From the CIHR Group in Protein Structure and
Function, Department of Biochemistry, University of Alberta, Edmonton,
Alberta T6G 2H7, Canada, the ¶ Department of Biochemistry,
University of Alberta, Edmonton, Alberta T6G 2H7, Canada,
Merck
KGaA, Department of Pharmaceutical Research, Frankfurter Straße 250, 64271 Darmstadt, Germany, and the ** Department of Physiology and
Biophysics, College of Medicine, University of Illinois-Chicago,
Chicago, Illinois 60612-7342
Received for publication, March 19, 2001, and in revised form, April 23, 2001
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ABSTRACT |
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Ca2+ binding to cardiac
troponin C (cTnC) triggers contraction in heart muscle. In heart
failure, myofilaments response to Ca2+ are often altered
and compounds that sensitize the myofilaments to Ca2+
possess therapeutic value in this syndrome. One of the most potent and
selective Ca2+ sensitizers is the thiadiazinone derivative
EMD 57033, which increases myocardial contractile function both
in vivo and in vitro and interacts with cTnC
in vitro. We have determined the NMR structure of the 1:1
complex between Ca2+-saturated C-domain of human cTnC
(cCTnC) and EMD 57033. Favorable hydrophobic interactions between the
drug and the protein position EMD 57033 in the hydrophobic cleft of the
protein. The drug molecule is orientated such that the chiral group of
EMD 57033 fits deep in the hydrophobic pocket and makes several key
contacts with the protein. This stereospecific interaction explains why
the ( In order to function properly, heart muscle must respond
efficiently to the transient increases in cytosolic Ca2+
levels in the myocardial cell. In the syndrome of heart failure, there
is strong evidence that the amount of Ca2+ available for
contraction is depressed as is the maximum force generating capability
of the myofilaments (for reviews, see Refs. 1-3). Treating this
condition by elevating intracellular Ca2+ has the potential
threat of inducing arrhythmias. In contrast, the ability to sensitize
cardiac muscle to Ca2+ and increase cardiac contractility
has the considerable advantage of increasing tension with little or no
change in intracellular Ca2+ and thus no increase in the
energy required to release and transport Ca2+ (for a
review, see Ref. 1). A logical target for such Ca2+
sensitizing drugs is cardiac troponin C
(cTnC)1 due to its role as
the Ca2+ binding receptor on the thin filament of cardiac muscle.
cTnC is a member of the EF-hand family of Ca2+-binding
proteins. Its structure represents a dumbbell with the N- and C-domains connected by a flexible linker in solution (4). Both domains contain a
core of hydrophobic residues. Once exposed, these hydrophobic residues
are essential for the binding of cTnI to cTnC and transmitting the
Ca2+ signal to other proteins in the thin filaments, and
ultimately signal the activation of the myosin-actin ATPase reaction
(for reviews, see Refs. 5 and 6). Structural studies have shown that
the apo N-domain of cTnC (cNTnC) adopts a "closed" conformation with most of its hydrophobic residues buried (7), like the apo N-domain
of sTnC (sNTnC) (8, 9). However, the binding of Ca2+ has
strikingly different structural consequences in cNTnC and sNTnC. In
sTnC, the N-domain switches from a closed to an "open" conformation upon binding Ca2+ (9), while the N-domain of
cTnC remains in a closed state in the Ca2+ bound state (7).
Consequently, a large hydrophobic surface is exposed in the
Ca2+-saturated sNTnC, but not in the
Ca2+-saturated cNTnC. This is mainly due to the fact that
sNTnC contains two functional Ca2+-binding sites, while
cNTnC contains only one (10).
The exposed hydrophobic surface on the Ca2+-saturated sNTnC
has been shown as the sTnI-binding site (11-14). Although
Ca2+ binding to cNTnC induces little structural changes, it
sets the stage for cTnI binding. In the end, both cNTnC and sNTnC adopt similar conformations in binding their respective TnI regions. Specifically, sTnI115-131 was found to bind to the
hydrophobic cleft of Ca2+-saturated sNTnC (13) and the
corresponding cTnI147-163 also interacts with the
hydrophobic cleft of Ca2+-saturated cNTnC and stabilizes
the opening conformation of cNTnC (15). This region of TnI has been
identified by many biological and biophysical studies to be the region
responsible for binding to the regulatory domain of TnC and this
interaction modulates the binding of the N-terminal and inhibitory
regions of TnI to the C-domain of TnC (for reviews, see Refs. 16 and
17).
Unlike the apo N-terminal domain, the apo C-terminal domain in both
sTnC and cTnC possesses a largely unstructured state. Upon binding two
Ca2+ ions, this domain folds into a compact globular
structure (18, 19) and exhibits a similar but slightly less open
conformation as that of the Ca2+-saturated sNTnC. Two
regions of TnI have been shown to interact with the
Ca2+-bound C-domain (12). These include the inhibitory
region corresponding to sTnI96-115 or
cTnI128-147, and the N-terminal region corresponding to
sTnI1-40 or cTnI34-71. The inhibitory region
is the critical functional region in the interaction of TnI with TnC
and its movement from TnC to actin-tropomyosin is believed to be the
major switch between muscle contraction and relaxation, while the
N-terminal region plays primarily a structural role (12). NMR studies
of the inhibitory peptides have yielded some structural information on
the interaction of inhibitory region with TnC (20-22), however, the
exact binding sites for the inhibitory region on TnC has been under
debate. The crystal structure of sTnC in complex with
sTnI1-47 has shown that sTnI3-33 forms a long
In view of the importance of the exposed hydrophobic surfaces on both
domains of cTnC for the binding of cTnI, it is clear that one way to
enhance the Ca2+ sensitivity of cardiac muscle would be to
stabilize the interaction of cTnC and cTnI by amplifying the
hydrophobic cTnI-binding interface on cTnC. This can be accomplished by
employing certain pharmacological agents that bind to the hydrophobic
cleft but do not interfere with cTnC-cTnI interaction. Indeed, a
variety of small hydrophobic compounds including the calmodulin
antagonists bepridil, trifluoperazine, and calmidazolium have been
shown to increase the Ca2+ sensitivity of cardiac muscle
preparations (for a review, see Ref. 1). However, most of these
compounds are not good Ca2+ sensitizers due to other
undesired properties. This led to a search for new generations of
Ca2+ sensitizing compounds, among which is the
thiadiazinone derivative EMD 57033 (24-26). This drug has been found
to increase the Ca2+ sensitivity of both myofibrillar
ATPase and force development by skinned muscle fibers (26, 27). Recent
in vivo studies have also demonstrated that EMD 57033 enhances cardiac contractile function without affecting
Ca2+ homeostasis (28-32). The myofibrillar
Ca2+ sensitizing effects of EMD 57033 are remarkably
stereo-specific. EMD 57033 is the (+)-enantiomer of a racemate. The
( In order to delineate the molecular basis of the interaction between
cTnC and EMD 57033, we have determined the NMR solution structure of
the 1:1 complex between the Ca2+-saturated C-domain of
human cTnC and EMD 57033. In the present structure, the drug molecule
is oriented such that the chiral group of EMD 57033 fits deep in
the hydrophobic pocket and makes several key contacts with the protein.
