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INTRODUCTION |
The skeletal muscle Ca2+ release channel (RYR1) is a
homotetrameric protein composed of four 565-kDa subunits. The
N-terminal four-fifths of the protein form the large cytoplasmic
"foot" region, and the C-terminal one-fifth of the protein spans
the sarcoplasmic reticulum
(SR)1 membrane and forms the
pore (1). RYR1 controls the release of Ca2+ from the SR,
which is necessary for muscle contraction. RYR1 is modulated by several
endogenous proteins (2) that bind within the cytoplasmic domain to
produce long-range, allosteric regulation of channel activity. Because
endogenous modulators affect RYR1 channel activity and ultimately
affect Ca2+ release from the SR, these modulators are
likely to affect excitation contraction coupling.
Calmodulin (CaM) is one of the endogenous modulators of the
Ca2+ release channel (3-5) and is a member of the
EF-hand calcium-binding protein family (6-8). CaM is a
dumbbell-shaped molecule consisting of N-terminal and C-terminal
domains separated by an eight-turn
-helix. The N- and C-terminal
domains each contain two Ca2+ binding sites (8). Upon
Ca2+ binding, CaM undergoes a global conformational change
that leads to the exposure of hydrophobic pockets that bind the
target protein (6, 7). Most target proteins of CaM require the
Ca2+-induced conformational change in CaM for binding and
activation. Some target proteins, including RYR1, bind and are
functionally altered by CaM in the absence of Ca2+ (9).
The major CaM-binding protein in skeletal muscle SR membranes is the
Ca2+ release channel (3, 5). At low Ca2+
concentrations, comparable to the levels in resting muscle, CaM binds
to and acts as a partial agonist of RYR1 (3, 10). At high
Ca2+ concentrations, such as those measured during a
calcium transient, CaM inhibits RYR1 (3, 10), suggesting that CaM plays
a role in closing the channel in response to a Ca2+
transient. We have shown that the conversion of CaM from an activator to an inhibitor of RYR1 is due to the binding of Ca2+ to
CaM (10). We have also shown that CaM binds with high affinity at low
and high Ca2+ concentrations to a single site on each
subunit of RYR1 (10, 11) and that these binding sites are lost rapidly
upon mild trypsin digestion of SR membranes (11). Furthermore, both
Ca2+-free CaM (apoCaM) and Ca2+CaM bound to
RYR1 protect sites at amino acids 3630 and 3637 from trypsin digestion
(11). These data suggest that there is either a common region for the
binding of both apoCaM and Ca2+CaM or that the binding of
apoCaM and Ca2+CaM to separate sites can allosterically
regulate the accessibility of this region of RYR1 (amino acids
3630-3637) to trypsin. The latter possibility seems unlikely because
both an activator of the channel (apoCaM) and an inhibitor of the
channel (Ca2+CaM) protect the same sites from trypsin
(11).
To determine whether the region of RYR1 that encompasses amino acids
3630 and 3637 binds apoCaM and Ca2+CaM, we prepared a
series of synthetic peptides matching sequences in this region. We
found that both apoCaM and Ca2+CaM bind to peptides
representing amino acids 3614-3643 of RYR1 but that different amino
acids within this region contribute to Ca2+CaM and apoCaM binding.
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EXPERIMENTAL PROCEDURES |
Materials--
TRANS35S-LABELTM (>1000 Ci/mmol) was
obtained from ICN Biomedicals, Inc. (Irvine, CA). Bovine serum albumin,
3-(N-morpholino)propane sulfonic acid,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid,
isopropyl-
-D-thiogalactopyranoside, and dithiothreitol were obtained from Sigma (St. Louis, MO).
Peptide Synthesis--
Peptides were synthesized in the core
facility at Baylor College of Medicine (Houston, TX) under the
direction of Dr. Richard Cook.
Expression and Purification of Calmodulins--
The mammalian
CaM cDNA was graciously provided by Dr. Z. Grabarek (Boston
Biomedical Institute, Boston, MA), and the cDNA for CaM B1234Q was
graciously provided by Dr. Kathy Beckingham (Rice University, Houston,
TX). Both CaM cDNAs were subcloned into the NdeI and
BPU 1102 sites of the pET3 vector (Novagen, Madison, WI).
