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J Biol Chem, Vol. 274, Issue 34, 23932-23939, August 20, 1999

Drug Binding to Cardiac Troponin C^*

Quinn Kleerekoper and John A. Putkey

From the Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston, Texas 77030

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Compounds that sensitize cardiac muscle to Ca²⁺ by intervening at the level of regulatory thin filament proteins would have potential therapeutic benefit in the treatment of myocardial infarctions. Two putative Ca²⁺ sensitizers, EMD 57033 and levosimendan, are reported to bind to cardiac troponin C (cTnC). In this study, we use heteronuclear NMR techniques to study drug binding to [methyl-¹³C]methionine-labeled cTnC when free or when complexed with cardiac troponin I (cTnI). In the absence of Ca²⁺, neither drug interacted with cTnC. In the presence of Ca²⁺, one molecule of EMD 57033 bound specifically to the C-terminal domain of free cTnC. NMR and equilibrium dialysis failed to demonstrate binding of levosimendan to free cTnC, and the presence of levosimendan had no apparent effect on the Ca²⁺ binding affinity of cTnC. Changes in the N-terminal methionine methyl chemical shifts in cTnC upon association with cTnI suggest that cTnI associates with the A-B helical interface and the N terminus of the central helix in cTnC. NMR experiments failed to show evidence of binding of levosimendan to the cTnC·cTnI complex. However, levosimendan covalently bound to a small percentage of free cTnC after prolonged incubation with the protein. These findings suggest that levosimendan exerts its positive inotropic effect by mechanisms that do not involve binding to cTnC.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Congestive heart failure results in desensitization of the myocardium to Ca²⁺ as well as depressed cardiac contractility. A promising approach to the treatment of congestive heart failure is the development of positive inotropic Ca²⁺-sensitizing agents that are capable of increasing the sensitivity of myocardium to Ca²⁺, thereby compensating for low cardiac output. Ideal calcium-sensitizing compounds would enhance the maximal force of muscle contraction without increasing Ca²⁺ ion flux into the cell or impairing relaxation in the failing myocardium. In addition, the compound should not inhibit phosphodiesterase activity (1) to minimize the possibility of heart arrhythmias and increased energy consumption in the heart (2).

Based on these criteria, cardiac troponin C (cTnC)¹ is an attractive target for potential Ca²⁺-sensitizing compounds. Cardiac troponin C is the EF-hand Ca²⁺ receptor in the thin filament of slow skeletal and cardiac striated muscle. The contraction relaxation cycle is regulated by the binding and release of Ca²⁺ ions from the N-terminal regulatory site II of cTnC. Compounds that bind with high affinity and selectivity to cTnC and increase the affinity of Ca²⁺ binding to site II could potentially increase the Ca²⁺ sensitivity of myocardial contraction without altering Ca²⁺ transients.

Knowledge of the structure and surface topology of cTnC is critical for an understanding of how existing Ca²⁺-sensitizing compounds interact with cTnC and to identify potential binding sites for new and more effective compounds. Previous structural models for cTnC based on known structures for Ca²⁺-bound fast skeletal troponin C (3, 4) and calmodulin (5) predicted that the N-terminal regulatory domain would have an open conformation with a large contiguous hydrophobic surface. Ca²⁺-sensitizing compounds such as bepridil, levosimendan, pimobendan, and trifluoperazine were thought to bind to this predicted hydrophobic surface (6-8). However, the recent NMR solution structures of intact Ca²⁺-bound cTnC and the apo and Ca²⁺-bound N-terminal domain of cTnC show the N-terminal regulatory domain to be partially closed in the presence of Ca²⁺ (9, 10). Hydrophobic surfaces in the N-terminal domain exist as discrete patches rather than a contiguous surface. Our recent study localized the binding site for bepridil and TFP in these N-terminal hydrophobic sites as well as in the C-terminal structural domain of cTnC (11).

Here we report studies on the binding of two newer generation Ca²⁺ sensitizers, EMD 57033 and levosimendan, to cTnC. EMD 57033 is reported to interact with the C-terminal domain of cTnC (12), but its Ca²⁺-sensitizing effects are thought to result primarily by affecting the actin myosin interface (13). EMD 57033 has an isoform-specific effect on phosphodiesterase activity (14). The Ca²⁺-sensitizing effects of levosimendan are thought to result from direct binding to cTnC (12, 15, 16). Our data confirm that EMD 57033 binds to the C-terminal domain of cTnC but that levosimendan does not appear to bind to either free cTnC or the cTnC·cTnI binary complex. However, levosimendan can form covalent adducts with cTnC after prolonged exposure.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Chemicals-- Levosimendan was kindly provided by Orion-Farmos (Espoo, Finland). Merck kindly provided EMD 57033. Bepridil was purchased from Sigma. L-[methyl-¹³C]Methionine, Tris-_d11, deuterium oxide, dimethyl sulfoxide-_d6, and methanol-_d were obtained from Cambridge Isotope Laboratories. (2,5)-Dihydroxybenzoic acid and trifluoroacetic acid were obtained from Aldrich Laboratories.

