 |
INTRODUCTION |
Troponin C (TnC)1 is the
calcium-binding protein in the thin filament of skeletal and cardiac
muscle and belongs to the EF-hand family of proteins. The structure of
TnC closely resembles that of calmodulin, a ubiquitous calcium sensor,
with an N-terminal and a C-terminal domain connected by a long central
-helix. Under physiological conditions the C-terminal domain of TnC
is always occupied by Ca2+ or Mg2+. In the
N-terminal domain the low affinity sites of TnC bind calcium ions when
they are released from the sarcoplasmic reticulum of skeletal or
cardiac muscle myocytes. The conformational changes brought about by
the calcium binding to the N-terminal domain of skeletal troponin C
(sTnC) have been followed by NMR (1-4). The data confirm the early
hypothesis of an open and a closed conformation (5-7). According to
the paradigm the Ca2+-induced conformational changes of the
N-terminal domain are transmitted to the other components of the
troponin complex and then to tropomyosin, triggering the muscle
contraction (8-12).
The sequence of the cardiac TnC (cTnC) is 70% identical to that of
sTnC, whose structure in the calcium-saturated form has been determined
by x-ray crystallography (7, 13) and by NMR (14). However, while the
N-terminal domain of sTnC contains two calcium-binding sites, the
N-terminal domain of cTnC has only one intact Ca2+-binding
site. The other site, often referred as a defunct site, does not bind
calcium. The functional meaning of this difference between the cardiac
and skeletal troponin C has not been explained in structural terms.
Even if there is no direct structural proof that the molecular
mechanism of action of cTnC is the same as for sTnC, evidence has been
given that also in cTnC the N-terminal domain is responsible for the
regulation. It is assumed that the C-terminal domain plays primarily a
structural role, since its calcium-binding sites are occupied even when
the intracellular calcium concentration drops to micromolar values (15,
16).
Recently, Sia et al. (17) working on cysteine (Cys-35 and
Cys-84) to serine-mutated cTnC, showed that, in its calcium-saturated form, the N-terminal domain is in the closed conformation. This result
implies that there is a much smaller conformational change to be
expected upon calcium binding than in sTnC as proved by the very recent
study by Spyracopoulos et al. (18). We aimed to confirm the
existence of the closed form of the N-terminal domain for the human
cTnC. Since the closed form of the N-terminal domain of cTnC was an
unexpected result, we wanted to assess the structural integrity by
dynamics measurements to get insight into a plausible conformational
exchange between the closed and open states.
 |
EXPERIMENTAL PROCEDURES |
Cloning and Expression of Recombinant Human Cardiac Troponin C
N-terminal Fragment--
The coding sequence of human cTnC was cloned
by using the reverse transcription-polymerase chain reaction technique
and human heart poly(A)+ RNA as a template and primers
(20). The amplified DNA fragment was digested, purified, and subcloned
to the pGEM3-vector (Promega). For protein expression the subcloned
insert was isolated and ligated to the glutathione
S-transferase fusion protein vector pGEX-2T (Pharmacia
PL-Biochemicals) (19). The bacterial expression for production of human
cTnC glutathione S-transferase fusion protein was carried
out in Escherichia coli DH5
-cells. The cells were grown
at 37 °C overnight in minimum medium according to Jansson et
al. (20), using ammonium chloride instead of ammonium sulfate. The
culture was diluted 1:25 in minimum medium containing
15N-labeled ammonium chloride (1 g/liter) and grown at
37 °C to the middle of the growth phase, prior to induction with
isopropyl
-D-thiogalactopyranoside (0.5 mM)
for 4 h. The cells were harvested by centrifugation, and an
aliquot of the collected cells was analyzed for estimation of the
amount of cTnC N-terminal fragment by SDS-polyacrylamide gel
electrophoresis.