This stereospecific interaction explains why the ( Sample Preparation--
The engineering of the expression
vector for the cCTnC-(91-161) protein was as described in
Chandra et al. (36). The expression and purification of
15N- and 15N/13C-labeled protein in
Escherichia coli follows the procedures as described
previously for sNTnC (37), except a further purification step was done
using a Superdex-75 column (Amersham Pharmacia Biotech) with a buffer
containing 0.15 M NaCl and 50 mM Tris, pH 8.0. The handling and characterization of the stock solutions of EMD 57033 are as described in Li et al. (35). Two stock solutions (94 and 53 mM, respectively) of EMD 57033 in
Me2SO-d6 (Cambridge Isotopes Inc.)
were prepared. The synthetic peptides, cTnI128-147, acetyl-TQKIFDLRGKFKRPTLRRVR-amide, and cTnI34-71,
acetyl-AKKKSKISASRKLQLKTLLLQIAKQELEREAEERRGEK-amide, were
prepared as described for sTnI96-115 and
sTnI1-40 by Tripet et al. (12). Solid peptides
were dissolved in double distilled water to make stock solutions. The
concentrations were 60 mM for cTnI128-147 and
32 mM for cTnI34-71, respectively, as
determined by amino acid analysis. All NMR samples were 500 µl in
volume. The buffer conditions were 100 mM KCl, 10 mM imidazole, 0.2 mM
2,2-dimethyl-2-silapentanesulfonic acid, and 0.01% NaN3 in
90% H2O, 10% D2O, and the pH was 6.7. For structure determination, NMR samples contained 1-2 mM
15N-cCTnC or 15N/13C-cCTnC
saturated with ~10 mM CaCl2. The EMD 57033 was titrated to the Ca2+-saturated cCTnC until ratios
reached 1:1. The NMR samples used for titrations with
cTnI128-147 or cTnI34-71 were prepared in a
similar manner as described above. The titrations of cCTnC by
cTnI128-147 or cTnI34-71 follow the
procedures as described previously (35).
Stability of EMD 57033 in Aqueous Solution--
EMD 57033 is
only marginally soluble in aqueous solutions. Once it is bound to
cCTnC, the complex is soluble and is stable for 4-5 days, which is
long enough for a typical three-dimensional NMR experiment.
Two-dimensional {1H,15N}-HSQC NMR spectra
acquired before and after every three-dimensional NMR experiment were
compared and their identity was taken as an indication of sample
stability throughout the three-dimensional NMR experiment. However, EMD
57033 tends to dissociate from the complex and precipitates out of the
aqueous environment after 4-5 days. This is reflected in the
disappearance of the EMD 57033-induced chemical shift changes (Fig. 1),
which, however, can be reproduced upon the addition of more EMD 57033. This indicates that the protein is still intact and it is the drug that
is disappearing from the solution. 15N-Filtered DIPSI
spectra of EMD 57033 in the cCTnC-EMD 57033 NMR sample detected no new
resonances except those of intact EMD 57033, implying that the drug is
not breaking down, but precipitating out of the solution. This
conclusion was further supported by evidence from mass spectrometry
measurements.2 1H
NMR spectra were used to check any possible modification of EMD 57033 by chemicals present in the NMR buffer, such as imidazole and
NaN3, which have been shown to interact with levosimendan (38), and the results show no sign of reaction between EMD 57033 and
the chemical reagents.
NMR Spectroscopy--
Most NMR data used in this study were
collected at 30 °C using Unity 600 MHz, Unity Inova 500 MHz, and
Inova 800 MHz spectrometers. All three spectrometers are equipped with
triple resonance probes and Z-pulsed field gradients (XYZ gradients for
the 800 MHz). Unity 300 spectrometer was also used to collect the
spectra of EMD 57033 in Me2SO-d6.
Two-dimensional {1H,15N}-HSQC NMR spectra
were acquired using the sensitivity enhanced gradient pulse scheme
developed by Lewis E. Kay and co-workers (39, 40). For cCTnC in the
cCTnC·EMD 57033 complex, the chemical shift assignments of the
backbone and side chain atoms and NOE interproton distance restraints
were determined using the two-dimensional and three-dimensional NMR
experiments described in Table I. For the bound EMD 57033, the proton
chemical shifts of the drug were assigned by using two-dimensional
15N/13C-filtered NOESY (80 ms mixing time) and
two-dimensional 15N/13C-filtered DIPSI
experiments (43.2-ms spinlock time). The pulse sequences for these two
experiments were based on Ogura et al. (41) with extensive
modifications done in this lab (Leo Spyracopoulos, University of
Alberta). One-dimensional 1H and two-dimensional DIPSI
spectra of EMD 57033 in Me2SO-d6
were obtained using the Unity 300 MHz spectrometer. The intermolecular proton distances were obtained using three-dimensional
15N/13C F1-filtered,
F3-edited NOESY HSQC experiments employing linear frequency
ramped broadband inversion pulses for 13C (80-ms mixing
time) (42).
