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INTRODUCTION |
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 (1). Levosimendan, a potent calcium sensitizer that
improves the force development of the muscle contraction without
increasing the cytosolic Ca2+ ion concentration (2), 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 (3).
This has also been proposed to be a potential binding site for calcium
sensitizers (4, 5). 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 (6-9). The simultaneous binding of cardiac
troponin I and Ca2+ to cNTnC, however, opens the structure
of the N-terminal domain (10, 11). 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 (5, 12).
However, the interaction of levosimendan with cTnC has been under
debate for some time. Pollesello et al. (5) 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 (4, 5). However, very
recently Kleerekoper and Putkey (13) 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.
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MATERIALS AND METHODS |
Levosimendan Samples--
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).
Protein Sample Preparation--
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 (8). 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 into
NcoI 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 (17). 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 (18) 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.
NMR Spectroscopy--
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 time
13C-HSQC spectra of the double-labeled cTnCC35S
were 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
CBCA[CO]NH were acquired from the sample of 15N/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 (19). All spectra were processed by Felix 97.0 software (Biosym Technologies, Inc.).
Small Angle X-ray Scattering--
For small angle x-ray
scattering measurements, a fine focus copper x-ray tube
in-line-focusing mode was utilized. Copper K
radiation
(1.54 Å) was monochromatized by using a Ni filter and a totally
reflecting glass block (Huber small-angle chamber 701). The intensity
curves were measured using a linear one-dimensional position-sensitive
proportional counter (M. Braun OED50M). The distance between the sample
and the detector was 152 mm, and the k range was from 0.03 to 0.7 1/Å. The magnitude of the scattering vector k is
defined as k = 4
sin
/
, where 2
is the
scattering angle, and
is the wavelength. The instrumental function
had a full width at half maximum of 0.35 and 0.005 1/Å in
vertical and horizontal directions, respectively. The protein solution (80 µl of 0.3 mM cTnCC35S solution) was
placed in a steel-framed cell with thin polyimide windows. Measuring
times of 2 h were used for cTnCC35S-levosimendan
sample and 3 times 2 h for cTnCC35S sample. The
background scattering due to solvent was measured separately and
subtracted from the intensity curves. The distance distribution
function was calculated by the indirect Fourier transform method using
the program Gnom (20).
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RESULTS |
Stability of Levosimendan in the Samples--
The stability of
levosimendan was monitored by recording one-dimensional proton NMR
spectra in the presence and absence of a reducing agent DTT and a
bacterial inhibitor sodium azide (NaN3). The effects of
these substances on the stability of levosimendan were tested because
they had been commonly used in the preparation of protein samples for
NMR analysis. A freshly made levosimendan sample (Fig.
1A) showed the characteristic
AA'BB' 1H signal pattern of para-disubstituted
phenyl ring in the aromatic region of the proton NMR spectrum. After a
20-h incubation in the absence of NaN3 and DTT at 40 °C,
the signal pattern remained the same (Fig. 1B).
Surprisingly, just a few minutes after the addition of NaN3
to a levosimendan solution, the 1H spectrum of the aromatic
protons of levosimendan showed a reduction in intensity combined with
the appearance of new resonances (Fig. 1C) indicative of a
formation of a new compound. In an NMR tube, this reaction proceeded to
completeness, and a single product was obtained. We suspect that the
cyano groups reacted with the azide group to form a cyclic adduct (Fig.
1F) by a 1,3-dipolar addition mechanism (21).

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Fig. 1.
Stability of levosimendan. A freshly
prepared levosimendan sample in water without additives (A)
and the same sample after an incubation of 20 h at 40 °C
(B) show the characteristic AA'BB' spectrum of levosimendan
in the aromatic region of the one-dimensional proton spectra acquired
at 600 MHz. Stability of the drug was tested in the presence of 0.05%
NaN3 (1 mM levosimendan) (C) and 8 mM DTT (1 mM levosimendan) (D). The
spectrum of levosimendan in the presence of NaN3 is
referenced according to the left doublet. Other spectra were referenced
to the water signal. Levosimendan molecule (E) and its
adduct with sodium azide (F) as well as a reaction product
of levosimendan in the presence of DTT (G) are shown on the
right.
