(Received for publication, July 13, 1995; and in revised form, September 15, 1995)
From the
Phospholamban (PLB) was rapidly isolated from canine cardiac
sarcoplasmic reticulum using immunoaffinity chromatography and prepared
by solid phase peptide synthesis. The two proteins are
indistinguishable when analyzed by SDS-polyacrylamide gel
electrophoresis and exhibit pentameric oligomeric states. They are
similarly detected on Western blots, are phosphorylation substrates,
have identical amino acid compositions that directly reflect their
predicted values, yield the same internal amino acid sequences upon
CNBr digestion, and have molecular mass values agreeing with the
expected value (6123 Da). Native and synthetic PLB reduced the
calcium sensitivity of Ca
ATPase, which is reversed by
anti-PLB antibody. A Cys-to-Ser PLB analog, where the cysteines (36,
41, and 46) were substituted by serines, is monomeric on
SDS-polyacrylamide gel electrophoresis, can be phosphorylated, and is
recognized by polyclonal antisera. PLB migrates with a sedimentation
coefficient of 4.8 S in sedimentation velocity ultracentrifugation
experiments, whereas Cys-to-Ser PLB does not sediment, consistent with
a monomeric state. Circular dichroism spectral analysis of PLB
indicates about 70%
-helical structure, whereas Cys-to-Ser PLB
manifests only about 30%. Because the physiochemical properties of
native and synthetic PLB appear identical, the more readily available
synthetic protein should be suitable for more extensive structural
studies.
Phospholamban (PLB) ()is an oligomeric membrane
protein present in stoichiometric amounts associated with the
Ca
ATPase of cardiac sarcoplasmic reticulum (SR). PLB
in its unphosphorylated state attenuates the catalytic activity of the
Ca
ATPase by reducing its apparent calcium
sensitivity. Phosphorylation of
PLB(1, 2, 3) , treatment with antibodies
directed against PLB(4, 5, 6, 7) ,
or mild trypsin proteolysis of PLB (8) reverses the decreased
calcium sensitivity, leading to an increase in
Ca
ATPase activity at submicromolar Ca
concentrations. Co-reconstitution of PLB and
Ca
ATPase into liposomes (9, 10) or
co-expression of the two proteins in COS cells (11) suggests
that PLB itself is sufficient to modulate the
Ca
ATPase activity. Chemical cross-linking studies (12, 13) and co-expression experiments with
Ca
ATPase and site-directed PLB mutants (14) indicate that the NH
terminus of PLB is
important for regulation of Ca
ATPase activity and
demonstrate the ability of monomeric PLB mutants to regulate
Ca
ATPase activity. Data supporting reversible and
direct regulation of the calcium pump by PLB are abundant, yet the
detailed mechanistic and structural requirements for PLB inhibition
remain to be described.
The primary structure of PLB has been
deduced from its cDNA(15) . It is highly conserved among
species, has an acetylated amino terminus, and consists of 52 amino
acids with a predicted molecular mass of 6123 Da. Several secondary
structure models (an example is shown in Fig. 1) have been
proposed (13, 14, 16) with the following
general features: (i) PLB is an amphipathic peptide with a hydrophilic
NH terminus, part of which is predicted to form an
-helix whose exact length and position are uncertain, (ii) serine
16 and threonine 17 are the sites for protein phosphorylation by
cAMP-dependent protein kinase (PKA) and calcium-calmodulin-dependent
protein kinase, respectively, and (iii) the hydrophobic COOH-terminal
amino acids (31-52) form a transmembrane
-helical segment
and are involved in protein oligomerization. The quaternary structure
of PLB under nondenaturing conditions is assumed to be pentameric. PLB
migrates as a homopentamer with an apparent M
of
28,000 in SDS-PAGE, which can be dissociated upon boiling into monomer
subunits with an apparent M
of 6,000. Other
oligomeric states are also detected. Pentameric complexes persist
following trypsin cleavage of the NH
-terminal 26 amino
acids of PLB(17) , implying that the NH
terminus is
not primarily responsible for association. Site-directed mutagenesis
studies of PLB (11, 18, 19, 20) have
identified numerous transmembrane amino acid residues that appear to be
required for oligomerization. For example, leucine or serine
replacement of the three cysteine residues located within the
transmembrane domain at 5-amino acid intervals (36, 41, and 46)
abolishes the protein's oligomeric properties(11) .
