Binding of Levosimendan, a Calcium Sensitizer, to Cardiac Troponin C

Levosimendan is an inodilatory drug that mediates its cardiac effect by the calcium sensitization of contractile proteins. The target protein of levosimendan is cardiac troponin C (cTnC). In the current work, we have studied the interaction of levosimendan with Ca2+-saturated cTnC by heteronuclear NMR and small angle x-ray scattering. A specific interaction between levosimendan and the Ca2+-loaded regulatory domain of recombinant cTnCC35S was observed. The changes in the NMR spectra of the N-domain of full-length cTnCC35S, due to the binding of levosimendan to the primary site, were indicative of a slow conformational exchange. In contrast, no binding of levosimendan to the regulatory domain of cTnCA-Cys, where all the cysteine residues are mutated to serine, was detected. Moreover, it was shown that levosimendan was in fast exchange on the NMR time scale with a secondary binding site in the C-domain of both cTnCC35S and cTnCA-Cys. The small angle x-ray scattering experiments confirm the binding of levosimendan to Ca2+-saturated cTnC but show no domain-domain closure. The experiments were run in the absence of the reducing agent dithiothreitol and the preservative sodium azide (NaN3), since we found that levosimendan reacts with these chemicals, commonly used for preparation of NMR protein samples. Levosimendan is an inodilatory drug that mediates its cardiac effect by the calcium sensitization of contractile proteins. The target protein of levosimendan is cardiac troponin C (cTnC). In the current work, we have studied the interaction of levosimendan with Ca2+-saturated cTnC by heteronuclear NMR and small angle x-ray scattering. A specific interaction between levosimendan and the Ca2+-loaded regulatory domain of recombinant cTnCC35S was observed. The changes in the NMR spectra of the N-domain of full-length cTnCC35S, due to the binding of levosimendan to the primary site, were indicative of a slow conformational exchange. In contrast, no binding of levosimendan to the regulatory domain of cTnCA-Cys, where all the cysteine residues are mutated to serine, was detected. Moreover, it was shown that levosimendan was in fast exchange on the NMR time scale with a secondary binding site in the C-domain of both cTnCC35S and cTnCA-Cys. The small angle x-ray scattering experiments confirm the binding of levosimendan to Ca2+-saturated cTnC but show no domain-domain closure. The experiments were run in the absence of the reducing agent dithiothreitol and the preservative sodium azide (NaN3), since we found that levosimendan reacts with these chemicals, commonly used for preparation of NMR protein samples. troponin C cardiac TnC N-terminal half of TnC troponin I cardiac troponin C with Cys-35 mutated to Ser cardiac troponin C with both cysteine residues 35 and 84 mutated to Ser N-terminal domain of cardiac troponin C dithiothreitol heteronuclear single-quantum coherence nuclear Overhauser effect NOE spectroscopy number of time increments number of transients matrix-assisted laser desorption ionization time-of-flight mass spectrometry bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane The number of patients suffering from heart failure is increasing along with the aging of population. Calcium sensitizers have been proposed as a treatment for congestive heart failure since they exert a positive inotropic effect without increasing the intracellular calcium concentration (1Endoh M. Gen. Pharmacol. 1995; 26: 1-31Crossref PubMed Scopus (80) Google Scholar). Levosimendan, a potent calcium sensitizer that improves the force development of the muscle contraction without increasing the cytosolic Ca2+ ion concentration (2Hasenfuss G. Pieske B. Castell M. Kretschmann B. Maier L.S. Just H. Circulation. 1998; 98: 2141-2147Crossref PubMed Scopus (251) Google Scholar), was discovered using troponin C as target protein. Troponin C (TnC)1 is responsible for the contraction trigger in the muscle. It belongs to the family of calcium binding EF-hand proteins and consists of two domains. The N-terminal half (NTnC) is responsible for the calcium-dependent regulation of the contraction, and the C-terminal half is a structural domain always loaded with divalent cations under physiological conditions. Troponin C interacts with troponin I (TnI), and this interaction is modulated by the binding of calcium. Studies of skeletal troponin C, a homologous protein, show that a hydrophobic patch is exposed in the open conformation of the calcium-loaded regulatory domain, which is a binding site for TnI (3Gagné S.M. Li M.X. McKay R.T. Sykes B.D. Biochem. Cell Biol. 1998; 76: 302-312Crossref PubMed Scopus (32) Google Scholar). This has also been proposed to be a potential binding site for calcium sensitizers (4Ovaska M. Taskinen J. Proteins. 