Cloning, Expression, and Purification of cTnI_33-211(A-Cys)-- A mouse cTnI cDNA was isolated from a mouse heart cDNA library by PCR using the following oligonucleotides: N-cTnI, 5'-GGGCCATGGCTGATGAAAGCAGCGATGCGG-3'; and C-TnI, 5'-CACAGTGTGGAAGCTTTGGCTCAGCC-3'.

Amplified DNA was purified and digested with NcoI and HindIII and subcloned into pET-23d (Novagen). The cTnI plasmid was transformed into E. coli strain Novablue (Novagen). Positive clones were confirmed by dideoxy DNA sequence analysis.

Both Cys⁸¹ and Cys⁹⁸ were changed to Ser using the Chameleon^TM double-stranded, site-directed mutagenesis kit from Stratagene and the following primers: C81S, 5'-CGCGTTCTGAGGACTCGTTCTCAGCCTTTGGAGTTGGATG-3'; C98S, 5'-GAAGAGCTTCAGGACTTATCTCGACAGCTTCACGCTCGG-3'; ScalI-MluI primer, 5'-CTGTGACTGGTGACGCGTCAACCAAGTC-3'; and MluI-ScaII primer, 5'-GCTTTTCTGTGACTGGTGAGTACTCAACCAAGTC-3'.

The monocysteine derivative cTnI(C81S) was produced in the first round of mutagenesis using the ScalI-MluI oligo as the selection primer. After selection, isolation, and confirmation of the sequence of cTnI(C81S), this plasmid DNA was used to introduce the Cys⁹⁸ to Ser⁹⁸ mutation in a second round of mutagenesis. The subsequent cTnI(A-Cys) plasmid was confirmed by restriction digestion and sequencing.

Generation of the truncated cTnI_33-211(A-Cys) plasmid and subsequent purification of the protein followed the method used by Guo et al. (17), with minor modifications. The coding sequence corresponding to amino acids 33-211 of cTnI(A-Cys) was amplified by PCR using the 5' primer reported by Guo et al. (17) and a 3' primer, the T7 terminator primer (Novagen). The subsequent PCR product was cloned into pET-23d using the NcoI and HindIII sites. After confirmation of the sequence, the construct was transformed into BL21(DE3) for overexpression. Purification of cTnI_33-211(A-Cys) protein was done essentially as reported previously for cTnI_33-211 (17).

NMR Sample Preparation-- The bacterial expression of cTnC(A-Cys) and cTnC3 and their labeling with [methyl-¹³C]Met and purification were reported previously (18-20). The NMR samples varied in protein concentration from 0.1 to 1 mM in NMR buffer consisting of either 20 mM Tris-d_ll, 200 mM KCl, and D₂O (pH 7.0) or 10 mM HEPES, 100 mM KCl, and D₂O (pH 7.0). Buffers used to prepare cTnC3 included five equivalents of dithiothreitol to reduce the Cys residues. In some experiments, the ionic strength or pH of the NMR samples was varied. Reconstitution of the binary complex with cTnC and cTnI was carried out by the method of Potter (21).

NMR Methods-- All spectra, except for those in Fig. 3, were collected at 40 °C on a Bruker AMX500 NMR spectrometer. HSQC (22) spectra were collected with 1024 complex data points in the t₂ domain and 72 increments in t₁. The experiments took approximately 26 min to complete per titration point. The ¹H and ¹³C spectra widths were 6012 and 600 Hz, respectively. ¹H and ¹³C chemical shifts were reported relative to the H₂O signal at 4.563 ppm and the [methyl-¹³C]Met signal at 14.86 ppm, respectively. All spectra were processed using the FELIX software package (Biosym Technologies, Inc.).

Drug Solutions-- Levosimendan is a light-sensitive drug; therefore, all stock solutions of the drug were prepared and stored in the dark. Stock solutions of levosimendan were prepared immediately before use in deuterated Me₂SO or as described previously (8). After adding the drug to the protein, the sample pH was adjusted when necessary. The stock solution of EMD 57033 was prepared in deuterated DMSO immediately before use. Bepridil was prepared as reported previously (11).