For expression, BL21-DE3 Escherichia coli transformed with
the pET3-CaM plasmid was induced with 0.3 mM
isopropyl-
-D-thiogalactopyranoside for 2 h at
37 °C. The cells were solubilized using the B-Per Reagent (Pierce,
Rockford, IL). Mammalian CaM was purified by phenyl-Sepharose chromatography, and B1234Q was purified by anion exchange
chromatography (10). The protein concentrations of CaM and B1234Q were
determined by the Bio-Rad protein assay using bovine brain CaM as the standard.
[35S]Methionine and Selenomethionine Labeling of
CaM--
The mammalian CaM cDNA subcloned into the pET3 vector was
expressed, metabolically labeled with TRANS35S-LABELTM, and
purified according to the procedure described previously (10). To
replace methionine residues with the unnatural amino acid
selenomethionine, CaM was expressed as described for the [35S]methionine labeling, except selenomethionine (7 µg/100 ml) was added to the minimal media at the time of induction in
place of the radiolabel.
SR Membrane Preparation--
SR membranes were prepared from
rabbit hind leg and backstrap skeletal muscle and then purified using
sucrose gradient centrifugation (12). Protein concentrations were
estimated by the method of Lowry (13) using bovine serum albumin as the standard.
Equilibrium 35S-CaM Binding
Assay--
35S-CaM binding to SR membranes (10 µg) was
as described previously (10). The binding buffer contained 50 mM 3-(N-morpholino)propane sulfonic acid, pH
7.4, 300 mM NaCl, 0.1%
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, 0.1 mg/ml bovine serum albumin, and 1 mM EGTA with 1.2 mM CaCl2 (high Ca2+ binding
buffer, 200 µM free Ca2+) or 1 mM
EGTA alone (low Ca2+ binding buffer, 10 nM free
Ca2+). Samples were incubated for 2 h at room
temperature. Samples were filtered through Whatman GF/F filters
and washed five times with 3 ml of the same buffer used for the binding incubation.
Nondenaturing Gel Electrophoresis--
The electrophoretic
mobility of CaM was evaluated by nondenaturing polyacrylamide gel
electrophoresis under discontinuous conditions as a modified technique
described by Laemmli (14). Nondenaturing gels (20% polyacrylamide)
were run at 30 mA under low Ca2+conditions (1 mM EGTA in all gel buffers) or high
Ca2+conditions (200 µM CaCl2 in
all gel buffers).
Fluorescence Spectroscopy--
To determine the relative
affinity of CaM for the CaM-binding peptide P3614-3643, tryptophan
fluorescence was monitored with increasing amounts of CaM (0-210
nM) in 150 mM NaCl, 50 mM
3-(N-morpholino)propane sulfonic acid, pH 7.4, and 1 mM EGTA. At nanomolar Ca2+ concentrations, the
final concentration of P3614-3643 was 100 nM. For high
Ca2+ conditions, CaCl2 was added to a final
concentration of 1.2 mM (200 µM free
Ca2+), and the P3614-3643 concentration was 30 nM. Fluorescence data were collected on an SLM 8000C
fluorometer (SLM Instruments, Urbana, IL). Excitation was at 295 nm
with a 2 nm bandwidth and filtered with a UV pass filter (Oriel 59152).
The emission was recorded at 340 nm with a 2.0 nm bandwidth and
filtered through a 340 nm bandpass filter (Corion 9L134). To monitor
the effects of selenomethionine CaM (Se-CaM), fluorescence data were
collected on a SpectraMax Gemini fluorescence plate reader (Molecular
Devices Corp., Sunnyvale, CA). CaM proteins and P3614-3643 were added
to final concentrations of 6 and 3 µM, respectively.
Tryptophan excitation was set at 280 nm, and emission spectra were
recorded from 310 to 400 nm. Final fluorescence data were obtained by
subtracting CaM and buffer effects from those of the CaM plus peptide
and then normalized to the amount of peptide. Under these conditions,
the observed fluorescence is attributed to the single tryptophan
residue in the CaM-binding peptide P3614-3643.