1991; 11: 79-94Crossref PubMed Scopus (29) Google Scholar, 5Pollesello P. Ovaska M. Kaivola J. Tilgmann C. Lundstöm K. Kalkkinen N. Ulmanen I. Nissinen E. Taskinen J. J. Biol. Chem. 1994; 269: 28584-28590Abstract Full Text PDF PubMed Google Scholar). Contrary to skeletal troponin C, the binding of Ca2+ to cTnC does not induce an opening of the conformation. Consequently there is, in vitro, no exposure of a hydrophobic region (6Spyracopoulos L. Li M.X. Sia S.K. Gagné S.M. Chandra M. Solaro R.J. Sykes B.D. Biochemistry. 1997; 36: 12138-12146Crossref PubMed Scopus (180) Google Scholar, 7Sia S.K. Li M.X. Spyracopoulos L. Gagné S.M. Liu W. Putkey J.A. Sykes B.D. J. Biol. Chem. 1997; 272: 18216-18221Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 8Pääkkönen K. Annila A. Sorsa T. Pollesello P. Tilgmann C. Kilpeläinen I. Karisola P. Ulmanen I. Drakenberg T. J. Biol. Chem. 1998; 273: 15633-15638Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 9Gaponenko V. Abusamhadneh E. Abbott M.B. Finley N. Gasmi-Seabrook G. Solaro R.J. Rance M. Rosevear P.R. J. Biol. Chem. 1999; 274: 16681-16684Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The simultaneous binding of cardiac troponin I and Ca2+ to cNTnC, however, opens the structure of the N-terminal domain (10Li M.X. Spyracopoulos L. Sykes B.D. Biochemistry. 1999; 38: 8289-8298Crossref PubMed Scopus (248) Google Scholar, 11Dong W.J. Xing J. Villain M. Hellinger M. Robinson J.M. Chandra M. Solaro R.J. Umeda P.K. Cheung H.C. J. Biol. Chem. 1999; 274: 31382-31390Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). This structural and functional difference between TnC in skeletal and cardiac muscle is still to be clarified. Levosimendan has been reported to bind to the regulatory domain of cardiac troponin C in a calcium-dependent manner (5Pollesello P. Ovaska M. Kaivola J. Tilgmann C. Lundstöm K. Kalkkinen N. Ulmanen I. Nissinen E. Taskinen J. J. Biol. Chem. 1994; 269: 28584-28590Abstract Full Text PDF PubMed Google Scholar, 12Haikala H. Kaivola J. Nissinen E. Wall P. Levijoki J. Linden I.-B. J. Mol. Cell. Cardiol. 1995; 27: 1859-1866Abstract Full Text PDF PubMed Scopus (299) Google Scholar). However, the interaction of levosimendan with cTnC has been under debate for some time. Pollesello et al. (5Pollesello P. Ovaska M. Kaivola J. Tilgmann C. Lundstöm K. Kalkkinen N. Ulmanen I. Nissinen E. Taskinen J. J. Biol. Chem. 1994; 269: 28584-28590Abstract Full Text PDF PubMed Google Scholar) report the binding of levosimendan to the Ca2+-saturated form of cNTnC. A possible binding site for calcium sensitizers in the vicinity of Asp-88 was located by using point-mutated and dansylated human recombinant cTnC, NMR, and molecular modeling (4Ovaska M. Taskinen J. Proteins. 1991; 11: 79-94Crossref PubMed Scopus (29) Google Scholar, 5Pollesello P. Ovaska M. Kaivola J. Tilgmann C. Lundstöm K. Kalkkinen N. Ulmanen I. Nissinen E. Taskinen J. J. Biol. Chem. 1994; 269: 28584-28590Abstract Full Text PDF PubMed Google Scholar). However, very recently Kleerekoper and Putkey (13Kleerekoper Q. Putkey J.A. J. Biol. Chem. 1999; 274: 23932-23939Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) reported that levosimendan did not bind to cTnC. To clarify this controversial situation we studied the stability of levosimendan and levosimendan-cTnC under various solution conditions and the interaction of levosimendan with cTnC by heteronuclear NMR spectroscopy and small angle x-ray scattering. The results are also of general importance to studies of the structure-activity relationship by NMR. For every experiment with levosimendan, a fresh 30 mm stock solution was prepared by dissolving dry levosimendan powder into 30 mm potassium carbonate. The solution was gently shaken for ∼30 s at room temperature until a clear solution was obtained. The stock solution was analyzed by high performance liquid chromatography and by mass spectrometry to ensure that no degradation had occurred during the course of the sample preparation. Levosimendan solutions were thereafter diluted in the same buffer solution used for protein samples (20 mm Bis-Tris, 10 mm CaCl2, pH 6.8). In this study, we used three different cTnC molecules. Recombinant 15N-labeled N-terminal fragment of human cardiac troponin C (residues 1–91) was cloned, expressed, and purified as previously described (8Pääkkönen K. Annila A. Sorsa T. Pollesello P. Tilgmann C. Kilpeläinen I. Karisola P. Ulmanen I. Drakenberg T. J. Biol. Chem. 1998; 273: 15633-15638Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The cDNA for cTnCA-Cys was generated by site-directed mutagenesis of the cTnCC35S cDNA previously subcloned into the pET23d+ expression vector (Novagen). The polymerase chain reaction-based gene