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In the presence of a large excess of DTT, sometimes a visible
precipitate appeared in the levosimendan sample, and the 1H
signals of the aromatic protons of levosimendan almost completely disappeared in a few minutes, as shown in Fig. 1D. The cyano
groups of levosimendan might react by a reductive addition mechanism with the reducing agent DTT. A possible reaction product with DTT is
presented in Fig. 1G. Notably, both levosimendan and DTT have two reactive groups, which can lead to formation of a polymer, causing the precipitation of the sample and the broad lines in the
1H spectrum (Fig. 1D).
Binding of Levosimendan to
(Ca2+)3-cTnC--
Two-dimensional
15N-HSQC spectra were acquired to determine the interaction
of levosimendan with the Ca2+-saturated form of cTnC. The
15N-HSQC spectrum (Fig. 2)
revealed changes in the chemical shifts of several cross-correlation
peaks. Some cross-peaks were split into two signals upon the addition
of levosimendan, whereas other cross-peaks experienced frequency
shifts. All resonance doublings took place in the N-terminal half of
the (Ca2+)3-cTnCC35S, whereas shift
changes appeared also in the C-terminal half.

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Fig. 2.
Binding of levosimendan to
Ca2+-saturated cTnC observed by 15N-HSQC
spectra at 800 MHz. The spectrum of cTnCC35S shows
chemical shift changes and resonance doublings upon levosimendan
binding (A). Expansions of the well resolved region of
Ca2+-saturated cTnCC35S (B),
cTnCA-Cys (C), and cNTnC (D) in the
presence (blue) and absence (red) of three
equivalents of levosimendan. Chemical shift changes are larger upon
levosimendan binding for Ca2+-saturated
cTnCC35S (B) than for cTnCA-Cys
(C) and also the isolated N-terminal fragment (1-91) of
cTnC (D).
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The observed resonance doublings and chemical shift changes might have
been due to at least two different processes. One process has slow
kinetics with a residence time of levosimendan >0.1 s, since it does
not result in any observable broadening of the two resonances. The
other employs fast kinetics with a residence time of <0.001 s,
resulting in shift changes of up to 20 Hz without any major line
broadening. In the following we will assume that the binding site with
the slow kinetics is the primary binding site and that the fast
kinetics are caused by binding to one or more secondary binding sites.
As can be seen from Fig.
3A, no resonance
doublings were observed beyond residue 92, and therefore, we concluded
that the primary binding site is in the N-terminal domain. A comparison
of the data in Figs. 2, B and C, shows that the
primary binding site did not exist in cTnCA-Cys. Evidently Cys-84, but not Cys-35, is important for levosimendan binding to the
primary site. It is also important to note that even though there are
no resonance doublings in the C-terminal half of cTnCC35S, this half of the molecule is essential for the binding to the primary
site, since no resonance doubling is observed in the isolated N-terminal half (1-91) of cTnC (Fig. 2D). These
observations may explain the discrepancies in the results obtained
earlier when studying the binding of levosimendan to cardiac troponin
C. In the C-domain of cTnCC35S and cTnCA-Cys,
there seems to be two secondary binding sites, judged by the relatively
small chemical shift changes (Fig. 3B and C). By
mapping these chemical shift changes to the cTnC structure (1AJ4 from
the Protein Data Bank), it appears that the two distinct interaction
sites in the C-domain of cTnC are not spatially related to each
other.

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Fig. 3.
Chemical shift changes due to levosimendan
binding to Ca2+-saturated cTnC. Levosimendan binding
to Ca2+-saturated cTnCC35S gave rise to
resonance doublings with large splittings (A) and to small
shift changes (B), presented as a function of amino acid
sequence. For cTnCA-Cys, the chemical shift changes are
small (C), and no resonance doublings are observed. The
splittings are presented as a distance in Hz between the
cross-correlation peaks of the cTnCC35S.