Mass Spectroscopy-- Matrix-assisted UV laser desorption mass spectroscopy spectra were acquired using a PerSeptive Voyager Elite Time of Flight mass spectrometer equipped with a delayed extraction and a nitrogen laser (337 mm). A saturated solution of (2,5)-dihydroxybenzoic acid in 0.1% trifluoroacetic acid in H₂O was used as a matrix. 0.5 µl of matrix was mixed with 0.5 µl of sample, placed on the target, and dried at room temperature while protected from light. The sample was a 1:10 dilution in H₂0 of the appropriate drug stock. The spectra in this study represent the summation of ~40 laser shots. In addition to the matrix-assisted UV laser desorption mass spectroscopy analysis, a Kratos MS50 fast atom bombardment was used to analyze the levosimendan provided by Orion-Farmos. 3-Nitrobenzyl alcohol was used as a matrix.

Drug Binding and Ca²⁺ Titration-- Dialysis was carried out at 25 °C using two-chamber equilibrium dialysis units from Sialmed, Inc. Each chamber had a 0.1 ml capacity. Prepared dialysis membranes (Life Technologies, Inc.) had a molecular mass cutoff of 12,000-14,000 daltons. Protein was added to one chamber at a concentration of about 0.2 mM to approximate the concentration used in NMR experiments. Protein was prepared in buffers of 20 mM Tris, pH 7.0, and 200 mM KCl or 20 mM HEPES, pH 7.0, and 100 mM KCl. Drug was added to the opposing chamber at concentrations between 0.2 and 0.4 mM in the corresponding buffer. Sufficient CaCl₂ was added to saturate all three Ca²⁺ binding sites on the protein. After equilibration (typically 16 h), aliquots of samples from both chambers were diluted at least 50-fold and assayed spectrophotometrically at 406 nm for levosimendan ( = 93,285 cm¹ M¹) and 296 nm for bepridil ( = 3,544 cm¹ M¹). After dilution, and at these wavelengths, the protein contributed insignificantly to total absorbance. The concentration of cTnC(A-Cys) in the samples was determined by the method of Bradford (23) and bicinchoninic acid assay (24).

A competitive dye binding assay to determine the relative Ca²⁺ binding properties of cTnC in the presence and absence of levosimendan was accomplished essentially as described previously by Linse et al. (25). Apo cTnC(A-Cys) was prepared by treatment with 10 mM EGTA followed by desalting into 3 mM Tris, pH 7.5, and 100 mM KCl. Samples used for titration contained 30 µM apo troponin C(A-Cys), 30 µM 5,5'-dibromoBAPTA, 3 mM Tris, pH 7.5, and 100 mM KCl. One ml of the protein solution without Ca²⁺ was titrated with 3 µl aliquots of the same solution made 2 mM in CaCl₂, and absorbance was monitored at 264 nm. Under these conditions, the concentrations of BAPTA and protein did not change during the titration, and there was no dilution effect on absorbance. The experimental data were fit to a Ca²⁺ binding isotherm as described by Linse et al. (25). The basal Ca²⁺ in the buffer/proteins sample was determined to be 7 µM.

Gel Filtration and HPLC Analysis-- NMR samples were analyzed by both gel filtration and HPLC chromatography to detect possible covalent binding of levosimendan to cTnC. Samples were run on a Bio-Gel P-6 column equilibrated in 20 mM Tris, 150 mM KCl, and 10 mM EDTA (pH 7.5) to remove free drug. Eluted protein was concentrated and stored in 6 M urea, 20 mM Tris, 150 mM KCl, and 10 mM EDTA (pH 8.0) overnight and then loaded onto a Bio-Gel P-6 column equilibrated in 3 M urea, 20 mM Tris-Cl, and 150 mM KCl (pH 7.5). These samples were analyzed by HPLC after exchanging the sample buffer by dialysis to remove the urea. The HPLC chromatographic system consisted of a Waters model 501 pump, a Waters model 680 automated gradient controller, and an Alltech Macrosphere^TM GPC 60 7 µm (250 × 4.6 mm, length × inner diameter) HPLC column. A Waters Lambda^TM model 481 liquid chromatography spectrophotometer was used to monitor the elution of levosimendan at 406 nm. The buffer consisted of 20 mM Tris and 150 mM KCl (pH 7.5). In addition, the samples were run on 15% SDS-polyacrylamide gel electrophoresis urea gels.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Effect of EMD 57033 on the Met Methyl Chemical Shifts of Free cTnC-- Using equilibrium dialysis and tyrosine fluorescence, Pan and Johnson (12) have shown that the thiadiazinone derivative EMD 57033 binds selectively to the C-terminal domain of cTnC (12). This contrasts with bepridil and TFP, which, as we (11) have shown, bind to both the N- and C-terminal domains of cTnC. To confirm the selectivity of EMD 57033 and as a further test of our methodology for detecting drug binding to cTnC, we determined the effect of EMD 57033 on the methionyl methyl chemical shifts of cTnC in the absence and presence of Ca²⁺. cTnC(A-Cys), which has both endogenous Cys residues converted to Ser and was chosen for these studies because the solution structure of this cTnC derivative is known (9), and it has been used in previous drug binding studies (11).