Data Analysis--
Scatchard analysis of 35S-CaM
binding data to SR membranes was analyzed by linear regression. In the
fluorescence studies, the EC50 and the Hill coefficient,
n, were determined by fitting the data to the Hill equation
by nonlinear regression (Sigma Plot 4.0; Jandel Scientific, San Rafael, CA).
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(1)
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FL is the fluorescence intensity after
subtracting nonspecific binding, R is the total binding site
concentration, and L is the concentration of CaM.
In the gel shift assays, densitometry was performed on the CaM band.
Optical density data obtained in the presence of peptide were
normalized to the optical density of the CaM band alone (the first lane
of each gel) and plotted as a function of the peptide:CaM ratio.
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RESULTS |
Identification of a Candidate Sequence within RYR1 for Both the
ApoCaM and Ca2+CaM Binding Sites--
We have previously
shown that CaM (either apoCaM or Ca2+CaM) bound to RYR1
protects sites at amino acids 3630 and 3637 from trypsin cleavage (11).
One explanation for this finding is that both forms of CaM bind
directly to this region of RYR1. To assess this possibility, we
synthesized a series of peptides (Table
I) matching sequences around this
region. A representative nondenaturing gel of the five different
peptides with CaM in 200 µM free Ca2+ is
shown in Fig. 1A. The
densitometer determinations of the intensity of the CaM bands in the
presence of increasing concentrations of peptide from three independent
experiments at a 200 µM freeCa2+ are shown in
Fig. 1B. The peptide-CaM complexes migrate as higher molecular weight bands than CaM. The peptides alone do not enter the
gel because they are positively charged. P3625-3644, P3614-3634, and
P3614-3643 all show significant binding to Ca2+CaM,
whereas P3614-3627 shows some binding to Ca2+CaM. Two of
the peptides (P3614-3627 and P3614-3634) form only a single band with
CaM regardless of the peptide:CaM ratio, whereas P3625-3644 and
P3614-3643 can form multiple complexes with Ca2+CaM. The
data suggest that Ca2+CaM interacts with these latter two
peptides at both the N- and C-terminal lobes, and, at these high
peptide concentrations, each CaM molecule binds to two peptides or both
ends of CaM interact simultaneously with a single peptide. However,
P3614-3627 and P3614-3634 may only have the binding site for one lobe
of CaM and therefore produce only a single band, even at high
peptide:CaM ratios. We interpret these findings to mean that both the
N- and C-terminal lobes of Ca2+CaM bind within the sequence
3614-3634.

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Fig. 1.
The ability of Ca2+CaM to bind to
RYR1 peptides. A shows a representative 20%
nondenaturing polyacrylamide gel of CaM (3.4 µM) with
each of the RYR1 peptides (6.8 µM) in 200 µM free Ca2+. The peptides alone do not enter
the gel. CaM bound to the peptide diminishes the intensity of the CaM
band. Lane 1, CaM alone; lane 2, P3636-3652;
lane 3, P3614-3627; lane 4, P3625-3644;
lane 5, P3614-3634; lane 6, P3614-3643. In
B, the densitometric analysis of the CaM (3.4 µM) band in the presence of an increasing amount of
peptide is summarized from three independent nondenaturing gel shift
assays under high Ca2+ conditions. P3636-3652, ;
P3614-3627, ; P3625-3644, ; P3614-3634, ; P3614-3643,
closed hexagons.
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We also examined the interactions of these peptides with CaM at
nanomolar concentrations of Ca2+ (Fig.
2). A representative nondenaturing gel is
shown in Fig. 2A, and the data from three experiments are
summarized in Fig. 2B. ApoCaM bound only P3625-3644 and
P3614-3643 (lanes 4 and 6), and its complexes
with these peptides did not enter the gel. ApoCaM does not bind to
either P3614-3634 or P3614-3627. As will be discussed below, the
apoCaM appears to cooperatively bind two molecules of P3614-3643,
suggesting that both the N- and C-terminal lobes of apoCaM bind
to the sequence between 3625 and 3643.

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Fig. 2.
The ability of apoCaM to bind to RYR1
peptides. A shows a representative 20% nondenaturing
polyacrylamide gel of CaM (3.4 µM) with each of the RYR1
peptides (17 µM) in 1 mM EGTA (<10
nM free Ca2+). The peptides alone do not enter
the gel. CaM bound to the peptide diminishes the intensity of the CaM
band. Lane 1, CaM alone; lane 2, P3636-3652;
lane 3, P3614-3627; lane 4, P3625-3644;
lane 5, P3614-3634; lane 6, P3614-3643.
