From the School of Molecular Biosciences and the
§ Department of Chemistry, Washington State University,
Pullman, Washington 99164
Received for publication, January 6, 2003, and in revised form, February 14, 2003
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ABSTRACT |
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Two distinct dimerization contacts in
calsequestrin crystals suggested a mechanism for Ca2+
regulation resulting from the occurrence of coupled Ca2+
binding and protein polymerization. Ca2+-induced formation
of one contact was proposed to lead to dimerization followed by
Ca2+-induced formation of the second contact to bring about
polymerization (1). To test this mechanism, we compared canine cardiac
calsequestrin and four truncation mutants with regard to their folding
properties, structures, and Ca2+-induced polymerization.
The wild-type calsequestrin and truncation mutants exhibited similar
K+-induced folding and end-point structures as indicated by
intrinsic fluorescence and circular dichroism, respectively, whereas
the polymerization tendencies of the wild-type calsequestrin differed markedly from the polymerization tendencies of the truncation mutants.
Static laser light scattering and 3,3'-dithiobis
sulfosuccinimidyl-propionate cross-linking indicated that
wild-type protein exhibited an initial Ca2+-induced
dimerization, followed by additional oligomerization as the
Ca2+ concentration was raised or as the K+
concentration was lowered. None of the truncation mutants exhibited clear stepwise oligomerization that depended on increasing
Ca2+ concentration. Comparison of the
three-dimensional structure of rabbit skeletal calsequestrin with a
homology model of canine cardiac calsequestrin from the point of view
of our coupled Ca2+ binding and polymerization mechanism
leads to a possible explanation for the 2-fold reduced Ca2+
binding capacity of cardiac calsequestrin despite very similar overall
net negative charge for the two proteins.
The sarcoplasmic reticulum
(SR),1 which is a specialized
elaboration of the endoplasmic reticulum, is adapted to supply
or remove Ca2+ rapidly to or from the myofilaments of
striated muscle. The total concentration of Ca2+ in the SR
is as high as 50 mM (2), but a large portion of this
Ca2+ is bound to a specific protein, calsequestrin.
Calsequestrin binds and releases large quantities of Ca2+
rapidly through its high capacity and relatively low affinity
interactions with Ca2+ (3). Due to this lumenal buffering
system, the concentration of free Ca2+ in the SR can be
maintained below the inhibitory level of the Ca2+ pump (1 mM), and simultaneously the SR can maintain the ability to
rapidly deliver the Ca2+ signal to the cytoplasm. Even
though the lumenal space is minuscule compared with the extracellular
space, the high concentrations of calsequestrin (100 mg/ml) make the SR
an efficient storage compartment for Ca2+ (4).
Calsequestrin is associated physically with the ryanodine receptor
(RyR) by a nucleation event that involves calsequestrin binding to the
basic lumenal domains of triadin (5) or junctin (6). These two proteins
interact with RyR in the junctional face region of the SR, and this
network of interacting proteins assures that high concentrations of
Ca2+ are stored very near to the site of Ca2+
release (1, 7-9).
The crystal structure of rabbit skeletal calsequestrin shows that this
protein is made up of three domains, each with a thioredoxin fold,
which is a five So far two isoforms of calsequestrin have been identified in mammalian
tissues, cardiac and skeletal. Among the ~360 residues in either
isoform of calsequestrins from various species, more than 110 residues
are either Asp or Glu, making calsequestrin one of the most acidic
self-folding proteins in existence. Despite the very similar net
negative charge for the two isoforms, cardiac calsequestrin appears to
bind only about half as much Ca2+ as does the skeletal
protein (12).
Previous studies (13) demonstrated that high capacity Ca2+
binding by calsequestrin is established by the formation of
Ca2+/protein complexes at relatively high calsequestrin and
Ca2+ concentrations. Under such conditions, two-thirds of
the total bound Ca2+ was associated with
Ca2+/calsequestrin aggregates, while one-third was
associated with the soluble form of calsequestrin. Even though weak
cooperativity of Ca2+ binding has been observed with the
soluble form of calsequestrin (14), strong Ca2+ binding
cooperativity accompanies the polymerization of calsequestrin into the
insoluble forms (13).