Fig. 1A shows that EMD 57033 did not bind to [methyl-¹³C]Met-labeled cTnC(A-Cys) in the absence of Ca²⁺, as evidenced by the absence of drug-dependent changes in ¹H-¹³C HSQC spectra. In the presence of Ca²⁺ (Fig. 1B), significant drug-induced changes in the positions of ¹H-¹³C correlations were observed for Met¹⁵⁷ and Met¹²⁰ located in the C-terminal domain of cTnC. Drug-induced changes in the chemical shifts of Met¹⁵⁷ and Met¹²⁰ were maximal at a drug:protein ratio of 1:1. Chemical shifts for N-terminal Met residues were unaffected by EMD 50733, even at a 2:1 drug:protein ratio (data not shown). These data agree with the results Pan and Johnson (12) and demonstrate that EMD 57033 binds selectively to the C-terminal domain of cTnC in the presence of Ca²⁺. The data also validate the use of the Met methyl chemical shifts as location-specific markers to monitor the binding of structurally distinct drugs to cTnC.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Titration of apo (A) and 3Ca2+-cTnC(A-Cys) (B) with EMD 57033. HSQC spectra were collected in the presence or absence of 1 equivalent of EMD 57033. Solid contours represent the initial cross-peaks in the absence of drug. The protein concentration was 1 mM. The subsequent addition of EMD 57033 to achieve a 2:1 drug:protein ratio (2 mM drug) resulted in no additional changes in the HSQC spectra.

Effect of Levosimendan on Met Methyl Chemical Shifts of Free cTnC-- Initial experiments showed that Met methyl chemical shifts of [methyl-¹³C]Met-labeled cTnC(A-Cys) were unaffected by an equimolar concentration of levosimendan in the presence or absence of Ca²⁺ (data not shown). Thus, the effects of higher concentrations of drug were determined. Fig. 2A shows that a 4-fold molar excess of levosimendan has generalized and minor effects on the methionyl methyl chemical shifts of cTnC(A-Cys) in the absence of Ca²⁺. In the presence of Ca²⁺ (Fig. 2B), only Met¹²⁰ and Met¹⁵⁷ exhibit significant chemical shift changes in the proton dimension upon the addition of 2-fold and 4-fold molar excesses of levosimendan. Observed changes for Met⁸¹ at high drug:protein ratios are restricted to the carbon dimension. However, the interpretation of these changes with respect to specific drug binding to cTnC must be made cautiously because they are observed only at drug:protein ratios of greater than 1:1. Binding of bepridil and TFP to cTnC(A-Cys) induced significant spectral changes at a drug:protein ratio of 0.5:1 (11). Even the fluorescent dye 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid, which binds to hydrophobic surfaces, induces large changes in the HSQC spectra of [methyl-¹³C]Met-labeled cTnC(A-Cys) (data not shown).

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Titration of apo (A) and 3Ca2+-cTnC(A-Cys) (B) with levosimendan. A, the HSQC spectra of apo [methyl-13C]Met cTnC(A-Cys) in the presence or absence of 4 equivalents of levosimendan. B, the methyl 1H-13C correlations observed for Ca2+-bound [methyl-13C]Met cTnC(A-Cys) in the presence of 0, 2, and 4 equivalents of levosimendan. In both panels, solid contours represent the initial peaks without drug. Significant chemical shift changes correspond to 0.01 and 0.2 ppm in the proton and carbon dimensions, respectively.

We next used isotope-filtered NMR techniques in an attempt to identify NOEs between aromatic protons in levosimendan and Met methyl protons in cTnC(A-Cys) that was metabolically labeled with [methyl-¹³C]Met and Phe-(D)₈. No NOEs were detected (data not shown). In contrast, similar experiments identified strong NOEs between bepridil and cTnC(A-Cys) (11).

cTnC(A-Cys) encodes Ser rather than Cys at positions 35 and 84. It is possible that these mutations affect the ability of levosimendan to bind to cTnC. To test this, we labeled cTnC3, which contains Cys³⁵ and Cys⁸⁴, with [methyl-¹³C]Met and monitored Met methyl chemical shifts upon the addition of levosimendan in the presence of Ca²⁺ as shown in Fig. 3. The Met methyl chemical shifts for cTnC3 in the presence of Ca²⁺ are slightly different than those for cTnC(A-Cys) but are identical to those reported previously for cTnC3 (19). The addition of levosimendan to cTnC3 at a probe:protein ratio of 1.6:1 had no effect on the N-terminal Met residues. Similar to what was observed for cTnC(A-Cys), levosimendan had minor effects on the proton chemical shifts of Met¹²⁰ (0.01 ppm) and Met¹⁵⁷ (0.02 ppm) in C-terminal domain.