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The 13C signals of the methionine methyl groups are
sensitive markers for binding of ligands to cTnC, and they have been
used to study the interaction between cTnC and cardiac troponin I (15, 22, and 23) and between cTnC and various drugs (13, 24). Constant time
13C-HSQC spectra were used to follow changes in these
groups, as the methionine methyls can be easily distinguished from
other methyls by their negative correlation peaks. Splitting of the methyl signals of N-terminal methionine residues Met-47, Met-81, and
Met-85 (Fig. 4) were observed. This is in
agreement with the result of the 15N-HSQC experiment, as
the amide proton signals of both Met-81 and Met-85 split into doublets
due to drug binding (Met-47 could not be assigned in the
15N-HSQC). In the C-terminal domain, the methyl groups of
Met-120 and Met-157 experience only small chemical shift changes
(Met-120,
1H 0.03 ppm;
13C, 0.04 ppm; and
Met-157,
1H 0.02 ppm).

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Fig. 4.
Binding of levosimendan to
Ca2+-saturated cTnCC35S followed by constant
time 13C-HSQC spectra at 800 MHz. 13C-HSQC
spectra of Ca2+-saturated cTnCC35S in the
absence (A) and presence (B) of three equivalents
of levosimendan. Assignments the for 1H-13C Met
methyl correlations are according to Lin et al. (25).
Met-47, Met-81, and Met-85 experience the largest changes in shifts.
For each of these residues the simultaneous presence of two
correlations reveals the presence of two conformations. Assignments of
the correlations in the complex are based on similarity to free
cTnCC35S.
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To gain further insight into the binding of levosimendan with
Ca2+-saturated cardiac TnC, two-dimensional
NOESY spectra of free levosimendan,
(Ca2+)3-cTnCC35S, and the
drug-protein complex were acquired (Fig. 5). For a small ligand like levosimendan
with a short rotational correlation time (
c), NOEs are
weak and negative but become stronger and positive when the ligand
binds to the target protein. In water, the intramolecular NOE signals
of levosimendan change their signs when the drug is titrated to the
protein solution. This shows that there is a pronounced change in the
correlation time of levosimendan in the presence of cTnC and, thus,
proves that levosimendan binds to cTnC. We also looked for cross-peaks
between unlabeled (Ca2+)3-cTnCC35S
and levosimendan with a 13C-labeled aromatic ring. In the
13C-edited two-dimensional NOESY spectrum, several NOE
correlations between labeled drug molecules and unlabeled protein were
observed (Fig. 5). However, due to the instability of the
protein-levosimendan samples, these NOEs could not be assigned by a
titration series. Nevertheless, they provide direct evidence of the
specific interaction between levosimendan and
Ca2+-saturated cTnCC35S.

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Fig. 5.
13C-edited NOESY of
Ca2+-saturated cTnCC35S with
13C-labeled levosimendan at 800 MHz. The
13C-edited NOESY of the 13C-labeled aromatic
ring of levosimendan with unlabeled cTnC was acquired at 40 °C.
Trace A through the low-field peak of the aromatic region is
indicated by the arrow. The levosimendan binding to
cTnCC35S leads to an increase of effective rotational
correlation time that gives rise to observable intramolecular NOEs
within the drug molecule (dashed lines). Weak correlations
seen at the high-field are thought to arise from the interaction
between levosimendan and cTnCC35S. Trace B is
from the same level of the reference NOESY spectrum of
13C-labeled levosimendan without cTnC in the same
conditions.
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All drug/protein samples were freshly prepared before collecting the
data of the levosimendan binding to cTnC. Mass spectrometric analyses
(MALDI-TOF) of the drug/protein samples were run in parallel with our
NMR experiments. According to the mass spectrometric data, no formation
of covalent complex was observed that could explain resonance doublings
in the 15N-1H correlation spectrum. After a
prolonged exposure of the protein to levosimendan, we observed some
modification of the sample as described by Kleerekoper and Putkey (13).
This modification prohibited longer NMR experiments required for the
complete structure characterization.
All the NMR data were collected immediately after the addition of
levosimendan to troponin C. Interestingly, we noticed that the new
signals and the shift changes induced by levosimendan binding
disappeared when the sample was stored for several days at 40 °C
(data not shown). Notably, the spectral changes reappeared upon the
addition of fresh levosimendan to the sample.