This Ca2+-induced precipitation leads to fibrils if induced
rapidly or to needle-shaped crystals if induced slowly (15-17).
Likewise, electron microscopy reveals the presence of electron-dense
fibrous arrays in SR microsomes at the junctional membranes, and these arrays are believed to be calsequestrin in its calcium-bound form (15,
18-21). Cross-linking SR lumenal proteins strongly suggest that most
of the calsequestrin in the SR microsomes is involved in direct
intermolecular calsequestrin/calsequestrin contacts (19, 20).
The Ca2+/calsequestrin aggregates are easily dissociated by
K+ (12, 22-24). pH changes may also be an important
regulator of aggregation in calsequestrin (25, 26). However, the
mechanistic dependencies among Ca2+, K+,
H+, and calsequestrin are poorly understood, and the role
of ion-induced structural changes in calsequestrin remains to be elucidated.
The association of calsequestrin monomers to form macromolecular
precipitates has a character similar to condensation or
crystallization, which provides an avenue for us to determine whether
the calsequestrin contacts in the crystal lattice are the same as the
contacts in SR membrane preparations. In the crystal, two different
dimerization contacts are observed: front-to-front and back-to-back.
These form the basis of a continuous, linear polymer that contains the major stabilizing forces in the crystal lattice. Not only does the
polymer in the crystal lattice have the linear morphology inferred for
the physiologically relevant aggregation, but also the two contacts
that stabilize this polymer have structural details that lend
themselves to control Ca2+ binding. That is, both of the
dimerization interfaces sequester large numbers of acidic residues and,
in addition, the back-to-back interface has a nearby polyanionic,
disordered tail. Furthermore, these dimerization interfaces do not look
like typical crystal contacts because large surface areas are buried in
the formation of intricate interactions. Given the intricacy, the
sequestration of so many negative charges, and the nearby polyanionic
tail, we proposed that these interfaces provide the basis for the
physiological mechanism of coupling Ca2+ binding with
polymerization and Ca2+ release with depolymerization (1,
8, 9).
Although the front-to-front and back-to-back contacts in our
calsequestrin crystals would appear to be favored by Ca2+
binding, attempts to directly visualize Ca2+ bound within
these interfaces have failed for technical reasons. Addition of
Ca2+ during crystallization leads to instant formation of
numerous needles (15-17) that have so far not been adequate for
structure determination. Therefore, we turned to alternative approaches to test our hypothesis. If the residues involved in the front-to-front and back-to-back interfaces are indeed involved in coupling
Ca2+ binding with calsequestrin polymerization, then
mutants lacking the key residues should show altered tendencies for
calcium-induced aggregation. Thus, we made a series of truncation
mutants of dog cardiac calsequestrin and compared their properties when
subjected to different Ca2+ levels.
Plasmid Constructs--
Expression plasmids, pET24b-cCSQ and
pTYB1-cCSQ were constructed using PCR strategy. Specific regions of
canine cardiac calsequestrin were amplified by PCR using the cDNA
clone as a template. A sense primer CAL5
(5'-CTGTCAACATATGGAAGAGGGGCTCAACTTCCCCA-3') that contains an
NdeI site and a start codon, and an antisense primer CAL3
(5'-CTGATGTCGACTATTACTCATCATCATCATCACTGTCGTC-3') that contains a
SalI site followed by two stop codons were used for
wild-type calsequestrin construct. A sense primer CAL5-2
(5-'CTGTCAACATATGGGGCTCAACTTCCCCACGT-3') and an antisense primer CAL3Im
(5'-CTAATGGCTCTTCCGCAACCCTCATCATCATCATCACTGTC-3'), and a sense primer
CAL5-13 (5'-ACTGTCAACATATGCGTGTGGTCAGTCTCACTGAGA-3') and CAL3Im were
used for Expression and Purification--
For the wild-type
calsequestrin, the Escherichia coli strain BL21 (DE3)
transformed with pET24b-cCSQ was grown at 37 °C in LB containing 50 µg/ml kanamycin to an A600 nm of 0.6 and then induced with 1 mM
isopropyl-
For the truncation mutants, the E. coli strain ER2566
transformed with pTYB1-cCSQs were grown at 37 °C in LB containing
100 mg/ml ampicillin to an A600 nm of 0.6 and
then induced with 1 mM
isopropyl-1-thio-
Purified wild-type and mutant calsequestrins were dialyzed against 300 mM KCl with 5 mM Tris-HCl (pH 7.5). The salt
and buffer concentration was gradually reduced in a stepwise manner.