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Gradient-enhanced HSQC spectra of 3Ca2+ cTnC3 in the presence and absence of levosimendan. The 1H-13C Met methyl correlations without drug are shown as solid contours. Even in the presence of 1.6 equivalents of levosimendan, no significant change is seen for any of the Met groups (dashed line). The spectra were collected on a Varian Unity-Plus 750 using a 5 mm HCN triple resonance probe. The gradient enhanced HSQC pulse sequence was provided by Varian.

Direct Binding Studies-- The results in Figs. 2 and 3 and the absence of NOEs between cTnC and levosimendan suggest either that the drug does not bind to cTnC or that it binds with very low affinity and preferentially to the C-terminal domain. Both of these interpretations are not consistent with the Ca²⁺-sensitizing effects of levosimendan, which is proposed to act via binding to cTnC (8, 15) and to be effective at concentrations as low as 0.03-1 µM levosimendan (26). An alternative explanation for the data is that levosimendan binds to cTnC but does not greatly affect the Met methyl chemical shifts. To address this, we used equilibrium dialysis to determine direct binding of levosimendan to Ca²⁺-saturated cTnC. The antianginal agent bepridil (27) was used as a positive control because it is known to bind to cTnC (7, 11). Protein and drug concentrations were selected to ensure that even low affinity binding would be detected.

Fig. 4A shows the results of equilibrium dialysis to detect the binding of bepridil to cTnC(A-Cys). The absorbance spectrum clearly shows significant binding of bepridil to cTnC(A-Cys), as evidenced by an accumulation of drug in the chamber containing protein. The apparent dissociation constant for binding of bepridil to cTnC(A-Cys) was approximately 20 µM, which agrees well with values of 10-20 µM that were reported using NMR and fluorescence techniques (7, 28). Fig. 4B shows the results of equilibrium dialysis to detect the binding of levosimendan to cTnC. It is clear that levosimendan does not bind significantly to cTnC because chambers with and without protein contained equal concentrations of levosimendan. Equilibrium dialysis experiments performed at pH 7.5, pH 7.0, and pH 6.8 and at different ionic strengths all showed no evidence of binding levosimendan to cTnC.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Equilibrium dialysis to detect the binding of bepridil (A) and levosimendan (B) to 3Ca2+-cTnC(A-Cys). cTnC(A-Cys) (0.17 mM) was placed in one dialysis chamber, and drug (0.35 mM) was placed in the other chamber. After equilibration, the concentration of bepridil or levosimendan on both sides of the dialysis membrane was determined by UV absorbance as described under "Materials and Methods." The black line represents the absorbance spectrum of free and bound drug in the chamber containing cTnC(A-Cys). The dashed line indicates the amount of free drug measured in the opposing chamber. Buffer conditions are given under "Materials and Methods."

Potential Effect of Levosimendan on Ca²⁺ Binding to cTnC(A-Cys)-- The potential effect of levosimendan on the Ca²⁺ binding affinity of cTnC was determined using Ca²⁺-sensitive dyes. Fig. 5 shows the absorbance of 5,5'-dibromoBAPTA when titrated with Ca²⁺ in the presence of cTnC(A-Cys) and in the presence or absence of levosimendan. Binding of Ca²⁺ to 5,5'-dibromoBAPTA causes a decrease in absorbance at 262 nm. Competition between 5,5'-dibromoBAPTA and cTnC for binding Ca²⁺ allows determination of the capacity and macromolecular binding constants for Ca²⁺ binding sites in cTnC. The line represents a fit of experimental data in the absence of levosimendan using the method of Linse et al. (25). It is clear from Fig. 5 that the presence of levosimendan had no effect on the Ca²⁺ binding properties of cTnC. The fractional change in absorbance was essentially identical for all level of Ca²⁺ in the presence and absence of levosimendan.

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Calcium binding properties of cTnC(A-Cys) in the presence and absence of levosimendan. Apo cTnC(A-Cys) was titrated with Ca2+ in the presence of 5,5'-dibromoBAPTA as described under "Materials and Methods." Absorbance was monitored at 264 nM. The percentage change in absorbance was plotted against total added Ca2+ and fitted to a Ca2+ binding isotherm as described by Linse et al. (25). The line represents a fit of data collected in the absence of levosimendan.