Figure 1:
Secondary structural model of
phospholamban. Two -helices, IA (amino acids 1-15) and II
(amino acids 31-52) are connected by an extended sequence IB
(amino acids 16-30). The two circled residues, serine 16
and threonine 17, are the phosphorylation sites for cAMP-dependent and
Ca
-calmodulin-dependent protein kinases,
respectively. The boxed residues are cysteines 36, 41, and 46.
The three CNBr proteolysis sites follow the initial methionine and
methionines 20 and 50.
Detailed structural information about PLB is lacking because of the limited availability of highly purified PLB and its poor solubility. A variety of purification schemes have been employed using canine cardiac SR (21, 22, 23, 24) with low PLB recoveries and sometimes disparate amino acid compositions. The low abundance of n-PLB in cardiac SR has lead to other strategies for obtaining PLB for structural studies. PLB expressed in Escherichia coli spontaneously aggregated into pentamers, but ultimately PLB expression led to cell lysis and was not suitable for large scale production(25) . Preliminary abstracts report the application of immunoaffinity chromatography for the purification of larger quantities of PLB either expressed in yeast (26) or in Sf21 cells using a baculovirus expression system (27) but lack a rigorous comparison with n-PLB. These expression systems are readily amenable for producing mutant PLB molecules for purification and characterization.
The chemical synthesis of the complete PLB protein
has been reported (28) ; however, a direct comparison with
n-PLB could not be performed. Synthetic PLB spontaneously aggregates
into pentamers, is primarily -helical, and has a phosphorylation
stoichiometry of approximately 1:1 (1 mol of P
to 1 mol of
PLB monomer). This report describes a detailed comparison of n-PLB with
s-PLB, a reverse-phase HPLC purification to yield homogeneous protein,
and a synthetic Cys-to-Ser PLB (
)analog that does not
oligomerize. These synthetic proteins provide a starting point for high
resolution structural studies of PLB, which should in turn lead to a
better understanding of the biological activity of this important
cardiac regulator.
Crude PLB (300 mg) was
dissolved in 20 ml of a 4:3:3 mixture of formic
acid/HO/isopropyl alcohol and purified on an Asahipak®
gel (Asahi Chemical Industries), ODP-200, 20-µm HPLC support
cartridge (47
300 mm) PrepPak® (Waters). A step gradient
(100-ml increments) was generated from 1 liter each of successively
increasing concentrations (10%) of mobile phase (solvent A, 60% formic
acid/H
O; solvent B, 20% formic acid/isopropyl alcohol). A
flow rate of 80 ml/min was used to elute the product. A distinct
component that eluted at 70% B contained the desired product. Fractions
containing purified PLB were pooled, concentrated, and lyophilized to
yield 27 mg of PLB.
PLB was purified from cardiac SR using previously described extraction and solubilization procedures(24) . The solubilized protein in Zwittergent 3-14 was applied to a mAb 1D11 immunoaffinity column with a 3.3-ml bed volume pre-equilibrated with column buffer (10 mM MOPS/KOH, pH 7.0, 0.2% Zwittergent 3-14). Following extensive washing including a high salt wash, PLB was eluted with 50 mM citric acid, pH 2, 0.2% Zwittergent 3-14, and the elutants were neutralized in tubes containing 1/20th volume of 2 M Tris/HCl, pH 9.5. The peak PLB fractions, as assessed by silver staining and Western blot analysis, were precipitated in 2 volumes of ice-cold acetone:ethanol (1:1). The precipitate was dried under vacuum and subsequently redissolved and applied to the reverse-phase HPLC column as described for s-PLB.