The final stage of dialysis was performed against distilled water.
Fluorescence Spectroscopy--
The intrinsic fluorescence of
wild-type and mutant calsequestrins was measured using 90 µl of
sample containing 2 µM of protein buffered in 10 mM Tris-HCl (pH 7.5). KCl ranging from 0 to 600 mM were used to assess the K+ dependence of
fluorescence. Measurement was performed with a MOS-250 spectrometer
(Bio-Logic) at 22 °C. Each protein was excited at 282 nm using 5-nm
slit width, and emission was measured at 331 nm using 20-nm slit width.
Circular Dichroism (CD)--
CD spectra were measured with an
Aviv 202SF spectropolarimeter (Protein Solutions, Inc.) in a 2-mm path
length cell at 22 °C. 5 µM of protein was buffered in
10 mM Tris-HCl (pH 7.5) with 300 mM KCl and 1 mM DTT. Spectra were recorded from 200 to 260 nm and
corrected for buffer contributions.
Size Exclusion Chromatography--
Prior to the experiment, a
TSK G3000SW column (TosoHaas) was equilibrated with running buffer
containing 10 mM Tris-HCl, pH 7.5, and 1 mM DTT
with varying concentrations of KCl and CaCl2. 100 µl of
each protein at 1 mg/ml, which was preincubated with the running
buffer, was injected onto a TSK G3000SW column (TosoHaas). The
chromatography was carried out at a flow rate of 1 ml/min using a
Acuflow series IV pump (Analytical Scientific Inst.). The eluate was
passed in tandem through an UV detector (GILSON), a refractometer
(Optilab DSP, Wyatt Tech.), and a multiangle laser light-scattering
detector (Dawn EOS, Wyatt Tech.). The chromatographic experiments were
performed at 22 °C.
Molecular Mass Determination by Multiangle
Light-scattering--
Scattering data were collected and analyzed
using the software ASTRA (Wyatt Tech.) supplied with the instrument.
Relative weight-averaged molecular masses were determined from the
scattering data collected for a given condition using the Zimm fitting
method, in which K*c/R(Q)
is plotted against
sin2(Q/2), where
Q is the scattering angle, R(Q) is the
excess intensity (I) of scattered light at the angle
Q, c is the concentration of the sample, and
K* is a constant equal to
4 Cross-linking--
20 µM of purified proteins were
preincubated for 10 min on ice with KCl and/or CaCl2 in the
presence of 150 mM KCl and 20 mM sodium
phosphate buffer (pH 7.5). The samples were then further incubated with
400 µM 3,3'-dithiobis sulfosuccinimidyl-propionate (DTSSP) (Pierce) for 2 h at 4 °C. The reactions were then
rapidly quenched through the addition of concentrated Tris-HCl (pH 8.0) to a final concentration of 50 mM, followed by incubation
for another 10 min. Reactions were mixed 1:1 ratio with 2× SDS-PAGE sample buffer in the absence of any reducing agent and subjected to
electrophoresis in 8% SDS-PAGE gels.
Calsequestrin Structure and Mutants Construction--
We
previously showed that our rabbit skeletal calsequestrin crystal
contains a ribbon-like linear polymer joined by two distinct dimerization contacts. A short, four-molecule segment of this polymer
is reproduced here (Fig. 2B), with annotation of several key
features, including the following: (i) the front-to-front and
back-to-back interfaces; (ii) the arm exchange, which is part of the
front-to-front interface; (iii) the cavities in the two interfaces;
(iv) the short (two-residue) disordered N-terminal residues, and (v)
the long (15-residue) disordered C-terminal tail. Both the N-terminal
two residues and the C-terminal tail are called disordered because of
the lack of sufficient electron density to trace the chain. Such lack
of electron density arises from structural inhomogeneity, which could
have static contributions, dynamic contributions or both.