Data Processing and Peak Calibration--
All two-dimensional
and three-dimensional NMR data was processed using NMRPipe (43), and
all one-dimensional NMR data were processed using VNMR (Varian
Associates). The spectra were analyzed using NMRView (44). For cCTnC in
the complex, intramolecular distance restraints obtained from the NOESY
experiments were calibrated according to Gagné et al.
(45). Intramolecular proton distances for EMD 57033 in the complex were
calibrated based on NOEs corresponding to known distances (neighboring
protons on aromatic ring are separated by 2.48 Å). Intermolecular NOEs
obtained from the 15N/13C-filtered/edited
experiment were categorized as either strong (1.8-3.0 Å), medium (1.8 to 4.0 Å), or weak (1.8 to 5.5 Å). Dihedral angle restraints were
derived from data obtained from HNHA, HNHB, and NOESY-HSQC experiments
according to Sia et al. (4).
Structural Calculations--
Using an initial set of
intramolecular NOE restraints for cCTnC, 100 structures of cCTnC
without EMD 57033 were calculated starting from an extended
conformation. The calculations were done using simulated annealing
protocol implemented in X-PLOR (46) with 10,000 high-temperature steps
(time step of 30 ps) and 6000 cooling steps (time step of 30 ps).
Approximately 50% of the initial structures converged. These
structures were used as templates for further rounds of refinements.
Dihedral angle restraints and 12 artificial distance restraints from
chelating oxygens to the two Ca2+ ions were added at later
stages of the refinement process. The structure of the cCTnC·EMD
57033 complex were calculated starting from the extended conformations
of cCTnC and EMD 57033 using simulated annealing protocol with the same
conditions as above. The calculations were carried out using the
distance and dihedral restraints for cCTnC, 12 distance restraints to
Ca2+ ions, 14 intermolecular distance restraints between
cCTnC and EMD 57033, as well as 8 intramolecular distance restraints
for the bound EMD 57033 molecule (see Table II). The final family of
solution structures presented in this article consists of 30 of the
lowest energy structures.
Coordinates--
The coordinates for the structure have been
deposited in the RCSB Protein Data Bank (1IH0). Chemical shifts
assignments for cCTnC and EMD 57033 have been deposited in the
BioMagResBank (4994).
Structure of the cCTnC·EMD 57033 Complex--
We have shown
clearly that EMD 57033 forms a 1:1 complex (KD = ~8 µM) with intact cTnC and the binding site resides in
the C-domain (35). For the purpose of this study, we have made a
complex between an isolated Ca2+-saturated C-domain
(residues 91-161) of human cTnC and EMD 57033. This reduces the NMR
spectral overlap and facilitates the structure determination process.
The two-dimensional {1H,15N}-HSQC NMR
spectrum of the Ca2+-saturated cCTnC, shown in Fig.
1, indicates that it is a well structured
domain characterized by the dispersion of amide proton signals.
Titration of EMD 57033 induces progressive shifts of the cross-peaks.
All the chemical shift changes fall into the fast exchange limit on the
NMR time scale. The linear movement of the cross-peaks indicates that
EMD 57033 binding to cCTnC occurs with a 1:1 stoichiometry. At the end
of titration, a stable 1:1 cCTnC·EMD 57033 complex
(KD = ~10 µM) was formed.
The two-dimensional {1H,15N}-HSQC NMR
spectrum (Fig. 1) of cCTnC in the cCTnC·EMD 57033 complex was highly
resolved, which allowed the chemical shifts of the backbone and the
side chain atoms to be readily assigned using 15N- or
15N/13C-labeled protein. Distance restraints
for cCTnC in the cCTnC·EMD 57033 complex were obtained by analyzing
three-dimensional 15N- or
15N/13C-NOESY experiments. Dihedral angle
restraints for cCTnC in the cCTnC·EMD 57033 complex were obtained
from three-dimensional HNHA and HNHB experiments. The NMR experiments
performed are summarized in Table I.
The proton NMR chemical shifts assignments and intramolecular distance
restraints for the bound EMD 57033 required the collection of
15N/13C filtered two-dimensional DIPSI and
two-dimensional NOESY experiments using unlabeled drug bound to
15N/13C-labeled cCTnC. These experiments
removed all of the resonances arising from
15N/13C-labeled cCTnC. One-dimensional
1H and two-dimensional DIPSI NMR spectra of free EMD 57033 in Me2SO-d6 were also analyzed to
aid the assignments. Intermolecular NOE correlations between cCTnC and
EMD 57033 were determined from three-dimensional
15N/13C-filtered,
15N/13C-edited NOESY data. These experiments
were optimized3 for detecting
NOEs between protons attached to 14N/12C and
protons attached to 15N/13C, thereby editing
out all the intramolecular NOEs. A total of 1000 experimental distance
restraints (approximately 14 restraints per residue) including 974 for
cCTnC, 8 for EMD 57033, 14 intermolecular contacts between cCTnC and
EMD 57033, 61 dihedral restraints for cCTnC, and 12 restraints to
Ca2+ were used to calculate the high resolution structure
of the cCTnC·EMD 57033 complex. Fig.