splicing by overlap extension strategy was used to incorporated base changes encoding for Ser at codon 84 (14). The cTnCA-Cys cDNA was subsequently subcloned intoNcoI and BamHI sites in the pET23d+ expression vector. Isotopically enriched cTnCC35S and cTnCA-Cys were expressed and purified as previously described (15 and 16). Protein samples were initially prepared in the presence of DTT to avoid disulfide formation (17Putkey J.A. Dotson D.G. Mouawad P. J. Biol. Chem. 1993; 268: 6827-6830Abstract Full Text PDF PubMed Google Scholar). Before the binding experiments, protein solutions containing DTT were washed with a large volume of DTT-free and NaN3-free buffer and concentrated by centrifuge ultrafiltration (3,000 Centricon, 5 °C, Sorvall SS-34 rotor, 7500 rpm). The washing buffer contained 20 mm Bis-Tris, 10 mm CaCl2 at pH 6.8. Protein concentrations, generally between 0.2 and 0.5 mm, were determined by the method of Bradford (18Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar) using bovine serum albumin as a standard. The NMR samples were prepared to the volume of 300 μl, containing 5% D2O, and the pH was adjusted to 6.8 at room temperature with a few microliters of dilute NaOH or HCl when necessary (pH was not corrected for deuteron effects). An aliquot from the levosimendan stock solution was instantly added after the preparation to the protein solution up to a 3-fold excess compared with the protein concentration, and pH was readjusted to 6.8 with dilute HCl. A small aliquot of the final Ca2+-saturated cTnC sample with levosimendan was kept at 40 °C and analyzed with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) at time points 0, 1, 4, 24, and 72 h. All spectra were acquired by a Varian Unity Inova 600- or 800-MHz spectrometers at 40 °C. One-dimensional proton spectra were collected to monitor the state of levosimendan under various experimental conditions. Two-dimensional 15N heteronuclear single-quantum correlation spectra (15N-HSQC) of cTnC and cNTnC were recorded at 800 MHz using 256 time increments (ni) and 16 transients (nt) and spectral widths of 11,000 Hz in proton dimension and 2,200 Hz in nitrogen dimension in the presence and absence of levosimendan. In addition, constant time13C-HSQC spectra of the double-labeled cTnCC35Swere recorded to measure chemical shifts of methionine methyl groups in the presence and absence of levosimendan (nt = 16, ni = 135, spectral widths of 12,000 Hz in proton dimension and 5,000 Hz in carbon dimension, 800 MHz). The 13C-edited NOESY spectra (ni = 256, nt = 48, spectral widths for both dimensions 10,000 Hz, 800 MHz) of selectively labeled levosimendan were acquired for the drug and protein-drug samples. Triple resonance spectra HNCACB and CBCACONH were acquired from the sample of15N/13C-labeled cTnCC35S complexed with levosimendan for the sequence-specific backbone assignment (ni = 64 for 13C, ni = 44 for 15N, nt = 16, spectral widths of 10,000 Hz in proton dimension, 12,000 Hz in carbon dimension, and 2,200 Hz in nitrogen dimension, 600 MHz). The 1H chemical shifts were referenced to the water signal (4.62 ppm), and the 13C and 15N chemical shifts were referenced indirectly relative to 3-(trimethylsilyl) propionate sodium salt (19Wishart D.S. Bigam C.G. Yao J. Abildgaard F. Dyson H.J. Oldfield E. Markley J.L. Sykes B.D. J. Biomol. NMR. 1995; 6: 135-140Crossref PubMed Scopus (2083) Google Scholar). All spectra were processed by Felix 97.0 software (Biosym Technologies, Inc.). For small angle x-ray scattering measurements, a fine focus copper x-ray tube in-line-focusing mode was was by using a and a The were using a The between the sample and the was mm, and the was from to The of the scattering vector is as = where is the scattering and is the The had a at half of and in and The protein solution of mm cTnCC35S was in a with of were used for sample and for cTnCC35S The scattering due to was and from the The was by the method using the A. Scopus Google Scholar). The stability of levosimendan was by proton NMR spectra in the presence and absence of a reducing agent DTT and a sodium azide The of these on the stability of levosimendan were they had been commonly used in the preparation of protein samples for NMR A levosimendan sample the 1H signal of in the region of the proton NMR a in the absence of and DTT at 40 °C, the signal the same a few after the of a levosimendan the 1H of the of levosimendan a in with the of indicative of a formation of a In an NMR this to and a was obtained. that the groups with the azide to form a by a J. and Scholar). In the presence of a large excess of a in the levosimendan and the 1H of the of levosimendan in a few as shown in The groups of levosimendan by a with the reducing agent A possible with DTT is in G. both levosimendan and