Because a clone of rabbit skeletal calsequestrin was not available when
this research was initiated, we used a clone of canine cardiac
calsequestrin instead. A sequence alignment of these two proteins is
shown in Fig. 2A. The regions associated with the arm,
domains I, II, and III, and the disordered tail are denoted. The
overall sequence identity between these two proteins is 70% (similarity 86%), suggesting with a near certainty that the two proteins have the same overall three-dimensional structure. Also, indicated are the negatively charged residues involved in
front-to-front (f or F) and back-to-back dimerization (b or B)
interfaces, with cavity residues (f or b) distinguished from non-cavity
interfacial residues (F or B).
Both the front-to-front and back-to-back interfaces contain cavities
that are fairly large, irregularly shaped, and open to the exterior.
The openings make it difficult to define the cavity volumes, but
approximate estimates indicate that the cavity within the
front-to-front interface is about 1600 Å3 (large enough
for about 50 water molecules), while that within back-to-back interface
is about 2400 Å3 (large enough for about 80 water
molecules). The back-to-back cavity contains the two negatively charged
residues at the tip of the arm as well as uncertain portions of the two
disordered tails.
The acidic and basic residues within the dimerization contacts
were determined by visual inspection of the three-dimensional structure
for the rabbit skeletal calsequestrin and by homology for the canine
cardiac calsequestrin (Fig. 2, Tables I
and II). The excess of 29 negative
charges in the front-to-front interface and the excess of 21 negative
charges in the back-to-back interface for rabbit skeletal calsequestrin
support the likely sensitivity of these interfaces to the presence of
divalent cations. Interestingly, the interfaces of canine cardiac
calsequestrin are also significantly negative, but greatly reduced in
net negative charge,
The alignment of the two sequences coupled with the three-dimensional
structure for rabbit skeletal calsequestrin provided the rationale for
the construction of four truncation mutants. The regions deleted for
each of these are indicated in Fig. 1. The deletion of the first two
residues ( Intrinsic Fluorescence and CD--
Monovalent or divalent cations
induce skeletal calsequestrin to undergo a conformational change from
an extended, random coil-like molecule to a compactly folded form. This
conformational change has been monitored both by circular dichroism and
by intrinsic fluorescence (3, 27). Here we compare the wild-type
calsequestrin and three of the truncation mutants by these two
spectroscopic methods.
Increasing the salt concentration using KCl caused a similar increase
in intrinsic fluorescence for the wild-type and the three truncation
mutants. Approximately 100 mM KCl was required for the
half-maximal increase in fluorescence, and the maximal signal was
achieved at about 300 mM KCl (see Supplementary Data at
http://www.jbc.org). These data suggest that wild-type and all three
truncation mutants are fully folded at the latter concentration. It is
unclear whether the slight differences observed at low salt concentrations are indicating slight differences in the early folding
steps or are simply due to experimental variations.
The far UV-CD spectra of the wild-type calsequestrin and the three
truncation mutants were compared in the same condition of 300 mM KCl, a concentration at which all should be fully folded (see Supplementary Data). The similar shapes and intensities for the
far UV CD spectra indicate that the wild-type calsequestrin and three
truncation mutants do indeed fold into a similar thee-dimensional structure.
Static Light-scattering Experiments--
Wild-type calsequestrin
and the four truncation mutants were studied by light scattering to
determine their tendencies to form oligomers. These light scattering
experiments were performed in 300 mM KCl, 10 mM
Tris-HCl (pH 7.5), so all the proteins are in fully folded state.
Variable concentrations of CaCl2 were included to assess
the effect of Ca2+ on polymerization.
Static Light-scattering of Wild-type Calsequestrin--
In 300 mM KCl without Ca2+, most of the wild-type
calsequestrin was monomeric with less than 1% of total population in
the form of dimers or higher oligomers (Fig.
3A). In 300 mM KCl
and 1 mM CaCl2, most of the population was in
the dimeric form (Fig. 3B). When the Ca2+
concentration was increased to 3 mM, most of the
calsequestrin population was in dimeric form, with small amounts of
tetramer starting to appear (Fig. 3C). The overall
dimer/tetramer pattern in 300 mM KCl, 3 mM
CaCl2 was unchanged up to ~5 mM
CaCl2. Beyond this CaCl2 concentration, the
calsequestrin starts to precipitate clogging the column and thus makes
further experiments impossible. This suggests that wild-type
calsequestrin has a transition point approximately at 5 mM
CaCl2.