2A depicts the ensemble of the
30 lowest energy structures of the cCTnC·EMD 57033 complex in stereo
view, with the overall structural statistics and conformational
energies for the ensemble of solution structures provided in Table
II. The ribbon and surface
representations of the structure of cCTnC·EMD 57033 complex are shown
in Fig. 2, B and C, respectively.
cCTnC in the present structure exhibits an overall fold resembling
other Ca2+-bound domains in the EF-hand family, such as the
C-domain of sTnC and N- and C-domains of CaM. The four helices, E, F,
G, and H, are well defined, superimposing with individual backbone
r.m.s.d.s of 0.23 ± 0.06 Å (E, residues 95-103), 0.27 ± 0.09 Å (F, residues 114-123), 0.36 ± 0.11 Å (G, residues
130-140), and 0.26 ± 0.07 Å (H, residues 150-156). The two
EF-hands are joined by a short twisted antiparallel Binding Interface between EMD 57033 and cCTnC in the cCTnC·EMD
57033 Complex--
Strip plots taken from the three-dimensional
15N/13C F1-filtered,
F3-edited spectrum of the cCTnC·EMD 57033 complex are
shown in Fig. 4A. In this
spectrum, only NOEs arising from the drug protons (attached to
12C) and terminating on protein protons (attached to
13C) are observed. For example, the H7# methyl protons from
the thiadiazinone unit show strong NOE contacts with the protons
attached to the methyl groups of Leu117 located in the
F-helix (Fig. 4B, c) and of Ile112 and
Ile148 located in the EMD 57033-induced Structural Changes in cCTnC--
The overall
fold of cCTnC in the cCTnC·EMD 57033 complex is similar to the
structure of the C-domain of intact cTnC determined by solution NMR
spectroscopy (4), with a r.m.s.d. of ~ 1.7 Å for backbone
overlay (Fig. 5A). There are
several EMD 57033-induced structural changes. Among the most notable
ones, the cCTnC in the cCTnC·EMD 57033 complex is more open,
i.e. the F/G helix unit is farther away from the E/H helix
unit, than the free C-domain of cTnC. This is mostly due to the strong
interactions of the drug with residues Leu117 and
Leu121 located on the F helix, which pulls helix F away
from helix E and toward helix G. This is quantified by a decrease in
the E/F interhelical angles (from 115 ± 4° for the C-domain in
cTnC to 92 ± 4° for cCTnC in the cCTnC·EMD 57033 complex).
The interhelical angle between G and H also showed a slight decrease
(from 121 ± 4° for the C-domain in cTnC to 113 ± 7° for
cCTnC in the cCTnC·EMD 57033 complex) (see Table
III). The slight opening of cCTnC and movement of the F/G helix unit away from the E/H helix unit was also
observed by the binding of the cTnI33-80 peptide to cCTnC
(18). It seems that these movements are necessary for the C-domain of
cTnC to provide a binding site for cTnI or a drug molecule. The degree
of EMD 57033-induced structural opening of cCTnC is also measured by an
increase of ~20 Å2 of exposed apolar surface area for
cCTnC in the cCTnC·EMD 57033 complex, compared with the free C-domain
of cTnC. Although EMD 57033 induced little G/H interhelical angle
changes, the multiple contacts between Leu136 of the G
helix with several ring protons of EMD 57033 resulted in a more
flexible G helix in the present structure than the C-domain in the cTnC
structure (r.m.s.d. of 0.36 Å for cCTnC in the cCTnC·EMD 57033 versus 0.24 Å for the C-domain in cTnC). Another
significant EMD-induced change on cCTnC is the unwinding of residues
156-161 at the end of helix H. This last helix has always been well
defined to the C-terminal end in many EF-hand domains such as sNTnC (9, 48), cNTnC (7), and CaM domains (49). However, in this complex, helix H
ends at residue 156, the last portion of the C-terminal residues
(157) forms an extended structure and appears to be more flexible
than usual. This can be attributed to the interactions between EMD
57033 and residue Met157 of cCTnC. Interestingly, the same
extended conformation at the end of the H helix was observed in the
C-domain of sTnC bound to the N-terminal region of sTnI
(sTnI1-47) (23). This suggests that the nature of the
interactions between EMD 57033 and cCTnC may be similar to that between
sTnI1-47 and sTnC. An overlay (Fig. 5B) of the
cCTnC in the cCTnC·EMD complex and sCTnC in the
sCTnC·sTnI1-47 complex shows that the backbones of cCTnC
and sCTnC adopt very similar conformations (r.m.s.d. = 1.3 Å). It
also shows that the binding of EMD 57033 to cCTnC possesses similar
features as the binding of sTnI1-47 to sCTnC. In fact,
residues Ile110, Leu115, Phe119,
Leu134, Ile146, and Met155
of sCTnC, corresponding to the six EMD 57033-contacting residues in
cCTnC, are all involved in the interaction with sTnI1-47 (23). It is interesting to note that binding of sTnI1-47 to sCTnC does not greatly perturb the fold of this domain from a
peptide-free conformation. In the free state, sCTnC exhibits E/F and
G/H interhelical angles of ~105° and ~115°, respectively (Table
III). These angles do not change very much upon sTnI1-47 binding to sCTnC. On the other hand, both EMD 57033 and
cTnI33-80 induce interhelical angle changes in cCTnC and
the bound cCTnC exhibits a more open conformation. This is analogous to
the ligand induced structural changes in the N-domain of sTnC and cTnC.
Ca2+ binding switches sNTnC from a closed to open
conformation (9) and the open sNTnC is ready to bind
sTnI115-131 with no need to open further (13). On the other
hand, Ca2+ binding to cNTnC induces little structural
changes but sets the stage for cTnI binding and cNTnC undergoes a large
closed to open transition upon binding cTnI147-163 (15) or
Bepridil (50). Thus, the interaction of both domains of cTnC with cTnI
would require more free energy than those of sTnC with sTnI, because cTnI has to overcome the energy barrier of opening the domains of cTnC
(see discussions in McKay et al. (51)). This energy cost may
be compensated by employing pharmacological agents, which bind to both
domains of cTnC and thereby help to stabilize the open conformation of
cTnC. The ideal situation would be that the interaction between the
drug and cTnC occurs without interfering with cTnI binding to cTnC. The
analysis presented herein raises issues about the effect of EMD 57033 on the interaction of cCTnC and cTnI. Since two regions of cTnI
(cTnI128-147 and cTnI34-71) were identified
to interact with the C-terminal domain of cTnC (12), we examined the
binding of these two synthetic peptides (cTnI128-147 and
cTnI34-71) to the cCTnC·EMD 57033 complex as discussed
below.