B shows the densitometric analysis of the CaM (3.4 µM) band in the presence of an increasing amount of
peptide from three independent nondenaturing gel shift assays under low
Ca2+ conditions. P3636-3652, ; P3614-3627, ;
P3625-3644, ; P3614-3634, ; P3614-3643, closed
hexagons.
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A mutant CaM, B1234Q, contains a single glutamate to glutamine
substitution at the -Z position in each of the four EF-hand Ca2+ binding domains (15). These substitutions cause a
substantial decrease in the ability of CaM to bind Ca2+.
Under the conditions used in these studies, B1234Q should not bind
Ca2+ and can be used as a model of Ca2+-free
CaM, even under high Ca2+ conditions. To confirm the low
Ca2+ results, we tested the ability of B1234Q to bind to
the peptides under high Ca2+ conditions. Fig.
3 shows the densitometric analysis of the
B1234Q CaM from three independent nondenaturing gel shift experiments at a micromolar concentration of Ca2+. Under this high
Ca2+ condition, B1234Q does not bind peptides P3636-3652,
P3614-3627, and P3614-3634 but does bind P3625-3644 and P3614-3643.
These results with B1234Q under high Ca2+ conditions are
consistent with those obtained with apoCaM (Table I). As with apoCaM,
the B1234Q-peptide complexes do not enter the gel. Taken together, the
results of all the gel shift assays suggest that the important
molecular determinants for apoCaM binding are within the C terminus of
the peptide P3614-3643, whereas Ca2+CaM binds toward the N
terminus of P3614-3643.

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Fig. 3.
The ability of the CaM mutant B1234Q to bind
RYR1 peptides under high Ca2+ conditions. Densitometer
analysis of the B1234Q (3.4 µM) band with increasing
amounts of peptide from three independent nondenaturing gels at 200 µM free Ca2+. P3636-3652, ; P3614-3627,
; P3625-3644, ; P3614-3634, ; P3614-3643, closed
hexagons.
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Intrinsic Tryptophan Fluorescence to Assess the Interaction of CaM
with P3614-3643--
The quenching of tryptophan fluorescence by
selenium incorporated into CaM as selenomethionine (Se-CaM) has
previously been used to characterize the interaction between CaM and
other CaM-binding peptides (16). To further evaluate the movement of
CaM from the C-terminal portion of P3614-3643 toward the N terminus
upon binding Ca2+, we assessed the ability of Se-CaM to
quench the intrinsic tryptophan fluorescence of the seventh amino acid
of P3614-3643 (corresponding to Trp3620 of RYR1) at low
and high Ca2+ concentrations. We tested the binding of CaM
(without selenium) to P3614-3643 at a low Ca2+
concentration (<10 nM free Ca2+) and found a
slight blue shift and enhancement of the tryptophan fluorescence (Fig.
4A). The complex of
P3614-3643 with Se-CaM displays a similar shift and enhancement in the
peak tryptophan fluorescence (Fig. 4A). At a high
concentration of Ca2+ (200 µM free
Ca2+), there is a large blue shift and enhancement in the
peak tryptophan fluorescence observed in the CaM-P3614-3643 complex
that is quenched when Se-CaM is complexed with P3614-3643 (Fig.
4B). These data support a model in which apoCaM binds to the
C-terminal portion of the CaM-binding peptide P3614-3643, and upon
binding Ca2+, Ca2+CaM shifts to bind the N
terminus of P3614-3643.

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Fig. 4.
Emission spectrum for the trytophan
fluorescence of P3614-3643. Changes in the steady-state
tryptophan fluorescence of P3614-3643 were monitored at low
Ca2+ (<10 nM; A) and high
Ca2+ (200 µM; B) concentrations.
P3614-3643 peptide alone, ; CaM-P3614-3643, ;
Se-CaM-P3614-3643, . The fluorescence data (RFU)
were corrected for CaM and buffer effects and are plotted as a function
of emission wavelength. Protein and peptide concentrations in the assay
were 6 and 3 µM, respectively.