Phospholamban samples (400 ng) were incubated with 40 units
of catalytic subunit of PKA in a reaction buffer containing 10 mM MOPS, 0.1% Zwittergent 3-14, and 5 mM MgCl for 10 min at 30 °C in a total volume of 80 µl. The
phosphorylation reaction was initiated by the addition of 20 µl of
ATP (final concentration, 100 µM) containing 2 µCi of
[
-
P]ATP. After 10 min, the phosphorylation
reaction was stopped by the addition of an equal volume of SDS sample
buffer. The gel was dried after staining and subjected to
autoradiography.
Figure 2:
Stimulatory effect of monoclonal antibody
1D11. Calcium dependence of calcium-stimulated ATP hydrolysis by
cardiac SR CaATPase (100 µg/ml) at 25 °C in
the absence (
) and presence (
) of 25 µg/ml 1D11 is shown
in A. In the absence of 1D11, the half-maximal calcium
concentration (pK
) was 0.511 µM (pK
= -6.29), and the V
was 124 nmol/mg/min. The addition of 1D11
decreased the calcium sensitivity to 0.259 µM (pK
= -6.59), without
significantly altering the V
(135 nmol/mg/min). B shows the calcium dependence of
Ca
uptake at 25 °C. The calcium sensitivities in the absence (pK
= -6.36 or 0.437
µM) and the presence of 1D11 (pK
= -6.68 or 0.209 µM) were similar to
the values obtained in A. The V
values,
67 and 74 nmol/mg/min in the absence and the presence of 1D11,
respectively, were not significantly
different.
Purified 1D11 antibody was coupled to an Affi-Gel support resin (Bio-Rad). Multiple applications of protein to the column have been made with very little loss in performance. Results of a representative PLB purification are summarized in Table 1. Cardiac SR was treated with sodium carbonate to extract calsequestrin with the loss of only trace amounts of PLB as detected by Western blot analysis. The extracted membranes were solubilized with deoxycholate, which was exchanged with Zwittergent 3-14 before being applied to the immunoaffinity column. The flow through fraction contained the majority of the protein with limited loss of PLB. A high salt wash removed several contaminants and only small amounts of PLB. The eluted fraction contained 1.4 mg of protein of which >80% was PLB. PLB readily precipitated upon addition of an equal volume of acetone/ethanol (1:1). This fraction was further purified using reverse-phase HPLC to yield a homogeneous PLB preparation.
Figure 3:
Characterization of native and synthetic
phospholamban. Samples of purified PLB were electrophoresed without
boiling or after boiling. The gel was silver-stained (left two
panels) or electroblotted onto nitrocellulose for Western blot
analysis (middle two panels). Alternatively, the samples were
prephosphorylated using [-
P]ATP and the
catalytic subunit of PKA, then were visualized by autoradiography (right two panels). The positions of molecular mass standards
are indicated on the far left side, and the positions of the
monomer, pentamer, and catalytic subunit (PKA) are indicated
on the right.
The purified proteins were subjected to amino acid analysis (Table 2). The results were then compared with the predicted amino acid composition derived from the canine PLB cDNA. Determinations of tryptophan and cysteine were not made. The amino acid yields were normalized to threonine and corrected to an internal standard. A comparison of n- and s-PLB finds virtually identical compositions with only small deviations from predicted values. These deviations can be attributable to incomplete hydrolysis of these sterically hindered amino acid residues (Val, Ile, Leu, and Phe), which diminishes their recovery. In addition, methionine is partially destroyed during acid hydrolysis. The amino acid compositions also agree closely with reported values(22) .