Competition of EMD 57033 with cTnI128-147 and
cTnI34-71--
Previously, we have shown that both EMD
57033 and cTnI128-147 bind with a 1:1 stoichiometry to
cTnC but do not compete for the same binding sites on cTnC (35). EMD
57033 binding is not affected by cTnI128-147 nor does the
drug affect cTnI128-147 binding. In the end, a stable
ternary cTnC·EMD 57033·cTnI128-147 complex is
formed. In the present work, similar titrations were performed on cCTnC
and similar conclusions were obtained (data not shown). Both EMD 57033 and cTnI128-147 can bind to cCTnC simultaneously and the
binding affinities (KD = ~10 µM for
EMD 57033 and KD = ~100 µM for
cTnI128-147) are similar to those determined from binding
to intact cTnC (35). However, unlike cTnI128-147, the
N-terminal region of cTnI, cTnI34-71, can displace EMD
57033 completely from cCTnC. When the 38-residue cTnI34-71
peptide is titrated to the Ca2+-saturated cCTnC, it binds
tightly (KD
It is necessary to put these results into perspective with respect to
the interaction of cTnC and cTnI. The noncompetitive binding sites of
cTnI128-147 and EMD 57033 on cTnC (35) or cCTnC (present
data) suggest that the inhibitory region of cTnI does not block the
binding of EMD 57033 to cTnC, and vice versa. This conclusion is
important in terms of addressing the Ca2+ sensitizing role
of EMD 57033 because the inhibitory region of cTnI constitutes a major
switch between muscle contraction/relaxation by moving between cTnC and
actin-tropomyosin (52). A good Ca2+ sensitizer would not
interfere with the inhibitory function of cTnI. A pertinent question is
whether this is also the case with intact cTnI, especially considering
the present clear-cut results that EMD 57033 cannot compete with
cTnI34-71 for cCTnC. The very tight association of
sTnI1-40 and the sCTnC have been shown by several groups.
In addition to the sTnC-sTnI1-47 crystal structure (23),
early functional studies of this region by Ngai and Hodges (53) have
shown that sTnI1-40 can effectively compete with sTnI or
sTnI96-115 inhibitory peptide for sTnC and a recent NMR
study has shown that sTnI1-40 binds strongly to sCTnC with
a KD of ~2 µM and can displace
sTnI96-115 completely (19). This raises questions
regarding how the inhibitory region binds to TnC to release inhibition
if the N-terminal region of TnI is always present. In order to
rationalize these results, two models have been proposed for the
interaction of TnC and TnI. One suggests that these two regions of TnI
share overlapping binding sites on the C-domain of TnC, which are
alternatively occupied by either one or the other depending on the
interactions between the N-domain of TnC and the C-domain of TnI or the
C-domain of TnT (12). The second model proposes that the N-terminal
region of TnI always binds to the C-domain of TnC, regardless of the Ca2+-dependent interactions between the
N-domain of TnC and the C-domain of TnI (23, 54, 55), while the
inhibitory region interacts with the central helix area (including part
of the D helix in the N-domain and part of the E-helix in the C-domain)
of TnC in a Ca2+-dependent manner. The later
model does not adequately explain the experimental data (20, 35,
56-59), supporting a binding site for the inhibitory region of TnI
primarily in the C-domain of TnC, especially when only isolated
C-domains of TnC were used in the study (19, 57). Based on our
titration data of cTnI128-147 binding to cTnC (35) and
cCTnC (present work) and the competitive binding of
sTnI1-40 and sTnI96-115 to sCTnC (19), we
suggest that cTnI128-147 binds primarily to the C-domain of cTnC and in order for this binding to occur, the interaction between
cTnI34-71 and cCTnC has to be weakened. This can be
accomplished by the mechanisms proposed for model 1 (12). Since cTnC
does not act alone, and the contractile proteins work in a highly
organized and cooperative manner in muscle contraction, it is possible
that cCTnC in myofilaments may have a lower affinity for
cTnI34-71 than it does in isolation. Although EMD 57033 alone is too small to compete with the extensive contacts between the
long Implications in the Ca2+ Sensitizing Effect of EMD
57033 in Cardiac Muscle Contraction--
The number of patients
suffering from congestive heart failure is rising accompanying the
aging of baby boomers. Ca2+ sensitizers have been proposed
as a treatment for this common disease. These agents increase
myocardial contractility by generating more force for a given amount of
cytosolic free Ca2+. This allows an achievement of positive
inotropic effect more economically as compared with other positive
inotropic drugs that exert effect by simply enhancing Ca2+
influx into myocytes and therefore add intracellular Ca2+
overload. Numerous studies have documented the Ca2+
sensitizing effects of this class of agents under in vivo
and in vitro conditions (for a review, see Ref. 1). Among
which, EMD 57033 is one of the most potent and selective
Ca2+ sensitizers available. Earlier physiological studies
have shown that EMD 57033 exerts detectable positive inotropic effects
on isolated cardiac myocytes at concentrations as low as 1 µM (27), and in skinned cardiac muscle fibers, less than
10 µM EMD 57033 induced significant Ca2+
sensitivity of force development (26). In a recent study, 0.3-1 µM EMD 57033 is shown to have positive inotropic effects
on both normal and failing cardiac myocytes (32). The cCTnC·EMD 57033 complex structure presented in this work provided a structural basis
for the understanding of the mechanism underlining the Ca2+
sensitizing effect of EMD 57033 in cardiac muscle contraction. In the
present structure of the cCTnC·EMD 57033 complex, the drug molecule
is orientated such that the chiral group of EMD 57033 fits deep in the
hydrophobic pocket and makes several key contacts with the protein.