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The affinity of CaM for Ca2+ is increased when it is
complexed with its target site (17). To determine whether the affinity of B1234Q for Ca2+ is increased when it is complexed with
P3614-3643, we assessed the Ca2+ dependence of the
interaction of CaM and B1234Q with P3614-3643 by monitoring the
intrinsic tryptophan fluorescence of P3614-3643. The
Ca2+CaM-P3614-3643 complex showed a greater enhancement in
tryptophan fluorescence than the apoCaM-P3614-3643 complex (Fig. 4,
B versus A). The EC50 for the change in
tryptophan fluorescence by CaM with increasing Ca2+
concentrations was 81.0 ± 1.0 nM (Fig.
5). The complex of B1234Q-P3614-3643 resulted in a similar blue shift and increase in the peak tryptophan fluorescence, as did apoCaM (data not shown). However, there was no
observable change in the peak tryptophan fluorescence with increasing
Ca2+ concentrations using B1234Q (Fig. 5). These data
suggest that at high Ca2+ concentrations, B1234Q remains
Ca2+-free when bound to the CaM-binding peptide
P3614-3643.

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Fig. 5.
Ca2+-dependent
enhancement of tryptophan fluorescence of P3614-3643. Changes in
the fluorescence (RFU) of the CaM-P3614-3643 complex
were monitored as a function of increasing Ca2+
concentrations (1 nM to 100 µM). Excitation
was at 280 nm, and emission was at 328 nm. The CaM-P3614-3643 complex
data were corrected for CaM and buffer effects. CaM alone, ; B1234Q
alone, ; P3614-3643 alone, ; CaM-P3614-3643, ;
B1234Q-P3614-3643, . Both CaM protein and peptide concentrations
were 3 µM.
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Affinity of CaM for P3614-3643 and RYR1--
To define a sequence
as the binding site for CaM, the peptide matching this sequence should
have an affinity for CaM that is similar to that of RYR1. Our previous
binding studies (11) used a CaM that had three additional amino acids
at the N terminus due to the expression vector (now designated
(N+3)CaM). Using metabolically labeled 35S-(N+3)CaM, we
determined that three to four CaMs bind per tetramer on RYR1 at both
nanomolar and micromolar concentrations of Ca2+. We have
now generated and metabolically labeled CaM that does not have these
three amino acids and analyzed its binding to SR membranes. The binding
of 35S-CaM to SR membranes is shown in Fig.
6 as representative Scatchard plots at
nanomolar and micromolar concentrations of Ca2+ and is
compared with 35S-(N+3)CaM in Table
II. At both nanomolar and
micromolar concentrations of Ca2+, 35S-CaM had
lower affinity than that found with the 35S-(N+3)CaM.
However, the Bmax values were similar. At a
nanomolar concentration of Ca2+, there are 2.9 ± 0.5 (n = 3) CaM binding sites per ryanodine binding site,
and at a micromolar concentration of Ca2+, there are
3.6 ± 0.5 (n = 3) CaM binding sites per ryanodine binding site. Because RYR1 binds one molecule of ryanodine, there are
three to four CaMs bound per RYR1. Furthermore, (N+3)CaM completely inhibits the binding of 35S-CaM at both low and high
Ca2+ concentrations (data not shown). These data indicate
that both CaM constructs bind to the same sites. The three extra amino
acids on the N terminus of (N+3)CaM are Gly-His-Ser. Why these amino acids increase the affinity of CaM for its binding site on RYR1 is
unclear but may involve the charge on the His residue.

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Fig. 6.
Scatchard analysis of 35S-CaM
binding at nanomolar and micromolar concentrations of
Ca2+. SR membranes (10 µg) were incubated for 2 h at room temperature with 5 nM 35S-CaM and
increasing amounts of cold CaM. , <10 nM free
Ca2+; , 200 µM free Ca2+. Data
were fit by linear regression.
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To determine the relative affinity between CaM and P3614-3643, we
measured the intrinsic tryptophan fluorescence of P3614-3643 as a
function of increasing CaM concentration at nanomolar and micromolar
concentrations of Ca2+ (Fig.