Because the amino termini are N-acetylated, the samples were cleaved using cyanogen bromide (CNBr) to obtain microsequencing data. Fig. 1indicates the position of the three methionine residues (1, 20, and 50) that constitute the CNBr cleavage sites. The cleaved samples were directly subjected to Edman microsequencing without purification. Two major internal PLB fragments (Table 3), recovered in roughly equimolar amounts, were discernible with some minor contaminants recovered in the first few cycles. The recovery yields of the first eight cycles are listed and separated according to the predicted peptide sequence. Recoveries in cycles 19 and 20 are listed showing a small amount of methionine 20 (cycle 19) followed by additional PLB sequence, due to incomplete CNBr digestion.
Laser desorption mass spectral analysis of the two PLB samples yielded molecular mass values of 6123 ± 6 and 6126 ± 6 Da for n- and s-PLB, respectively (Fig. 4). These values agree with the predicted value of 6123 Da for acetylated PLB.
Figure 4: Laser desorption mass spectra of phospholamban. Native phospholamban (top panel) and synthetic PLB (bottom panel) are shown. Samples were processed as described under ``Experimental Procedures.'' Insulin (5733 Da) was added to synthetic phospholamban as an internal calibration standard. Each sample had a major peak with masses of 6123 ± 6 and 6126 ± 6 Da for native and synthetic phospholamban, respectively.
A synthetic full-length PLB molecule in which the three cysteine residues were replaced with serine was also chemically synthesized using the same methods. Unlike s-PLB, Cys-to-Ser PLB does not oligomerize into pentamers, and only monomers are observed on SDS-polyacrylamide gels (Fig. 5). This PLB analog is still detectable by Western blot analysis and can be phosphorylated using the catalytic subunit of PKA. Its amino composition was confirmed as described above.
Figure 5:
Characterization of synthetic and
Cys-to-Ser phospholamban. Samples were electrophoresed without boiling
or after boiling, then electroblotted onto nitrocellulose, and analyzed
by Western blot (left panels). In addition, the samples were
phosphorylated using [P]ATP and the catalytic
subunit of PKA. The labeled proteins were visualized by autoradiography (right panels).
Figure 6:
Circular dichroic spectra of phospholamban
in CE
. Spectra are shown for native PLB (solid line), synthetic PLB (long dashed line), and
the Cys-to-Ser PLB (short dashed line). The data were
collected at 20 °C.
PLB samples were also examined by analytical ultracentrifugation to determine their association state under conditions that more closely mimic a membrane environment than those used for SDS-PAGE. In a sedimentation velocity experiment, PLB diffusely migrated with a mean sedimentation coefficient of 4.8 S suggestive of an oligomeric species. Under the same conditions, the Cys-to-Ser PLB did not sediment, as predicted for monomeric PLB, implying little or no self-association.
Co-expression studies (14) have provided some insight
into the nature of the interaction between PLB and
CaATPase, including identification of the regions of
both proteins involved in PLB's regulation of
Ca
ATPase activity. Ultimately, knowledge of the
three-dimensional structure of PLB and the Ca
ATPase
will be required for understanding the reversible inhibition elicited
by PLB. Detailed structural information on intact native PLB is
lacking, even though its small size makes it an attractive molecule for
structural analyses. Major limitations have included the lack of
availability of large amounts of highly purified PLB and its poor
solubility. The ability to chemically synthesize a native form of PLB
should eliminate the first problem. Because it is critical to verify
the biochemical integrity of s-PLB by comparison to native protein,
sufficient quantities of highly purified n-PLB are necessary to
establish its biochemical properties.