This stereospecific interaction explains why the ()-enantiomer of EMD 57033 is inactive. Titrations of the
cCTnC·EMD 57033 complex with two regions of cardiac troponin I
(cTnI34-71 and cTnI128-147) reveal that the
drug does not share a common binding epitope with
cTnI128-147 but is completely displaced by
cTnI34-71. These results have important implications for
elucidating the mechanism of the Ca2+ sensitizing effect of
EMD 57033 in cardiac muscle contraction.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-helix and binds to the hydrophobic groove of sCTnC (23). The
corresponding region of cTnI33-80 has also been shown to
bind within the hydrophobic patch of cCTnC (18).
)-enantiomer, EMD 57439, exhibits no Ca2+ sensitizing
activity but acts as a pure phosphodiesterase III inhibitor (26,
27). The molecular mechanism underlining the Ca2+
sensitizing effects of EMD 57033 is not well understood. It is possible
that the compound interacts directly with cTnC and increases its
affinity for Ca2+. It is also possible that it exerts an
effect on the interface of myosin-actin. An earlier fluorescence study
has demonstrated that EMD 57033 interacts with isolated cTnC in a
Ca2+-dependent and stereoselective manner (33).
A NMR study has shown that EMD 57033 induces chemical shift changes of
Met methyl groups in the C-domain of cTnC (34). Using NMR chemical
shift mapping, we have demonstrated that the EMD 57033-binding site is
in the hydrophobic pocket of the C-domain of cTnC and that this drug
does not interfere with the binding of the inhibitory region of cTnI to
cTnC (35). These findings suggest that EMD 57033 may exert its positive
inotropic effect by binding to the structural domain of cTnC,
modulating the interaction between cTnC and other thin filament
proteins, rather than directly enhancing Ca2+ binding to
the regulatory domain of cTnC.
)-enantiomer of EMD
57033 is inactive. This structure provides a structural basis for the
Ca2+ sensitizing effect of EMD 57033. We also examined the
possible competition of the drug with cTnI peptides and found that the drug does not share the common binding epitope with the inhibitory region of cTnI but is displaced completely by the N-terminal region of
cTnI. These results have important implications in understanding the
mechanism underlining Ca2+ sensitizing effects of EMD 57033 in cardiac muscle contraction.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (20K):
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Fig. 1.
Titration of Ca2+-saturated cCTnC
with EMD 57033. Two-dimensional
{1H,15N}-HSQC NMR spectra arising from the
backbone NH and side chain NH2 groups of cTnC are
superimposed for various EMD 57033 additions. Each titration point
represents the addition of 1 µl of 94 mM EMD 57033 stock
solution to a NMR sample containing 0.92 mM
15N-labeled cCTnC. Assignments of the cross-peaks are
indicated. Cross-peaks corresponding to free cCTnC with no drug added
are shown as multiple contours. Cross-peaks from subsequent spectra are
shown as single contours. The arrow indicates the direction
of the movement of cross-peak for Gly140 with increasing
EMD 57033 concentrations.
NMR spectra acquired and experimental conditions used to obtain
assignments and NOE restraints
View larger version (37K):
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Fig. 2.
A, the solution structure of the
cCTnC·EMD 57033 complex. The backbones (N, C , and C') of a family
of 30 structures are shown in blue. The assembly of EMD
57033 structures is shown in red. The Ca2+ ions
are shown as green spheres. B, the ribbon representation of
the cCTnC·EMD 57033 complex. The protein is shown in dark
red and Ca2+ ions are shown as cyan
spheres. The thiadiazinone functional group of the drug is colored
in blue; the tetrahydroquinolyl group of the drug is colored
in gold; and the dimethoxybenzoyl group is shown in
gray. C, molecular surface of cCTnC in the cCTnC·EMD 57033 complex. The side chain atoms of hydrophobic residues (Ala, Ile, Leu,
Met, Phe, and Val) are shown in yellow. Negatively charged
residues (Asp and Glu) in red, positively charged residues
(Arg and Lys) in blue. Polar residues (Ser, Thr, and Tyr)
are shown in cyan. EMD 57033 is embedded in the hydrophobic
cleft of the protein. The orientation of the complex is the same as in
B. C was created with the program GRASP
(47).
Structural statistics of the family of the 30 structures calculated
-sheet. The two
Ca2+-binding sites are relatively well defined with
backbone r.m.s.d. of ~0.61 Å. The
-sheet (residues 111-113,
147-149) is well defined with a backbone r.m.s.d. of 0.29 ± 0.08 Å. The N- and C-terminal residues (residues 91-94, and 158-161) are
less well defined (r.m.s.d., 1.4 ± 0.4) than the helices and the
-sheet. EMD 57033 consists of three main organic groups, which are
the thiadiazinone (A ring in Fig.
3A), the tetrahydroquinolinyl
(B and C rings in Fig. 3A), and the dimethoxybenzoyl (D ring
in Fig. 3A) moieties, respectively. The three functional
groups have rigid conformations, however, the bonds that connect the
thiadiazinone-tetrahydroquinolinyl units and the
tetrahydroquinolinyl-dimethoxybenzoyl units can rotate freely (see Fig.
3A). The drug molecule is completely assigned and the
chemical shifts for all the protons are labeled in Fig. 3A.
Eight intramolecular NOEs of the bound drug were observed and shown in
Fig. 3, B and C. The relative orientations of the three moieties in the cCTnC·EMD 57033 complex are determined by both
the intramolecular NOEs within the drug molecule and the intermolecular
NOEs between cCTnC and the drug. In the ensemble of structures (Fig.