7). The EC50 for apoCaM
interaction with P3614-3643 was 20 ± 1 nM
(n = 3). The EC50 for the interaction of
Ca2+CaM with P3614-3643 was 9 ± 1 nM.
Therefore, the affinity of CaM for the CaM-binding peptide P3614-3643
is on the same order of magnitude as that seen for RYR1 in SR
membranes, suggesting that the sequence from 3614-3643 of RYR1
constitutes an apoCaM and Ca2+CaM binding site. From these
data, it can be seen that at low Ca2+ concentrations, the
maximal enhancement of tryptophan fluorescence is at a molar
stoichiometry of 2:1 (peptide:CaM). This is also evident at high
Ca2+ concentrations, although to a lesser extent. At both
high and low Ca2+ concentrations, the Hill coefficients are
greater than 1, suggesting that two peptides bind to each CaM molecule
and that the binding of the first molecule greatly enhances the
affinity for the second. The actual values of the Hill coefficients are
difficult to determine because we are working with peptide
concentrations that are greater than the Kd of CaM
for the peptide.

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Fig. 7.
Calmodulin binding to P3614-3643 at low and
high Ca2+ concentrations. The intrinsic tryptophan
fluorescence of P3614-3643 was measured while titrating with CaM
(0-250 nM) as described under "Experimental
Procedures." Data, which were corrected for CaM and buffer effects,
are plotted as fluorescence units (RFU) as a function
of the total CaM concentration and fit to Eq. 1. A,
P3614-3643 (100 nM) at low Ca2+,
EC50 = 20 ± 1 nM (n = 3);
B, P3614-3643 (30 nM) at high Ca2+,
EC50 = 9 ± 1 nM (n = 3).
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DISCUSSION |
Both apoCaM and Ca2+CaM modulate RYR1, but the
functional consequences of the binding of these two forms of CaM to
RYR1 are completely different: apoCaM is a channel activator, whereas
Ca2+CaM is a channel inhibitor (3, 10). The molecular
mechanism of activation and inhibition of RYR1 by CaM is unknown. We
have previously shown with RYR1 (10, 11, 18, 19) that: 1) apoCaM and
Ca2+CaM each bind to three to four sites per RYR1 tetramer,
2) apoCaM and Ca2+CaM binding sites are destroyed by
trypsin at the same rate, 3) apoCaM and Ca2+CaM protect
their own and each other's binding sites from destruction by trypsin,
4) the trypsin cleavage sites protected by both apoCaM and
Ca2+CaM are located at amino acids 3630 and 3637, 5) both
apoCaM and Ca2+CaM can protect their own and each other's
binding site from oxidation (which destroys binding of both apoCaM and
Ca2+CaM), 6) both apoCaM and Ca2+CaM can
protect the apoCaM site from alkylation with
N-ethylmaleimide (NEM), whereas NEM destroys only
apoCaM binding, 7) the cysteine protected from alkylation with
NEM by bound apoCaM or Ca2+CaM is cysteine 3635, and
8) the binding of 35S-B1234Q (the mutant CaM that cannot
bind Ca2+) is completely inhibited by Ca2+ CaM
and vice versa. These data support a model in which both apoCaM and Ca2+CaM bind to an overlapping region on RYR1,
but there are different determinants within the RYR1 sequence for
apoCaM and Ca2+CaM binding. To our knowledge, there are no
other examples of a single region of a protein that can bind both
Ca2+CaM and apoCaM using different determinants within the
same region. We have also previously shown that this region is at a
subunit-subunit interface within the RYR1 tetramer (20), suggesting the
possibility that CaM may regulate RYR1 function by regulating the
interactions between subunits.
We now present additional data to support this model and to map the
molecular determinants of the CaM binding site on RYR1. We demonstrate
that a peptide corresponding to amino acids 3614-3643 of RYR1 can bind
both apoCaM and Ca2+CaM. ApoCaM and B1234Q (a model of
apoCaM even at high concentrations of Ca2+) bind to
peptides P3625-3644 and P3614-3643 with similar affinities, suggesting that the middle and C-terminal half (amino acids 3625-3643) of the longer peptide are required for apoCaM binding.
Ca2+CaM binds to both of these peptides and to a shorter
peptide corresponding to amino acids 3614-3634, suggesting that the
Ca2+CaM site is N-terminal to the apoCaM binding site.