The use of a 1D11 immunoaffinity column facilitated the purification of n-PLB by dramatically decreasing isolation time and improving the yield of highly purified protein as compared with procedures using sulfhydryl affinity chromatography(21, 22, 24) . The immunoaffinity column proved to be very efficient at purifying solubilized PLB because more than 90% of the PLB bound to the column was recovered. PLB comprises about 0.3% of the total cardiac SR protein(40) , which makes it a poor source of material for large scale purification. In addition, solubilization of PLB from the SR membrane is inefficient, with at least a third of the PLB remaining in the insoluble SR pellet. The overall protein recovery from the immunoaffinity column of 0.36% is better than the previously reported values of 0.2 (40) and 0.19%(22) , and 10-fold more protein was used. Some minor contaminants were also recovered requiring the use of a reverse-phase HPLC column to obtain a homogeneous PLB sample. The purity of n-PLB was verified by mass spectral analysis, amino acid composition, and microsequencing. Final recovery was 0.2%. The PLB immunoaffinity column will allow rapid purification from richer sources as well as mutant PLBs from expression systems. In a preliminary report, the immunoaffinity column has been successfully used to purify PLB expressed in yeast(26) , although solubilization of PLB from the yeast membrane limits the yield.
The structure of the NH terminus
has not been clearly defined. Secondary structural models (1, 13, 14) predict helices of varying
lengths. Based upon data from homonuclear NMR spectroscopy, chemical
shift assignments have recently been obtained for PLB
in 30% trifluoroethanol/H
0(44) . Using
sequential and medium range nuclear Overhauser effect connectivities
and secondary C
-H shifts as criteria, residues
1-17 appear to form a regular
-helix. Other work has also
shown that the NH
terminus is capable of forming
-helical structure in trifluoroethanol (PLB
)
and PLB
(46) ) and in charged detergents
(PLB
). The NH
-terminal PLB peptide,
PLB
, possesses little structure in aqueous buffer
solution(40, 44) . Likewise, PLB
(45) showed predominantly disordered structure. Nevertheless,
it has been reported that PLB
(9, 46) and PLB
(47) can regulate purified and reconstituted
Ca
ATPase. Presumably, interaction with itself,
membranes, or the Ca
ATPase may stabilize the
secondary structure of the NH
-terminal region facilitating
functional association.
Cys-to-Ser PLB possesses about
half of the -helical content of natural PLB in
C
E
. Upon first analysis, this result was
somewhat surprising because serine and cysteine are thought to be
indifferent helix formers. Although there is as yet no entirely
reliable predictive algorithm for determining peptide secondary
structure, neither the Chou and Fasman (48) or Kyte and
Doolittle (49) methods predict a major disruption in helix
structure for these substitutions. Serine can make shared H bonds
within neighboring backbone NH or CO groups or more typically with
solvent in exposed locations on turns and loops that can destabilize
the
-helix(50) . Assuming that the NH
-terminal
region is unaffected, the serine replacements appear to essentially
abolish formation of the putative transmembrane
-helix. Thus, the
finding that Cys-to-Ser PLB does not oligomerize is easily
rationalized.
The relative sedimentation profiles of PLB and Cys-to-Ser PLB indicate that the results from SDS-PAGE experiments accurately reflect actual solution conditions. The sedimentation results are in agreement with those obtained by Vorherr et al.(28) , who examined a variety of aggregate complexes under various experimental conditions. Attempts to form monomeric PLB or to disrupt the PLB aggregates by boiling the sample in the presence of 10 mM dithiothreitol prior to velocity ultracentrifugation were unsuccessful. However, Cys-to-Ser PLB, which ran solely as a monomer in SDS-PAGE, failed to sediment under the same conditions used for natural PLB, suggesting that the protein is monomeric.
Note Added in Proof-A
similar analysis of the secondary structure of PLB has
been published (Hubbard, J. A., MacLachlan, L. K., Meenan, E., Salter,
C. J., Reid, D. G., Lahouratate, P., Humphries, J., Stevens, N., Bell,
D., Neville, W. A., Murray, K. J., and Darker, J. G.(1994) Molec.
Membr. Biol.11, 263-269). Full-length PLB in
supported bilayers was 64-67%
-helical which decreased to
54% upon phosphorylation (Tatulian, S. A., Jones, L. R., Reddy, L. G.,
Stokes, D. L., and Tamm, L. K.(1995) Biochemistry34, 4448-4456).