2A), the interplanetary angles for the
thiadiazinone-tetrahydroquinolinyl and the
tetrahydroquinolinyl-dimethoxybenzoyl units are 44° ± 21° and
69° ± 14°, respectively. When the heavy atoms of EMD 57033 superimpose onto the average structure in the cCTnC·EMD complex, the
average r.m.s.d. is 0.29 ± 0.07 Å. This r.m.s.d. increases to
1.15 Å when the backbone atoms of residues 95-158 of cCTnC in the
ensemble of solution structures for the cCTnC·EMD 57033 complex are
superimposed onto the average cCTnC structure. As a result, the
stereochemical quality of the drug molecule assembly is slightly lower
than the structures of cCTnC in the cCTnC·EMD 57033 complex. This is
due in part to the loosely imposed intermolecular restraints between
cCTnC and EMD 57033 because of a lack of calibration for those NOEs
(see "Experimental Procedures").
View larger version (19K):
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Fig. 3.
A, the chemical structure of EMD 57033. The four rings that form the three functional groups are labeled as
A-D. The two bonds that connect the three units are
indicated by curved arrows to emphasize their ability to
rotate freely. The number designation and the chemical shift
assignments of most protons of the drug are indicated. The residues of
cCTnC, which are involved in interacting with protons of EMD 57033, are
also indicated. B and C, strips from the
two-dimensional 15N/13C-filtered-NOESY
NMR experiment showing the intramolecular NOE contacts of EMD
57033.
-sheet of cCTnC (Fig. 4B,
a); the H9, H12, and H13 protons from the B ring (Fig.
3A) show NOE contacts with the protons attached to the
methyl groups of Leu136 located in the G-helix (Fig.
4B, b); and the two H17 protons from the C ring (Fig.
3A) show NOE contacts with the protons attached to the
methyl groups of Met157 located in the H-helix (Fig.
4B, d). These experimental intermolecular restraints serve
to orient the drug molecule in the cCTnC·EMD 57033 complex. As a
result, the thiadiazinone group is located at the bottom of the cCTnC
pocket in a small hydrophobic cavity (Fig. 2C). It seems
that the strong contacts between the chiral methyl group and three
residues of cCTnC (Ile112, Leu117, and
Ile148) serve as the primary anchoring site for this drug
molecule (see below for biological implications). The dimethoxybenzoyl
group protrudes toward outside of the hydrophobic pocket (Fig.
2C). Although most interactions between cCTnC and EMD 57033 are hydrophobic in nature, the fact that the chiral methyl (H7#) group
delves deep into the hydrophobic pocket of cCTnC while the two methoxy moieties, despite their hydrophobic nature, stick out to indicate the
interaction is not nonspecific. Another important interaction is
between the methyl group of Met157 located in the
C-terminal G-helix and H17# protons from ring C (Fig. 3A) of
EMD 57033. This contact serves mainly to hold the plane formed by rings
B and C of the drug in an orientation that is almost perpendicular to
the plane formed by ring A, and to allow the residues on helix G
(Leu136) and at the end of helix H (Met157) to
interact with the same proton (H9) on ring B. The contacts between the
protons (H12 and H13) on ring B and the methyl groups of
Leu117 and Leu136 help the drug bound to the
protein firmly. Leu121 located on the C-terminal of F-helix
makes only weak NOE contacts with aromatic protons on both ring B (H12)
and ring D (H20, H23, and H24) of EMD 57033, which also play a role in
binding the drug to cCTnC. Overall, the protons from EMD 57033 make 14 NOE contacts with protons of the methyl groups from six hydrophobic
residues (Ile112, Leu117, Leu121,
Leu136, Ile148, and Met157) of
cCTnC and these interactions keep the drug molecule fit snuggly in the
hydrophobic cavity of Ca2+-saturated cCTnC (Fig.
2C). The total "exposed" nonpolar accessible surface
area for residues 95-158 of cCTnC, excluding the drug, in the
cCTnC-EMD 57033 complex is ~ 2689 Å2, as measured
by GRASP (47) and the drug molecule shields ~740 Å2 of
the apolar surface from the solvent.
View larger version (27K):
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Fig. 4.
NOE contacts between the protein and the
drug. A, strip plots from 13C portion of
the three-dimensional
15N/13C-filtered/edited-NOESY NMR experiment
illustrating the NOEs between cCTnC and EMD 57033. The carbon chemical
shift is shown on the left. The cCTnC proton to which the
strip corresponds is labeled on the right. The proton
chemical shifts of the drug are indicated at the top. The
two peaks circled in the
Leu121,C 2 strip are artifacts in the
spectra. B, detailed views of the interactions between EMD
57033 and cCTnC. Dotted lines represent the NOE distances
observed in A.
View larger version (37K):
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Fig. 5.
A, the backbone overlay of cCTnC in the
cCTnC·EMD 57033 complex (dark red) and the free C-domain
of cTnC (green), PDB accession code 1AJ4. The two
Ca2+ ions are shown as cyan-colored spheres. The
three units of the drug are colored in the same scheme as Fig.
2B. Note the slight opening of helix E and F as well as the
unwinding of the C-terminal helix. B, the backbone overlay
of cCTnC in the cCTnC·EMD 57033 complex (dark red) with
the C-domain of skeletal TnC (purple), bound to
sTnI1-47 (light green), PDB accession code
1A2X. The Ca2+ ions and EMD 57033 are colored in the same
scheme as in A. The overlay shows that the binding of EMD
57033 to cCTnC mimics the binding of sTnI1-47 to
sCTnC.
Interhelical angles of various EF hands
1 µM) to form a stable
cCTnC·cTnI34-71 complex. Fig.