DTT have two which to formation of a the of the sample and the in spectra were acquired to the interaction of levosimendan with the Ca2+-saturated form of cTnC. changes in the chemical shifts of were into two the of levosimendan, All resonance in the N-terminal half of the changes also in the C-terminal The resonance and chemical changes have been due to at two different has slow with a time of levosimendan since it does not in of the two The fast with a time of in changes of up to 20 Hz without In the we that the binding site with the slow is the primary binding site and that the fast are by binding to or secondary binding be from no resonance were and we that the primary binding site is in the N-terminal A of the in and C, that the primary binding site did not in cTnCA-Cys. but not is for levosimendan binding to the primary It is also to that there are no resonance in the C-terminal half of cTnCC35S, this half of the is for the binding to the primary site, since no resonance is in the N-terminal half of cTnC the in the results when the binding of levosimendan to cardiac troponin C. In the C-domain of cTnCC35S and cTnCA-Cys, there to be two secondary binding by the small chemical changes and these chemical changes to the cTnC structure from the Protein it that the two interaction sites in the C-domain of cTnC are not to The 13C of the methionine methyl groups are for binding of to cTnC, and they have been used to the interaction between cTnC and cardiac troponin I and and between cTnC and various (13Kleerekoper Q. Putkey J.A. J. Biol. Chem. 1999; 274: 23932-23939Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, Q. Liu W. Putkey J.A. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). time13C-HSQC spectra were used to changes in these as the methionine be from by correlation of the methyl of N-terminal methionine residues and were observed. This is in with the of the as the proton of both and into due to drug binding not be in In the C-terminal domain, the methyl groups of and small chemical changes and To into the binding of levosimendan with Ca2+ cardiac NOESY spectra of levosimendan, and the were acquired For a small levosimendan with a correlation time are and but and positive when the to the target protein. In the NOE of levosimendan when the drug is to the protein This that there is a in the correlation time of levosimendan in the presence of cTnC that levosimendan to cTnC. also for between levosimendan with a In NOESY NOE between labeled drug and protein were However, due to the of the these not be by a they of the specific interaction between levosimendan and Ca2+-saturated All samples were prepared the of the levosimendan binding to cTnC. (MALDI-TOF) of the samples were run in with NMR to the mass no formation of was that resonance in the correlation a exposure of the protein to levosimendan, we some of the sample as described by Kleerekoper and Putkey (13Kleerekoper Q. Putkey J.A. J. Biol. Chem. 1999; 274: 23932-23939Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). This NMR experiments for the structure All the NMR were collected after the of levosimendan to troponin C. we that the and the changes by levosimendan binding when the sample was for at 40 °C not the spectral changes the of fresh levosimendan to the angle x-ray scattering of the Ca2+-saturated form of cTnCC35S and its with levosimendan are The at the and there is no of protein A. G. of Scholar). is a small in the form of the when levosimendan to cTnCC35S The from 5 to 5 and the of from 0.5 to The determined of 0.5 for troponin C without levosimendan, is in a with a on troponin C T. T. Scholar). that levosimendan reacts with in protein solutions used in NMR studies the for studies of the stability of used in binding In the drug strategy structure-activity relationship by NMR 274: PubMed Scopus Google Scholar, 1997; PubMed Scopus Google Scholar, T. M. J. Chem. 1999; PubMed Scopus Google Scholar), for it of importance to the stability of the to be at the experimental conditions which are The binding of levosimendan to cardiac troponin C has been under debate for some time. studies (5Pollesello P. Ovaska M. Kaivola J. Tilgmann C. Lundstöm K. Kalkkinen N. Ulmanen I. Nissinen E. Taskinen J. J. Biol. Chem. 1994; 269: 28584-28590Abstract Full Text PDF PubMed Google Scholar, 12Haikala H. Kaivola J. Nissinen E. Wall P. Levijoki J. Linden I.-B. J. Mol. Cell. Cardiol. 