Effect of Levosimendan on Met Methyl Chemical Shifts in cTnC(A-Cys) when Bound to cTnI(A-Cys)_33-211-- Together, the data in Figs. 2-5 demonstrate that levosimendan at concentrations of less than 1 mM does not bind to free Ca²⁺-bound cTnC. The modest chemical shift changes seen in Fig. 2 at high concentrations of levosimendan (4 mM) may be due to nonspecific or very weak binding. However, it is possible that levosimendan binds to cTnC only when it is bound to cTnI. To address this possibility, we monitored drug-induced chemical shift changes when cTnC(A-Cys) was bound to cTnI. For these experiments, we generated mouse cTnI(A-Cys)_33-211 in which amino acids 1-33 were deleted and in which Cys⁸¹ and Cys⁹⁸ were converted to Ser. A derivative of mouse cTnI in which the first 33 amino acids had been deleted was shown previously by Guo et al. (17) to be functional and has been used for structural studies (29, 30). We elected to convert both Cys residues to Ser to prevent formation of intramolecular disulfide bonds in native cTnI.² A derivative of cTnI in which both Cys residues were converted to Ser was shown to be functional (31).

Table I lists the chemical shifts for the 10 methionyl methyl ¹H-¹³C correlations cTnC(A-Cys) when free and when associated with cTnI(A-Cys)_33-211. All Met methyl chemical shifts in the C-terminal domain of cTnC(A-Cys), except Met¹³⁷, were significantly affected by cTnI(A-Cys)_33-211. The absolute chemical shift values for Met residues in the C-terminal domain of cTnC(A-Cys) in the presence or absence of cTnI(A-Cys)_33-211 are in excellent agreement with those of Krudy et al. (29), who used cTnC(C35S) and cTnI_33-211. The chemical shifts of Met⁸⁵, Met⁸⁰, and Met⁶⁰ in the N-terminal domain are not affected by cTnI(A-Cys)_33-211. Met⁴⁵ and Met⁸¹ show a small effect, whereas Met⁴⁷ shows a significant change in the ¹H dimension of 0.09 ppm. These data differ from those of Krudy et al. (29),who reported that only methionyl methyl ¹H chemical shifts of Met⁸¹ and Met⁸⁵ in the N-terminal domain of cTnC(C35S) were altered upon association with cTnI_33-211.

Fig. 6 shows the effect of levosimendan on the cTnC(A-Cys)·cTnI(A-Cys)_33-211 complex in the presence of Ca²⁺. The ¹H-¹³C correlations attributed to Met groups located in the N-terminal domain (Fig. 6A) are shown separately from those in the C-terminal domain (Fig.6B). Solid contour lines indicate cross-peaks in the absence of drug, whereas dashed contour lines indicate cross-peaks upon the addition of 1 equivalent of drug. The lack of change in the chemical shifts indicates that levosimendan does not bind to the cTnC·cTnI complex.

View larger version (11K): [in this window] [in a new window]   Fig. 6.   Effect of levosimendan on Ca2+-saturated cTnC(A-Cys)·cTnI(33-211)(A-Cys) complex. Chemical shift changes for Met residues located in the N-terminal domain (A) are plotted separately from those in the C-terminal domain (B). Solid and dashed contour lines represent data collected in the absence and presence of levosimendan, respectively.

Evidence for a Covalently-bound Drug-Protein Complex-- During the course of our experiments, we observed that incubation of [methly-¹³C]Met-labeled cTnC(A-Cys) with levosimendan at 40 °C for 24 h resulted in the splitting of a number of ¹H-¹³C correlations. This phenomenon is illustrated for apo cTnC(A-Cys) in Fig. 7. No splitting was seen for the C-terminal Met residues. However, splitting is observed for Met⁴⁵, Met⁴⁷, and Met⁸⁵ (see boxes in Fig. 7). The more intense of the split peaks, which corresponds to the untreated protein, represents about 85% of the total protein. This observation suggests the presence of more than one conformational state for the N-terminal domain of cTnC that is induced by prolonged exposure of the protein to levosimendan.

View larger version (19K): [in this window] [in a new window]   Fig. 7.   HSQC spectrum of apo cTnC(A-Cys) after incubation with levosimendan at 40 °C for 24 h. Boxes indicate 1H-13C split Met methyl correlations. The methyl 1H-13C correlation for Met81 is at a 2-fold lower contour level.

Conformational heterogeneity can result from relatively high affinity binding of a ligand such that it is in slow exchange between the bound and free form on the NMR time scale. Such splitting of the C-terminal Met resonances in cTnC(C35S) was observed when the high-affinity C-terminal Ca²⁺-binding sites are partially filled with Ca²⁺. However, Figs. 2-6 show that levosimendan does not bind to cTnC. Thus, we reasoned that the split resonances could be due to a slow covalent attachment of levosimendan to cTnC, likely in the N-terminal domain. This conclusion was supported by the observation that a distinct yellow color characteristic of levosimendan co-migrated with cTnC(A-Cys) when NMR samples were analyzed by SDS-polyacrylamide gel electrophoresis.