Purification of Recombinant Human 15N-Labeled cTnC
N-terminal Fragment--
The collected bacterial paste of the 5-liter
culture was diluted in 50 ml of 16 mM
Na2HPO4, 4 mM
NaH2PO4, 150 mM NaCl, 1% Triton-100, pH 7.3, buffer containing 0.2 mM
phenylmethanesulfonyl fluoride and 25 µl of bentzonuclease. The cells
were disrupted by sonication on ice. The suspension was clarified by
centrifugation and used as the starting material for purification on
the glutathione Sepharose (10 ml; Pharmacia) affinity column. The
recombinant human cTnC was further purified by HPLC anion exchange
chromatography (Mono-Q HR 5/5) as described by Pollesello et
al. (19). The free calcium was removed using Chelex-100 affinity
chromatography (30 ml; Bio-Rad) equilibrated in water/ammonia solution,
pH 8.0, and the eluted purified protein was freeze-dried. The amount of protein was estimated according to Bradford (21). The purity of the
purified human cTnC fragment was analyzed by reversed phase HPLC
(C1 TSK TMS 250 column, 0.46 × 4 cm) (19), and by
SDS-polyacrylamide gel electrophoresis followed by Coomassie Brilliant
Blue staining (22). The reversed-phase chromatography-purified protein
peak was further analyzed by matrix-assisted laser desporption
isonization time-of-flight mass spectrometry.
NMR Spectroscopy--
Samples of the freeze-dried N-terminal
fragment of cTnC (10 mg) were dissolved in 500 µl of a solution
containing 7.7 mM dithiotreitol in H2O.
Dithiothreitol was added to prevent potential dimerization of cNTnC due
to a possible disulfide formation between Cys-35 or Cys-84. The pH was
adjusted at 6.0 with the addition of microliters of NaOH 0.5 M. Thereafter, CaCl2 was added to a final
concentration of 3.9 mM. Finally, D2O and
perdeuterated trifluoroethanol (TFE) were added in order to obtain 750 µl of a 1.3 mM protein solution in 82% H2O,
9% D2O, 9% perdeuterated TFE, at pH 6.0 ± 0.1 (not corrected for the deuteron effect). 15N-Labeled sample (1.3 mM) was prepared to 300 µl in a Shigemi microcell.
NMR spectra were acquired mostly with a 600-MHz Varian Unity NMR
spectrometer, and certain spectra were taken with a 500-MHz Varian
Unity and a 400-MHz ARX Bruker spectrometer. For the nonlabeled sample
homonuclear two-dimensional spectra, correlation spectroscopy (COSY),
relayed COSY, total correlation spectroscopy (TOCSY with mixing times
of 30, 50, 80, and 110 ms), double quantum and nuclear Overhauser
enhancement (NOESY with mixing times of 50, 100, 150, and 200 ms), were
collected at 30 °C and at 40 and 50 °C (with mixing times 150 and
200 ms, respectively). For the 15N-labeled sample,
heteronuclear single quantum correlation spectroscopy spectra were
taken at temperatures from 20 to 50 °C with 2 °C increments.
15N-Edited TOCSY (50 and 90 ms) and NOESY (180 ms) as well
as T1 (with relaxation delays 42, 168, 336, 589, 841, 1114, 1514, 2102, and 2691 ms), T2 (16, 17, 64, 95, 127, 159, 223, 286, and 382 ms) and heteronuclear NOE spectra
were recorded at 30 °C. In the CPMG spin echo pulse sequence the
delay (2
) was 900 µs (23). The 15N spin lock strength
in T1
measurements (4, 8, 15, 30, 46, 61, 76, 106, and 122 ms) was limited to 1.7 kHz due to experimental restrictions. The cross-correlation rate (
) was determined by the
relaxation interference measurement (24). The relaxation delay (2
)
was 70 ms. The data were processed with Felix 95 software.