1995; 27: 1859-1866Abstract Full Text PDF PubMed Scopus (299) Google Scholar, and J. Pollesello P. Kaivola J. Tilgmann C. Sorsa T. Annila A. Kilpeläinen I. J. Mol. Cell. Cardiol. Full Text PDF PubMed Scopus Google Scholar) for levosimendan However, results that show no binding have also been reported (13Kleerekoper Q. Putkey J.A. J. Biol. Chem. 1999; 274: 23932-23939Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). In in the course of the of levosimendan, some was observed. The pH of the protein-drug solution during the experiments, and levosimendan of the protein solution as a it to the not have found that commonly used in protein DTT and with levosimendan azide an with levosimendan, and it was no added to protein samples. To cTnC samples without DTT was of of the possible formation of and disulfide However, we no disulfide formation in DTT-free cTnCC35S samples after a of of as analyzed by Moreover, disulfide are not possible in cTnCC35S, with cysteine The controversial results of levosimendan binding to cTnC are to by drug under different experimental conditions. that the to some also from the that various protein have been In the recombinant N-terminal fragment of human cTnC two cysteine Cys-35 and In full-length cTnCC35S, Cys-35 is mutated to serine, and in full-length cTnCA-Cys, both cysteine residues are to The residues Cys-35 and of cTnC are various importance for the of the protein. However, it has been previously reported that the of to does not calcium binding to cTnC but the structure of cTnC, as by changes in its binding (17Putkey J.A. Dotson D.G. Mouawad P. J. Biol. Chem. 1993; 268: 6827-6830Abstract Full Text PDF PubMed Google Scholar). The spectra show that binding of levosimendan to Ca2+-saturated of cTnCC35S and cTnCA-Cys are The small chemical changes to the secondary binding sites are but the resonance in the N-domain of cTnCC35S are from of the form of cTnC. This that the a difference in levosimendan binding to the primary binding that the primary binding site on The N-terminal fragment of cTnC also interaction with levosimendan However, the binding to be different compared with the full-length The N-terminal fragment does not an primary binding site for levosimendan. This is is a few residues from the chain at It be very to the primary binding site of levosimendan in the cTnC. This is, however, not possible since there are all the N-terminal half of cTnC. The that of the residues in the N-domain of cTnCC35S show that the binding of levosimendan to the primary site a conformational of cNTnC. The exchange for this conformational is 10 since we not in two the open and The exchange between open and is on the NMR time and the the form (8Pääkkönen K. Annila A. Sorsa T. Pollesello P. Tilgmann C. Kilpeläinen I. Karisola P. Ulmanen I. Drakenberg T. J. Biol. Chem. 1998; 273: 15633-15638Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 9Gaponenko V. Abusamhadneh E. Abbott M.B. Finley N. Gasmi-Seabrook G. Solaro R.J. Rance M. Rosevear P.R. J. Biol. Chem. 1999; 274: 16681-16684Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). An for be that levosimendan to the open but there is a large difference between and as compared possible is that levosimendan to both but to the open since in the presence of levosimendan the a for the two The exchange between the two of cTnC is in the presence of levosimendan. the of the work, we were not to It is to the levosimendan binding to troponin C with the binding of and Q. Liu W. Putkey J.A. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google it has been shown by x-ray that the structure of cNTnC opens in to binding Putkey J.A. C. A. PubMed Scopus Google Scholar). bind to cTnCA-Cys to the N-terminal half and opens its and the two the TnI binding to the C-terminal The chemical changes by levosimendan binding are by binding not In to the of binding to cTnC, small angle x-ray scattering show no of a domain-domain in the presence of levosimendan it that levosimendan binding to the primary binding site, located to the of the from to and the this be by a levosimendan structural in the regulatory domain of cTnC. In cTnC is not to a large of the troponin This is in with the regulatory However, the final be the structure of the in solution is show interaction sites for levosimendan on the Ca2+-loaded form of cardiac troponin C Levosimendan does bind to cTnCC35S, but in the absence of and which degradation of levosimendan. the current the between studies of levosimendan binding to cTnC. results that the primary binding site is located in the regulatory domain of cTnC and that there are two secondary binding sites at the C-terminal half of cTnC to the of three binding to cTnCA-Cys Putkey J.A. C. A. PubMed Scopus Google Scholar). levosimendan to the opening of the regulatory However, levosimendan does not a domain-domain closure. we are not to the of the primary binding site on the N-terminal domain due to the changes in the spectra levosimendan However, results from experiments with cTnCA-Cys show that the presence of is of importance for levosimendan The results a to that the binding of levosimendan to the regulatory domain of cTnC is the its