We used analytical HPLC gel filtration to investigate what appeared to be levosimendan covalently bound to cTnC(A-Cys). Elution profiles of various samples were monitored at the absorbance maxima for levosimendan (406 nm). Fig. 8A shows the chromatographic profile of cTnC(A-Cys) that had not been exposed to levosimendan. No peak is observed because the protein does not absorb light at 406 nm. The arrow in Fig. 8A shows the elution position of cTnC(A-Cys) detected at 280 nm. Fig. 8B shows the elution profile of levosimendan. Fig. 8C shows the elution profile of an NMR sample of cTnC(A-Cys) that had been exposed to levosimendan for an extended period of time. Fig. 8D shows the elution profile of the NMR sample after removal of free drug by desalting in the presence of 6 M urea and EGTA. Comparison of these elution profiles demonstrates that a chromophore, which absorbs at 406 nm, remains associated with cTnC(A-Cys) after extended exposure to levosimendan and subsequent removal of free drug under denaturing conditions. This data, as well as the data in Fig. 7, and SDS-polyacrylamide gel electrophoresis analysis of levosimendan-treated protein strongly suggest the covalent binding of the drug to cTnC(A-Cys).

View larger version (23K): [in this window] [in a new window]   Fig. 8.   Detection of a covalently bound levosimendan·cTnC(A-Cys) complex. Chromatograms of cTnC(A-Cys) (A), levosimendan (B), the NMR sample containing apo cTnC(A-Cys) and 1 equivalent of levosimendan (see Fig. 5) (C), and the same NMR sample after removal of unbound drug (D). Conditions used are given under "Materials and Methods."

Mass Spectra of Levosimendan-- Both fast atom bombardment and matrix-assisted UV laser desorption mass spectroscopy were used to determine whether breakdown products or derivatives of levosimendan, which could covalently bind to protein, accumulate in organic or aqueous solutions. Levosimendan was first analyzed immediately after preparation in Me₂SO as shown in Fig. 9A. The peak at m/z 281.0 agrees well with the expected mass of levosimendan. The peak at m/z 550.5 is not identified but was seen in all spectra to varying extents. Levosimendan was stable in Me₂SO because no change in the mass species was apparent after several days of storage (data not shown).

View larger version (22K): [in this window] [in a new window]   Fig. 9.   Matrix-assisted UV laser desorption mass spectra of levosimendan. A-C, spectra of levosimendan stock prepared under previously published conditions (8, 15). A is the drug prepared in Me2SO. A single major peak with corresponding to levosimendan (expected (M + H)+ = 281.0) is observed visible. B is levosimendan freshly prepared in 0.5 M NaOH and neutralized. C shows the spectra of the same diluted stock as in B after 72 h.

Levosimendan was next prepared in 0.5 M NaOH as described by Pollesello et al. (8) and analyzed immediately (Fig. 9B) or 72 h (Fig. 9C) after dilution into water. The major peak at m/z 302.9 corresponds well to the mass of the monosodium form of levosimendan. After 72 h in aqueous solution, a number of higher mass peaks appear (Fig. 9C). Peaks at m/z 561.0, m/z 583.2, and m/z 601.1 correspond well to the mass of (2 M), (2 M + Na²⁺), and (2 M + 2Na²⁺) forms of levosimendan. These data suggest that levosimendan can dimerize in solution. A time-dependent accumulation of higher molecular mass species was also seen when levosimendan was prepared in Tris or HEPES buffers (data not shown).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The primary purpose of the present study was to identify binding sites for levosimendan on cTnC. As part of this overall study, we also report the identification of binding sites for EMD 57033 on cTnC.

EMD 57033 is structurally distinct from bepridil and TFP. It has positive inotropic effects on cardiac myocytes (13) but is believed to act at the actin myosin interface (13). It is reported to bind selectively to the C-terminal domain of cTnC with a K_d of approximately 40 µM (12) but does not increase the Ca²⁺ affinity of the regulatory site II (13). Our interest in EMD 57033 was to determine whether it can induce domain-specific changes in the Met methyl chemical shifts that would be consistent with selective binding to the C-terminal domain of cTnC. The data show a single binding site for EMD 57033 in the C-terminal domain of cTnC. Chemical shifts of Met methyl groups in the N-terminal domain of cTnC are unaffected, even at high concentrations of EMD 57033. It is likely that binding of EMD 57033 to the C-terminal domain of cTnC will be inhibited by cTnI because both bepridil and TFP are displaced from their C-terminal binding sites when cTnC binds to cTnI.³ This would be consistent with the hypothesis put forth by Solaro et al. (13) that EMD 57033 acts at the actin/myosin interface downstream of cTnC.