When KCl was decreased to 70 mM with 1 mM
Ca2+, however, various oligomeric species up to hexamer
became evident (Fig. 3D). As a control experiment, the
condition of 500 mM KCl but without Ca2+ was
also used. All populations in this condition were monomers (Fig.
3E). The solution containing 700 mM KCl also
showed the same result (data not shown).
Static Light-scattering of Mutants--
The
Despite the small size of the deletion,
The Cross-linking--
To confirm the results of the light-scattering
experiments, we performed cross-linking experiments of Mechanisms of Ca2+ binding by calsequestrin have no
reason to mimic the Ca2+ binding mechanism of either the
Ca2+ pump or TnC (9). Ca2+ binding sites of
calsequestrin need to be made and broken, but not over the low
cytosolic Ca2+ concentration range or with the same
stoichiometry and precision as those formed and subsequently disrupted
in the Ca2+ pump or those that are intrinsic to the EF hand
structure of TnC. Ca2+ binding by calsequestrin is very
likely nonspecific. It is, therefore, unlikely that there are specific
Ca2+ sites, although the first few ions that bind at low
Ca2+ concentrations may have some specificity.
We are proposing that Ca2+ regulation by calsequestrin
involves an interplay among protein folding, Ca2+ binding,
and calsequestrin polymerization. This interplay is shown schematically
in Fig. 6. In summary, the polymerization of calsequestrin is promoted by Ca2+ and inhibited by
K+. Both the N-terminal arm and the acidic, C-terminal tail
are necessary for the Ca2+-dependent linear
polymerization. N-terminal arm exchange inhibits random aggregation of
the protein. Deletion of the N-terminal arm may disrupt specific
orientation of the front-to-front interaction, resulting in random
interactions among calsequestrin monomers. Deletion of the acidic,
C-terminal tail reduces the negative charge in the back-to-back
interface, resulting in low concentrations of counter ion for
establishing the back-to-back interactions.
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-strand sandwiched by four
-helices (1). Each
domain of calsequestrin has a hydrophobic core with acidic residues on
the exterior, generating electronegative potential surfaces. Individual
domains are connected by short sequences located interior to the
domains themselves. These connecting loops and the secondary structural
elements that fill the interdomain space contain mostly acidic
residues, making the overall center of the protein hydrophilic rather
than hydrophobic. Therefore, cations are required to stabilize the
acidic center of calsequestrin. Divalent cations, which can provide
cross-bridging, would be expected to be more effective in this regard
than monovalent cations. This cationic stabilization of the acidic core
likely provides at least part of the explanation for observations
indicating that calsequestrin is more extended and more susceptible to
protease digestion at low salt concentrations and then collapses as the
ionic strength is raised (1, 3, 10, 11).
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C27 (amino acids 3-391) and
N13 (amino acids 14-391),
respectively. CAL5 and an antisense primer CAL3-27Im (5'-CTAATGGCTCTTCCGCACCCGTCATCCCCCTCTT-3'), and CAL5 and an antisense primer CAL3-38Im (5'-ACGAAGGCTCTTCCGCAACCAGTGTTTATCTTTCCAG-3') were
used for
C27 (amino acids 1-364) and
C38 (amino acids 1-353), respectively (Figs. 1 and
2A). NdeI site for
all sense primers and SapI site for all antisense primers
were introduced to make truncation mutants. PCR-amplified products were
digested with NdeI/SalI or
NdeI/SapI and ligated into the expression vector pET24b (Novagen) for the wild-type calsequestrin and pTYB1 (New England
Biolabs) for the truncation mutants. The sequence of each construct was
confirmed by DNA sequencing.
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Fig. 1.
Schematic diagram of wild-type and mutant
calsequestrin expression constructs. Front and end
shaded areas indicate N-terminal arm and
C-terminal DE-rich tail. N2,
N13,
C27, and
C38 each
represents N-terminal two-amino acid truncation, N-terminal 13-amino
acid truncation, C-terminal 27-amino acid truncation, and C-terminal
38-amino acid truncation, respectively.
