mg/ml 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 1040% density gradient at 60,000
x g for 12 h to confirm successful reconstitution.
Reconstitution of PLBDimyristoylphosphatidylcholine (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
PLBCo-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 25 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 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 1040% 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 PLBFollowing 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 MeasurementsSpecific 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 MeasurementsAll 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
R of 7000 Hz using a standard spin-echo pulse-sequence
with rotorsynchronized
pulses applied at the frequency of 15N
(31). Rotational resonance
experiments (32) were carried
out at sample spinning frequencies of 1100012500 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 56 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).
|
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 (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 PLBA 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 -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
-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.
|
The results of the H/D exchange experiment are shown in
Fig. 3, BD. The
control REDOR NMR spectrum showed superimposed contributions from PLB in the
carbonyl region (170 ppm) and two partially overlapping peaks from the
C 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
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
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 PLBThe 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 13C13C and
13C15N 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 -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.
|
Rotational resonance experiments were carried out to measure
13C13C distances between the labeled carbonyl and
C sites. The sample spinning frequency
R, was
adjusted to the first order rotational resonance condition for each pair of
13C labels (
R =
, where
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
13C13C 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 -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.
|
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
Ala25Gln26 distance was beyond the limits of
detection and greater than 5 Å (Fig.
5A). The distance expected for an -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
-helix (Fig. 5).
|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 PLBDespite 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 -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 13C15N 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 13C15N 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
and
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.
|
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 Pro21Gln26, and the
two domains switch from a perpendicular arrangement to a continuous
-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+-ATPaseIf 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 |
---|
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; OG,
n-octyl-
-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.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|