Structure Generation--
Spin systems were assigned from the
through bond correlation spectra and sequence specific assignments were
deduced from the NOEs between the adjacent spin systems. The spectral
overlap was partly unraveled by 15N editing and exploiting
temperature dependence of HN resonances. Distance restraints were
extracted from the homonuclear NOE data by fitting a second-order
polynomial to integrated cross-peak volumes (I) of the NOE
series. Additional distance restraints were extracted from NOEs
observable in the 15N-edited spectra according to
intensity. The intramethylene and helical
NHi-NHi+1 NOEs served for the calibration.
Distances were given +20 and
50% uncertainties. There was no
objectives to tighten the distance limits because of a likely presence
of conformational exchange and spin diffusion in the
15N-edited NOE spectrum. When a distance could not be
extracted from the build-up curve, owing to a overlap, a poor
signal-to-noise ratio or disturbances, the distance was restrained to
be at most 5.0 Å. The upper bounds were extended by 1.0 Å for each
pseudo atom in methyl groups and 1.5 Å for pseudo atoms in aromatic
rings. Backbone
dihedral angles characterized by small
JNH
, measured from COSY spectra, were
restrained to the helical conformation (±30 degrees) on the basis of
the Karplus relation and NOEs (25). Dihedral angles characterized by
intermediate JNH
were not constrained. The
temperature dependence of HN
NH(T) was
measured from two-dimensional spectra,
proton chemical shifts
(
HA) and JNH
served to
recognize secondary structures. A residue was constrained by
NHi-COi+4 hydrogen bond (±0.2 Å) provided that at least for three subsequent residues
NH(T) < 0.005 ppm/ °C and
HA were smaller by 0.1 ppm than the corresponding random coil values and JNH
< 6 Hz. The calcium
binding site was constrained by six distances between the calcium-ion
and carbons in the coordinating carboxyl groups (2.1-3.9 Å). The
short
-sheet between the calcium binding loop and the pseudo site
was constrained by two hydrogen bonds.
Structures were generated by distance geometry followed by restrained
simulated annealing. The structures were iteratively refined. At each
iteration, distance restraints corresponding to degenerate NOEs were
included provided that it was possible to exclude all but one
alternative based on clearly longer distances. The procedure was
finished when no more restraints could be imposed. The assignment and
structure calculations were carried out by Felix and InsightII 95 software. A final set of structures was analyzed for RMSD, energy,
backbone dihedrals, exposed surface, distance violations by PROCHECK
and InsightII programs. Difference distance matrices (DDM) were
calculated with a program modified to compute DDMs for families of
structures.
The relaxation and heteronuclear NOE data were analyzed in terms of the
commonly used Lipari-Szabo model-free spectral density (26) with the
programs by Palmer (27). The overall correlation time
m was
estimated from R2/R1
ratios (28). The cross-correlation rate between the dipole dipole and
chemical shift anisotropy (29) was measured by a modified HSQC
1H-15N correlation experiment with a relaxation
period prior to the 15N evolution time, as described by
Tjandra et al. (24). During the relaxation period (2
) the
two 15N doublets relax with rates proportional to the
square of the sum and difference of double dipole and chemical shift
assay interactions. The in- and anti-phase components are subsequently
chosen separately for the detection yielding two spectra. The
cross-correlation rate (
) is obtained from the ratio of the signal
intensities (IA and IB) of the
two spectra by
= (1/2
)tanh
1
(IA/IB) (24).
 |
RESULTS |
Structure--
The structure of the calcium-loaded form of the
N-terminal domain of cTnC (Fig. 1)
comprises five helices, i.e. N (residues 4-10), A (14-29),
B (43-47), C (57-64), and D (74-89). The A and B helices and the C
and D helices form a pair of EF-hands connected by the central loop
between the B and C helices. The defunct site is between the A and B
helices and the calcium binding site between the C and D helices. The
two sites are spatially close, and there are interstrand hydrogen bonds
between the backbone amide protons and carbonyl oxygens (residues 36 and 72). The spatially adjacent N, A, and D helices are approximately
orthogonal to each other, and they form a compact structural unit, the
NAD-unit (18).