Previous investigations on the interactions between calmodulin and
target peptides have made use of fluorescence spectroscopy of the
tryptophan residue within the target peptide (21, 22). We have taken
advantage of this technique to monitor the interaction of CaM with the
RYR1 CaM-binding peptide, P3614-3643. This peptide has a tryptophan
residue seven amino acids from its N terminus. We have shown that upon
binding both apoCaM and Ca2+CaM, the peak intrinsic
tryptophan fluorescence of P3614-3643 is enhanced and blue shifted.
These effects are much more pronounced for Ca2+CaM. This
effect has also been observed for tryptophan residues on other
CaM-binding peptides (16, 21) and can be attributed to the tryptophan
moving from a solvent exposed to a more buried hydrophobic position
upon the binding of CaM. However, the possibility of other types of
conformational changes occurring in the peptide-CaM interaction cannot
be eliminated. Through monitoring the changes in the fluorescence yield
of P3614-3643 upon the addition of CaM, we have shown that the
relative affinity of CaM for P3614-3643 is in the same range as we
have observed between 35S-CaM and RYR1. These data suggest
that this stretch of amino acids within RYR1 constitutes the apoCaM and
Ca2+CaM binding site.
In other studies using tryptophan fluorescence to characterize CaM
binding to its target peptide, Yuan et al. (16) were able to
determine the binding orientation of CaM with a peptide encompassing
the CaM binding region of skeletal muscle myosin light chain kinase.
These investigators made use of the unnatural amino acid
selenomethionine in place of methionine to quench the tryptophan
fluorescence of myosin light chain kinase. The CaM-binding peptide of
RYR1, P3614-3643, contains a single tryptophan residue near its N
terminus. Data from our gel shift assays suggest that the determinants
for apoCaM binding lie in the C-terminal portion of P3614-3643,
whereas those for Ca2+CaM are more N-terminal to this on
P3614-3643. If this is correct, then the use of Se-CaM at low
Ca2+ concentrations should not significantly alter the
tryptophan fluorescence of the Se-apoCaM-P3614-3643 complex compared
with that of the apoCaM-P3614-3643 complex. However, at high
Ca2+ concentrations, the binding of Se-CaM to P3614-3643
should quench the tryptophan fluorescence compared with that of the
CaM-P3614-3643 complex. This is what we observed, supporting a model
in which CaM, upon binding Ca2+, slides toward the N
terminus of the RYR1 CaM-binding peptide, P3614-3643.
Previous studies have shown that CaM can bind two peptides under some
conditions (23). Our fluorescence binding data show Hill coefficients
greater than 1, suggesting that each CaM molecule can bind two
molecules of the peptide P3614-3643. Apparently, under the conditions
of the peptide binding assays used for this study, this interaction is
favored over the engagement of both ends of the CaM molecule by a
single peptide. This phenomenon was also seen in the nondenaturing
gels. However, it is expected that a single CaM molecule will bind to
this sequence in the intact RYR1. The studies with the peptides
strongly indicate that the sequence has the components necessary for
binding both the N- and C-terminal lobes of the CaM molecule.
In summary, our studies suggest that both apoCaM and
Ca2+CaM interact with a sequence on RYR1 that includes
amino acids 3614-3643. Our peptide data indicate that the
Ca2+CaM site on RYR1 overlaps the apoCaM binding site, but
different amino acids within these regions contribute to the binding of the two forms of CaM. This interpretation is supported by our previous
findings that alkylation of cysteine 3635 inhibits apoCaM binding but
not Ca 2+ CaM binding (19). Our findings also support a
model in which Ca2+CaM shifts toward the N terminus of its
binding site upon binding Ca2+ (Fig.
8). Because Ca2+CaM is known
to inhibit the channel, whereas apoCaM activates it, the conformation
of the Ca2+CaM binding site would be expected to differ
substantially from that of the apoCaM site.

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Fig. 8.
A model for the interaction of CaM with RYR1
at amino acids 3614-3643. ApoCaM binds to RYR1 amino acids
3625-3643. The Ca2+CaM binding site is shifted toward the
N terminus of RYR1 relative to the apoCaM site.
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