All data lead to the conclusion that levosimendan does not bind to cTnC or the cTnC·cTnI complex. This conclusion is in contrast to previous reports. Haikala et al. (15) showed that the retention time of levosimendan on a cTnC affinity column was increased in the presence of Ca²⁺ or Mg²⁺. From this it was concluded that levosimendan binds to both the N- and C-terminal domains of cTnC. However, significant effects on retention time were observed only at concentrations of Ca²⁺ greater than 1-3 mM, and no maximal retention time appeared to be reached even at 30 mM Ca²⁺. Solution phase equilibrium dialysis shown in Fig. 4 failed to show direct binding of levosimendan to cTnC at a variety of pH values and ionic strengths, whereas bepridil binds to cTnC under similar conditions.

Pollesello et al. (8) studied an N-terminal fragment of cTnC using homonuclear ¹H NMR, and reported NOEs between levosimendan and Met⁸¹, Met⁸⁵, and Phe⁷⁷ in cTnC. However, these NOEs were weak, and the sequence-specific assignment of Met methyl groups was tentative. Our experiments used full-length cTnC(A-Cys) and cTnC3, coupled with high-resolution heteronuclear NMR and unambiguous assignments of the Met methyl groups derived from mutagenesis (19) and the solution structure (9) of full-length cTnC(A-Cys). Our data show that the ¹H chemical shifts of N-terminal Met residues in free cTnC(A-Cys), free cTnC3, and the cTnC(A-Cys)·cTnI(A-Cys) binary complex are unaffected by all concentrations of levosimendan tested. Moreover, no NOEs were observed between free cTnC(A-Cys) and levosimendan. The chemical shifts for Met¹⁵⁷ and Met¹²⁰ in free cTnC are affected by levosimendan, but the observed changes are significant only at drug:protein ratios of greater than 1:1, which equates to millimolar concentrations of the drug, and this effect is not seen for the binary complex. These data, coupled with equilibrium dialysis experiments shown in Fig. 4, support the conclusion that levosimendan does not bind to the N-terminal regulatory domain of cTnC. Potential interactions with the C-terminal domain that affect the chemical shifts of Met¹⁵⁷ and Met¹²⁰ may be due to a nonspecific or very weak association that is prevented when cTnI binds to cTnC.

Pollesello et al. (8) reported that levosimendan increased the Ca²⁺ binding affinity of site II in cTnC as evidenced by Ca²⁺-dependent changes in fluorescence from dansylated cTnC in the presence and absence of drug. We have not directly addressed this issue; however, we see no evidence that levosimendan alters the Ca²⁺ binding properties of cTnC(A-Cys) using a competitive dye assay in which the protein is not covalently modified.

Our data show that levosimendan, or a dansylated, can covalently link to cTnC with apparent specificity for the N-terminal domain, as evidenced by the splitting of resonances for N-terminal Met residues after prolonged incubation with the drug. NMR data sets were collected in 20-30 min to minimize the extent of modification. Covalent modification of cTnC by levosimendan provides a plausible mechanism to account for the levosimendan-dependent effects observed by Pollesello et al. (8).

Our demonstration that levosimendan does not bind to cTnC does not question the positive inotropic effects of this compound that have been reported by different groups (26, 32), it simply questions the mechanism by which this effect is achieved. Several groups have reported that the positive inotropic effects of levosimendan result, at least in part, from inhibition of phosphodiesterase activity and accumulation of cAMP (26, 33-35). Our data would be consistent with this, or with other mechanisms of action.

	ACKNOWLEDGEMENTS

We are indebted to Dr. Edward Nickonowicz from Rice University for helpful advice and for providing us with access to his NMR facility. We are also grateful for the dedicated help of Dr. Wen Liu, who prepared the labeled cTnC samples.

	FOOTNOTES

^* This work was supported in part by National Institutes of Health Grant RO1-HL45724, Robert Welch Foundation Grant AU-1144, and American Heart Association Grant 9750496N (all to J. A. P.).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.

To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Texas Medical School, 6431 Fannin St., P. O. Box 77030, Houston, TX 77030. Tel.: 713-500-6061; Fax: 713-500-0652; E-mailjputkey@bmb.med.uth.tmc.edu.

² Q. Kleerekoper and J. A. Putkey, unpublished observations.

³ Q. Kleerekoper and J. A. Putkey, manuscript in preparation.

	ABBREVIATIONS

The abbreviations used are: cTnC, cardiac troponin C; cTnI, cardiac troponin I; TFP, trifluoperazine; HPLC, high pressure liquid chromatography; NOE, nuclear Overhauser effect; HSQC, heteronuclear single-quantum coherence; 5,5'-dibromoBAPTA, 5,5'-dibromo-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid..

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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