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Fig. 2.
Calsequestrin sequence and
structure. In A, the indicated structural features in
the three-dimensional structure are mapped onto the rabbit skeletal
calsequestrin amino acid sequence, and this sequence is aligned with
the canine cardiac calsequestrin amino acid sequence. In B,
ribbon diagram representations of the three-dimensional structures of
four rabbit skeletal calsequestrins are shown, with indications made to
identify the disordered tails at the C termini
(dotted) and the two distinct dimerization contacts,
front-to-front and back-to-back.
-D-thiogalactopyranoside for 4 h. The
expressed wild-type calsequestrin was purified as previously described
(11).
-D-galactopyranoside for 6 h at
room temperature. The truncation mutants were purified through a
chitin affinity column (New England Biolabs) followed by an anion
exchange column. Briefly, cells were pelleted and suspended in
sonication buffer (10 mM Tris-HCl (pH 9.0), 500 mM NaCl, 1 mM EDTA, 1% Triton-X100), sonicated
on ice, and centrifuged for 15 min at 20,000 × g.
Supernatant was applied to a chitin column and washed with a 10-column
volume of sonication buffer without Triton-X100. On-column cleavage was performed by incubating the chitin resin with cleavage buffer containing 10 mM Tris-HCl (pH 9.0), 1 M NaCl, 1 mM EDTA, and 90 mM DTT. After overnight
incubation, the cleaved calsequestrin proteins were eluted with elution
buffer (cleavage buffer without DTT). The elution was loaded onto an
Uno-Q12 (BioRad) column after concentrating and substituting the buffer
to 20 mM Tris-HCl (pH7.5), 20 mM NaCl with an
ultrafiltration unit (Amicon). The column was then washed with a linear
gradient from 100 mM to 1 M NaCl. The mutant
calsequestrins eluted at ~400-600 mM NaCl were
concentrated with Centriplus-10 (Amicon).
2n2(dn/dc)2/
04NA
(where n = solvent refractive index,
dn/dc = refractive index increment of
scattering sample,
0 = wavelength of
scattered light and NA = Avogadro's number).
Extrapolation of a Zimm plot to zero angle gives an estimate of the
weight-averaged molecular mass (Mw). Mw is
defined in Equation 1 as:
for c moles of i different species with an
individual molecular weight Mi.
(Eq. 1)
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16 for the front-to-front interface and just
11 for the back-to-back interface. Of the 50 (29 + 21) negative
charges in the two interfaces of rabbit skeletal calsequestrin, 18 are
changed in the canine cardiac calsequestrin. Of these changes, two
conserve the negative charge (one Asp to Gly and one Gly to Asp,
seven are negative to polar or non-polar (Asp or Glu to Asn, Thr, Ser,
Gln, or Gly), and nine are negative to positive (Asp or Glu to Arg,
Lys, or His).
Total numbers of charged amino acids appear on the sequence of rabbit
skeletal calsequestrin and canine cardiac calsequestrin
Total numbers of amino acids involved in the specific dimerization
interactions
N2) removes two residues negatively charged residues (EE)
that are both disordered and located within the back-to-back cavity.
Deletion of the first 13 residues (
N13) leads to removal of the arm
and therefore would be expected to disrupt the major interaction for
the front-to-front contact. Deletion of the last 27 residues (
C27) would remove a large portion of the disordered tail,
which is mostly negatively charged. Finally, deletion of the last 38 residues (
C38) would remove almost all of the disordered tail. These
two C-terminal truncation mutants would be expected to make the
back-to-back contact less sensitive to divalent cations.
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Fig. 3.
Multiangle light scattering of
wild-type calsequestrin. Typical elution profiles are shown as
molecular weight versus elution volume with varying
concentrations of KCl and CaCl2. The solid lines
represent refractive index on an arbitrary scale, which is
proportional to protein concentration. Colored dots indicate
calculated molecular mass. The different colors indicate different
polymerization states of the calsequestrins. Numbers in the each
profile show salt concentrations in addition to the running
buffer.
C38 mutant showed a
consistent pattern of monomeric and dimeric populations throughout the
conditions that were tested. The monomer peaks from 300 mM (or 500 mM) KCl solution without Ca2+ ions were broad and delayed when they were compared
with those with 300 mM KCl with Ca2+ (Fig.