6A shows a superimposition of
the two-dimensional {1H,15N}-HSQC NMR
spectra of cCTnC and the cCTnC·cTnI34-71 complex. Similar to sTnI1-40 binding to sCTnC (19),
cTnI34-71 binding to cCTnC occurs with slow exchange
kinetics on the NMR time scale. Thus, as the titration progresses, the
resonance peaks corresponding to cCTnC becomes less intense while those
corresponding to cCTnC-cTnI34-71 grow. When the
cTnI34-71:cCTnC ratio reaches 1:1, all cross-peaks
corresponding to cCTnC have completely disappeared while those
corresponding to cCTnC-cTnI34-71 attain maximum intensity.
When cTnI34-71 is titrated to the cCTnC·EMD 57033 complex, EMD 57033 is displaced completely by cTnI34-71
and a stable cCTnC·cTnI34-71 complex forms
(KD
1 µM). The binding behavior of
cTnI34-71 to the cCTnC·EMD complex is very similar to
that of cTnI34-71 to cCTnC (Fig. 6A) and is
illustrated in Fig. 6B, which shows a superimposition of the
two-dimensional {1H,15N}-HSQC NMR spectra
of cCTnC-EMD 57033 and cCTnC-cTnI34-71. The only
difference between Fig. 6, A and B, is the
starting spectra. The former started with the spectrum of cCTnC and the
latter started with the spectrum of cCTnC-EMD 57033. The end spectra
are identical and represent that of the cCTnC·cTnI34-71
complex. Thus, cTnI34-71 associates tightly with cCTnC
regardless of the presence of EMD 57033.
View larger version (22K):
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Fig. 6.
Two-dimensional
{1H,15N}-HSQC NMR spectra of the titrations
of (A) cCTnC with cTnI34-71,
(B) the cCTnC·EMD 57033 complex with
cTnI34-71. A, the cross-peaks
corresponding to free cCTnC are shown in multiple contours, whereas the
peaks corresponding to the cCTnC·cTnI34-71 complex are
shown as single contours. B, cross-peaks corresponding to
the cCTnC·EMD 57033 complex are shown as multiple contours, whereas
cross-peaks corresponding to the cCTnC·cTnI34-71 complex
are shown as single contours.
-helix of cTnI34-71 and cCTnC, the fact that EMD 57033 interacts with many of the same residues on cCTnC as
sTnI1-47 on sCTnC suggests that the drug may play a role
in disrupting and therefore weakening the interaction of
cTnI34-71 with cCTnC in the myofilaments, and
consequently, in enhancing the binding of the inhibitory region of TnI
to TnC. Since this interaction is
Ca2+-dependent, the apparent Ca2+
sensitivity of the contractile system can be modulated by EMD 57033.
)-enantiomer of EMD
57033 is inactive (27). Since the methyl group attached to the chiral
carbon of EMD 57033 makes extensive contacts with methyl groups of
Ile112, Leu117, and Ile148, these
contacts may be weakened or lost if the stereospecificity of the chiral
carbon is changed. This is especially true for the interactions between
residues on the short
-sheet and the drug. These interactions would
be eliminated if the chirality of the drug has reversed, not even the
rotation of ring A can completely restore all the contacts between the
drug and these residues. Interestingly, the (
)-enantiomer of EMD
57033, EMD 57439 is also capable of stimulating muscle contraction, but
through a different mechanism. EMD 57439 is a potent phosphodiesterase
III inhibitor, and as such, is capable of producing an increase in cAMP
level inside the cell. This will result in the activation of protein kinase A and ultimately, an increase in Ca2+ concentration
in cardiac muscle cells (26, 28). The fact that EMD 57439 has no
Ca2+ sensitizing activity and EMD 57033 is only a weak
phosphodiesterase III inhibitor points to the sensitivity of protein
toward stereospecificity of ligands.
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ACKNOWLEDGEMENTS |
---|
We are indebted to Pascal Mercier for tremendous help with the NMRView program and Dr. Carolyn Slupsky for constructing the structure and parameter files of EMD 57033. We gratefully acknowledge Dr. Truong Ta and Prof. Liang Li in the Department of Chemistry for Mass spectrometric measurements of the drug/protein samples. We thank Gerry McQuaid for maintaining the NMR spectrometers, David Corson for expression and purification of the cCTnC proteins, the Protein Engineering Network Center of Excellence (PENCE) for the use of their Unity 600 NMR spectrometer, and the National High Field Nuclear Magnetic Resonance Center (NANUC) for the use of their Inova 800 NMR spectrometer.
![]() |
FOOTNOTES |
---|
* This work was supported in part by the Canadian Institutes of Health Research, the Heart and Stroke Foundation of Canada, and the National Institutes of Health.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.
§ Supported by an Alberta Heritage Foundation for Medical Research Studentship.
To whom correspondence should be addressed. Tel.: 403-492-5460;
Fax: 403-492-0886; E-mail: brian.sykes@ualberta.ca.
Published, JBC Papers in Press, April 24, 2001, DOI 10.1074/jbc.M102418200
2 T. Ta, unpublished data.
3 L. Spyracopoulos, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: TnC, troponin C; cTnC, cardiac troponin C; cCTnC, C-domain of cTnC; cNTnC, N-domain of cTnC; sTnC, skeletal troponin C; sNTnC, N-domain sTnC; sCTnC, C-domain of sTnC; cTnI, cardiac troponin I; cTnI128-147, synthetic peptide (residues 128-147) of cTnI; cTnI34-71, synthetic peptide (residues 34-71) of cTnI; sTnI, skeletal troponin I; sTnI96-115, synthetic peptide (residues 96-115) of sTnI; sTnI1-40, synthetic peptide (residues 1-40) of sTnI; CaM, calmodulin; HSQC, heteronuclear single-quantum coherence; NMR, nuclear magnetic resonance; NOESY, nuclear Overhauser effect spectroscopy; TOCSY, total correlation spectroscopy; DIPSI, decoupling in the presence of scalar interactions; r.m.s.d., root mean square deviation.
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