Small angle x-ray scattering of the Ca2+-saturated form of
cTnCC35S and its complex with levosimendan are rather
similar. The scattered intensity obeys Guinier law at the smallest
k values, and there is no sign of protein aggregation (Fig.
6A) (26). There is only a
small change in the form of the distance distribution function,
P(r), when levosimendan binds to
cTnCC35S (Fig. 6B). The maximum distance
increases from 63 ± 5 to 70 ± 5 Å, and the radius of
gyration increases from 20.2 ± 0.5 to 21.7 ± 0.6 Å. The
determined radius of gyration, 20.2 ± 0.5 Å, for troponin C
without levosimendan, is in a good agreement with a previous study on
troponin C (27).

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Fig. 6.
Small angle x-ray scattering data.
A, scattered intensities (I) of troponin C
(solid line) and troponin C in the complex with levosimendan
(filled circles) as a function of the magnitude of the
scattering vector k. For clarity, only every third measuring
point is plotted in the figure. The Guinier region is shown in the
inset. Error bars are based on statistical
precision of the experimental intensity curves. B,
experimental distance distribution functions,
P(r), of troponin C (solid line) and
troponin C with levosimendan (filled circles) and
P(r) calculated from the coordinates of
bepridil-cTnCA-Cys (1DTL from the Protein Data Bank)
(dotted line). The difference in the shapes of the
P(r) functions indicates that levosimendan only
slightly affects the relative orientation of the two domains of
troponin C.
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DISCUSSION |
Our present finding that levosimendan reacts with common additives
in protein solutions used in NMR studies clearly shows the need for
careful studies of the stability of molecules used in binding
experiments. In the drug discovery strategy structure-activity relationship by NMR (28-30), for example, it appears now of utmost importance to know the stability of the compounds to be tested at the
experimental conditions which are used.
The binding of levosimendan to cardiac troponin C has been under debate
for some time. Previous studies (5, 12, and 31) gave evidence for
levosimendan binding. However, contradictory results that show no
binding have also been reported (13). In our hands, in the course of
the titration of (Ca2+)3-cTnCC35S
with levosimendan, some odd behavior was observed. The pH of the
protein-drug solution changed during the experiments, and sometimes
levosimendan precipitated out of the protein solution as a bright
yellow precipitate, making it difficult to reproduce the measurements
(data not shown). We have now found that commonly used additives in
protein samples, DTT and NaN3, react with levosimendan (Fig. 1). Sodium azide forms an adduct with levosimendan, and consequently it was no longer added to our protein samples. To prepare
cTnC samples without DTT was of concern because of the possible
formation of intra- and intermolecular disulfide bonds. However, we
observed no intermolecular disulfide bond formation in our DTT-free
cTnCC35S samples after a couple of days of incubation, as
analyzed by MALDI-TOF. Moreover, intramolecular disulfide bonds are not
possible in cTnCC35S, with only one cysteine residue.
The controversial results of levosimendan binding to cTnC are difficult
to explain only by drug reactivity under different experimental
conditions. We believe that the differences to some extent also
originate from the fact that various protein sequences have been used.
In fact, the recombinant N-terminal fragment of human cTnC contains two
cysteine residues, Cys-35 and Cys-84. In full-length chicken
cTnCC35S, Cys-35 is mutated to serine, and in full-length
chicken cTnCA-Cys, both cysteine residues are changed to
serines. The residues Cys-35 and Cys-84 of cTnC are conserved among
various species, suggesting their importance for the function of the
protein. However, it has been previously reported that the conversion
of cysteines to serines does not alter calcium binding to cTnC but
might modify the structure of cTnC, as indicated by changes in its dye
binding properties (17). The 15N-HSQC spectra show that
binding of levosimendan to Ca2+-saturated forms of
cTnCC35S and cTnCA-Cys are different. The small
chemical shift changes attributed to the secondary binding sites are
similar, but the observed resonance doublings in the N-domain of
cTnCC35S are completely missing from the
15N-HSQC spectrum of the A-Cys form of cTnC. This
observation proves that the C84S mutant makes a difference in
levosimendan binding to the primary binding site. We therefore conclude
that the primary binding site critically depends on Cys-84. The
isolated N-terminal fragment of cTnC also shows interaction with
levosimendan (Fig. 2D). However, the binding seems to be
different compared with the full-length cTnCC35S. The
N-terminal fragment does not contain an intact primary binding site for
levosimendan. This is reasonable because Cys-84 is only a few residues
away from the chain end at Gly-91.
