Calcium Binding to Calmodulin Leads to an N-terminal Shift in Its Binding Site on the Ryanodine Receptor*

George G. Rodney, Catherine Porter Moore, Barbara Y. Williams, Jia-Zheng Zhang, Jack Krol, Steen E. Pedersen, and Susan L. HamiltonDagger

From the Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas 77030

Received for publication, September 28, 2000, and in revised form, October 12, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The skeletal muscle calcium release channel, ryanodine receptor, is activated by calcium-free calmodulin and inhibited by calcium-bound calmodulin. Previous biochemical studies from our laboratory have shown that calcium-free calmodulin and calcium bound calmodulin protect sites at amino acids 3630 and 3637 from trypsin cleavage (Moore, C. P., Rodney, G., Zhang, J. Z., Santacruz-Toloza, L., Strasburg, G., and Hamilton, S. L. (1999) Biochemistry 38, 8532-8537). We now demonstrate that both calcium-free calmodulin and calcium-bound calmodulin bind with nanomolar affinity to a synthetic peptide matching amino acids 3614-3643 of the ryanodine receptor. Deletion of the last nine amino acids (3635-3643) destroys the ability of the peptide to bind calcium-free calmodulin, but not calcium-bound calmodulin. We propose a novel mechanism for calmodulin's interaction with a target protein. Our data suggest that the binding sites for calcium-free calmodulin and calcium-bound calmodulin are overlapping and, when calcium binds to calmodulin, the calmodulin molecule shifts to a more N-terminal location on the ryanodine receptor converting it from an activator to an inhibitor of the channel. This region of the ryanodine receptor has previously been identified as a site of intersubunit contact, suggesting the possibility that calmodulin regulates ryanodine receptor activity by regulating subunit-subunit interactions.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha -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.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-beta -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-beta -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).


F<SUB><UP>L</UP></SUB>=R/(1+(K/L)<SUP>n</SUP>) (1)
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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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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Table I
Summary of the interaction of Peptides with CAM and B1234Q



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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, black-square; P3625-3644, black-triangle; P3614-3634, black-diamond ; P3614-3643, closed hexagons.

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, black-square; P3625-3644, black-triangle; P3614-3634, black-diamond ; P3614-3643, closed hexagons.

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, black-square; P3625-3644, black-triangle; P3614-3634, black-diamond ; P3614-3643, closed hexagons.

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, black-square; CaM-P3614-3643, ; Se-CaM-P3614-3643, open circle . 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.

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, open circle ; B1234Q alone, ; P3614-3643 alone, black-triangle; CaM-P3614-3643, ; B1234Q-P3614-3643, black-square. Both CaM protein and peptide concentrations were 3 µM.

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. open circle , <10 nM free Ca2+; , 200 µM free Ca2+. Data were fit by linear regression.


                              
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Table II
Binding of 35S-CAM and 35S-(N+3)CAM to SR membranes

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).



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.



    FOOTNOTES

* This work was supported by a grant from the Muscular Dystrophy Association and National Institutes of Health Grants AR41802 and AR44864 (to S. L. H.).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.

Dagger To whom correspondence should be addressed: Dept. of Molecular Physiology and Biophysics, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030.

Published, JBC Papers in Press, October 16, 2000, DOI 10.1074/jbc.M008891200


    ABBREVIATIONS

The abbreviations used are: SR, sarcoplasmic reticulum; CaM, calmodulin; apoCaM, Ca2+-free calmodulin; Se-CaM, selenomethionine calmodulin; NEM, N-ethylmaleimide; RFU, relative fluorescence units.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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