4A). The
C27 mutant showed
Ca2+-dependent dimerization in both 1 and 3 mM CaCl2 with 300 mM KCl, but it
did not polymerize further as the wild-type protein did (Fig.
4B). Consistent dimer peaks indicate that dimerization is not dependent on the concentration of Ca2+. The dimers seen
on these mutants seem to be a front-to-front type of dimer because both
C-terminal truncation mutants have intact N termini. But we
could not rule out the possibility of back-to-back dimers. Since
C-terminal truncation greatly reduces the charge repulsion on
back-to-back interaction, its dimerization could be very well
independent of Ca2+ as shown and might be due to
intermolecular helix-helix interaction as seen in the crystal structure
of rabbit skeletal calsequestrin.
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Fig. 4.
Multiangle light scattering of
C38 (A),
C27 (B),
N13 (C), and
N2 (D) mutants. Elution
profiles are shown as molecular weight versus elution
volume. The solid lines represent refractive index on an
arbitrary scale, which is proportional to protein concentration, and
the dots indicate calculated molecular mass. Each color
represents different salt conditions in addition to the running buffer
as indicated in the caption.
N2 mutant exhibited only a
slight tendency to undergo Ca2+-induced dimerization (Fig.
4C). Furthermore, unlike wild-type protein, the monomer and
dimer peaks were not distinct following the addition of
Ca2+, suggesting that the removal of these two residues
leads to destabilization of the Ca2+-induced dimer and a
resulting rapid equilibrium between the monomer and dimer forms.
N13 mutant formed high molecular weight polymers (~16-20
mer), which elute from the column after only ~11 min. This profile was consistent throughout the various conditions that we have tested
(Fig. 4D).
N13 mutant,
C38 mutant, and wild-type calsequestrin. The wild-type protein
clearly showed Ca2+-dependent polymerization. 2 mM CaCl2 without KCl produced an increased
amount of very large molecular weight polymers compared with other
conditions. Addition of 150 mM KCl inhibits this
polymerization (Fig. 5). Cross-linking
buffer alone produced very weak dimer and tetramer bands. Both C- and
N-terminal truncation mutants do not show any
Ca2+-dependent polymerization, which is
inferred from the same band patterns in all conditions. However,
compared with the band patterns of the
C38 mutant, those of the
N13 mutant indicate the existence of very high molecular polymers
together with a reduced intensity of the monomer band. These results
are consistent with the results of the light-scattering experiments
where very high molecular weight aggregates are apparent in
N13 mutant and no polymerization further than dimers are apparent in
C38 mutant.
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Fig. 5.
DTSSP cross-linking of wild-type calsequestrin
(lanes 1-4), C38 mutant (lanes 5-8),
and
N13 mutant (lanes 9-12). Each protein sample
was preincubated with additional 2 mM CaCl2
(lanes 1, 5, and 9), 150 mM KCl (lanes 2, 6, and
10) and 2 mM CaCl2, 150 mM KCl (lanes 3, 7, and
11) before cross-linking. Lanes 4, 8,
and 12 are the negative controls without DTSSP cross-linker.
Lane 13 is a molecular weight marker.
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Fig. 6.
The model of calsequestrin folding and
polymerization based on our crystal structure and experiments performed
on this paper. In the absence of ionic strength, calsequestrin
exists in an unfolded state due to charge repulsion (A). As
the ionic strength is increased, calsequestrin domains begin to fold
(B). Finally, the three domains come together as the charge
repulsion is shielded (C). As the divalent cation
concentration is increased, the front-to-front interaction occurs first
since the N-terminal region has fewer charged amino acid residues
(D). The back-to-back interaction occurs last since more
cations are required to shield the acidic residues including the
DE-rich tail (E). Calsequestrin eventually forms a linear
polymer with the front-to-front and back-to-back interactions
(F).