It would naturally be very interesting to localize the primary binding
site of levosimendan in the cTnC. This is, however, presently not
possible since there are effects all over the N-terminal half of cTnC.
The fact that most of the residues in the N-domain of
cTnCC35S show resonance-doubling indicates that the binding of levosimendan to the primary site causes a conformational change involving most of cNTnC. The exchange rate for this conformational change is slow, k < 10 s
1,
since we do not observe any line-broadening effects.
(Ca2+)3-cTnC exists in two conformations,
i.e. the open and closed states. The exchange between open
and closed conformations is intermediate on the NMR time scale, and the
equilibrium favors the closed form (8, 9). An obvious
explanation for peak doublings would be that levosimendan binds only to
the open conformation, but there is a large difference between the
kon and koff values, the
koff being significantly slower as compared with
kon. Another possible explanation is that
levosimendan binds to both forms, but preferably to the open one, since
in the presence of levosimendan the equilibrium reaches about a 50:50
ratio for the two states. The exchange between the two states of cTnC
is significantly slower in the presence of levosimendan. At the present
stage of the work, we were not able to completely rule out either possibility.
It is interesting to compare the levosimendan binding to troponin C
with the binding of other molecules (e.g. bepridil,
EMD57033, and trifluoperazine (24, 32)). Recently, it has been shown by
x-ray crystallography that the structure of cNTnC opens in response to
bepridil binding (33). Three bepridil molecules bind to one
cTnCA-Cys molecule. One bepridil molecule binds to the
N-terminal half and opens its structure, and the other two bepridil
molecules mimic the TnI binding to the C-terminal half. The chemical
shift changes induced by levosimendan binding are smaller than those
caused by bepridil binding (data not shown). In contrast to the case of
bepridil binding to cTnC, our small angle x-ray scattering data show no
sign of a domain-domain closure in the presence of levosimendan (Fig.
6). If anything, it seems that levosimendan binding to the primary
binding site, located close to the end of N-domain, prevents the
domains from moving closer to each other and might actually increase
the maximal distance (rmax). Alternatively, this
might be caused by a levosimendan induced structural change in the
regulatory domain of cTnC. In vivo cTnC is not expected to
experience a large spatial reorganization of domains within the
troponin complex. This is in accordance with Ca2+
sensitizers, affecting only the regulatory domain. However, the final
evidence will be obtained once the structure of the
levosimendan-(Ca2+)3-cTnCC35S
complex in solution is determined.
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CONCLUSIONS |
Our data unambiguously show several interaction sites for
levosimendan on the Ca2+-loaded form of cardiac troponin C
(cTnCC35S). Levosimendan does bind to
cTnCC35S, but only in the absence of NaN3 and
DTT, which cause degradation of levosimendan. Thus, the current
observations explain the discrepancies between earlier studies of
levosimendan binding to cTnC. Our results suggest that the primary
binding site is located in the regulatory domain (cNTnC) of cTnC and
that there are two secondary binding sites at the C-terminal half of cTnC possibly analogous to the case of three bepridil molecules binding
to cTnCA-Cys (33). Likewise, levosimendan may contribute to
the opening of the regulatory domain. However, levosimendan does not
cause a domain-domain closure. At present, we are not able to determine
the precise locus of the primary binding site on the N-terminal domain
due to the numerous changes in the spectra upon levosimendan binding.
However, results from experiments with cTnCA-Cys show that
the presence of Cys-84 is of critical importance for levosimendan
binding. The results presented give us a reason to believe that the
binding of levosimendan to the calcium-saturated regulatory domain of
cTnC is the mechanism behind its known Ca2+-sensitizing effect.