From Table I, we notice that rabbit skeletal calsequestrin has a net
charge of 75, while canine cardiac calsequestrin has a net charge of
64. Despite this small overall difference in net charge, skeletal
calsequestrin binds about twice the amount of Ca2+ compared
with the cardiac calsequestrin (12). If our coupled binding and
polymerization mechanism were correct, then one would expect to observe
2-fold more negative charge within the skeletal as compared with the
cardiac calsequestrin interfaces. Table II shows this expectation to be
true: skeletal calsequestrin has a net charge of
18 and
32 for a
total of
50 within the front-to-front and back-to-back interfaces
(including the cavity-lining residues), while cardiac calsequestrin has
just
7 and
20 for a total net charge of
27 within the two interfaces.
Since about 40 calcium ions are bound by skeletal calsequestrin
compared with about 20 calcium ions by the cardiac calsequestrin, the
interfacial charges of 50 and
27 would be insufficient to balance
the positive charges arising from the bound calcium ions. Charge
balance could result to a significant degree from the polyanionic C-terminal tails. In addition, the large cavities observed within the
front-to-front and back-to-back interfaces could play a crucial role by
providing the space needed for negative counter ions to help balance
the positive charge. If charge balance by negative counter ions were
indeed significant, then the amount of bound Ca2+ would be
reduced if a large counter ion such as gluconate were used in place of
a smaller ion such as chloride. This conjecture could be tested by
measuring the number of (radioactive) Ca2+ ions bound per
calsequestrin molecule with either gluconate or chloride as the sole anion.
The biological significance of this dynamic polymerization of
calsequestrin can be explained in several ways. The front-to-front and
back-to-back contacts between the calsequestrin monomers permit formation of a ribbon-like linear polymer. Ca2+ largely
fills the electronegative pockets formed in these two contacts
cross-bridging the monomers. In this paper, we report Ca2+-dependent oligomerization. Considering the
high concentration of calsequestrin (100 mg/ml) and physiologically
varying concentrations (opposite direction) of K+ and
Ca2+ in SR, the dynamic formation and destruction of
calsequestrin oligomer in SR can be well explained from our
experimental data. Growing calsequestrin polymers provides a highly
charged surface onto which calcium can be adsorbed. The attractive
forces exerted by such an extended surface would have a longer range
than those from an isolated molecule. A sparingly soluble ion such as
Ca2+ would tend to spread over the surface of the
calsequestrin ribbon forming a readily exchangeable film. The
propensity of Ca2+-bound calsequestrin to form linear
structures makes Ca2+ dissociation and diffusion a more
rapid event than would be the case if crystallization were to occur in
three dimensions at lumenal Ca2+ concentrations. The use of
Ca2+ as a cross-linker rather than as a tightly bound form
free of H2O also speeds dissociation. Although the acidic
residues are highly concentrated in pockets, the surfaces of the
calsequestrin polymers are also highly acidic. Thus Ca2+
diffusion from calsequestrin to the Ca2+ release channel is
likely to involve surface diffusion, a more rapid process than
diffusion through liquid (28). Calsequestrin, probably in a
Ca2+ bound form, forms regular arrays that appear
crystalline in the lumen of the sarcoplasmic reticulum (18, 19, 29).
Calsequestrin is associated physically with RyR by a nucleation event
that involves calsequestrin binding to the basic lumenal domains of
triadin (5) or junction (6). These two proteins interact with RyR in
the junctional face region of the sarcoplasmic reticulum, and the
network of interacting proteins assures that high concentrations of
Ca2+ are stored very near to the site of Ca2+
release. Ca2+ release from calsequestrin through the
Ca2+ release channel is regulatory but not limiting.
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FOOTNOTES |
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* The original research described in this paper was supported by grants (to C. H. K. and A. K. D.) from the National Science Foundation, the American Heart Association, and the Murdock Charitable Trust.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains K+-dependent intrinsic
fluorescence changes of wild-type and mutant calsequestrins and far
UV-CD scans of wild-type and mutant calsequestrins.
¶ To whom correspondence should be addressed. Tel.: 509-335-1409; Fax: 509-335-9688; E-mail: chkang@wsunix.wsu.edu.
Published, JBC Papers in Press, February 19, 2003, DOI 10.1074/jbc.M300120200
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ABBREVIATIONS |
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The abbreviations used are: SR, sarcoplasmic reticulum; RyR, ryanodine receptor; DTT, dithiothreitol; DTSSP, 3,3'-dithiobis sulfosuccinimidyl-propionate.
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