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Fig. 1.
Solution structure of the calcium loaded
N-terminal domain of human cardiac troponin C (91 amino acids).
Calcium binding site is between C and D helices. The defunct site is
between A and B helices. The sequence is MDDIYKAAVE QLTEEQKNEF
KAAFDIFVLG AEDGCISTKE LGKVMRMLGQ NPTPEELQEM IDEVDEDGSG TVDFDEFLVM
MVRCMKDDSK G.
|
|
The structural statistics are given in Table
I. The overall fold is defined by 224 long range NOEs. The backbone heavy atom RMSD is 1.1 ± 0.2 Å.
The helices and the calcium binding site are well defined. Three
residues at each termini, the defunct site, and the central loop have
larger RMSD (
2 Å). The all heavy atom RMSD is 1.8 ± 0.2 Å.
The extensive spectral overlap in the aliphatic region prevented us
from deriving stereospecific assignments for side chain methylene
groups. The RMSD per residue reflects approximately the distribution of
the number of restraints (on the average 19) per residue.
The orientation of the A and B helices is based on the observation of
20 NOEs. These NOEs are primarily between aliphatic protons. For
example, we find an NOE between Phe-20 and Met-45, which are in the
middle of the A helix and the B helix, respectively. In an open
conformation these distances would be clearly longer, and NOEs except
in the vicinity of the loop would be beyond detection. However, the
uncertainty in the side chain to side chain distance bounds involving
particularly the pseudo atoms of methyl groups do not allow us to
determine the interhelical angles precisely. Due to the overlap of in
the two-dimensional homonuclear spectra, few NOEs between the A and B
as well as between the C and D helices were not assigned. The loop
between the A and B helices, i.e. the defunct site, is quite
irregular and not particularly well defined. We identify 17 NOEs from
the defunct loop. In the central loop between the B and C helices the
prolines 52 and 54 are both in the trans configurations. We
did not find any evidence for cis-trans isomerism.
Dynamics--
The rate constants of the 15N
longitudinal relaxation (R1) are nearly constant
(1.5 ± 0.2 s
1) over the entire sequence. Only for
Gly-91, the R1 is substantially smaller. The
variation in the rate constant of the transverse relaxation
(R2) is larger. For the helices and the calcium
binding site the R2 values are 10 ± 2 s
1 and toward the N- and C termini the
R2 values drop for a few residues. Furthermore,
for residues Val-28, Ser-37, Thr-38, Lys-39, Glu-40, Ile-61, and Val-64
the transverse relaxation proceeds so fast that the rate constants
(>15 s
1) could not be determined reliably. The values of
R1
for residues 37-40 were smaller than the
corresponding R2 values but owing to the
off-resonance effects (30) quantitative comparisons were not made. The
15N line widths for these residues measured from the HSQC
spectrum recorded at 30 °C are indeed larger than on the average
(Fig. 2). Whereas at increased
temperature (40 and 50 °C), the line widths are only 1-2 Hz wider
than on the average. At lower temperatures (10 and 20 °C), also the
lines for residues Ile-36, Leu-41 and Gly-42 become broader than the
average (Fig. 3). The rate of the 15N cross-correlation (
) between the dipole and chemical
shift anisotropy was insensitive to the conformational exchange. The comparison of
with R2 reveals residues
experiencing fast transverse relaxation (Fig. 3). For the residues
(Val-28, Thr-38, Lys-39, Ile-61, and Val-64) with very fast transverse
relaxation, no values of
were obtained due to the insensitivity of
the cross-correlation experiment.

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Fig. 2.
15N line widths color-coded from
blue to red as the width of the ribbon.
Residues with large 15N line widths are mostly at the
defunct site. The broad red part of the ribbon represents
residues with line broadening.
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Fig. 3.
A, 15N line widths were
measured at 20 ( ), 30 ( ), and 40 °C ( ) versus
residue. Residues 52 and 54 are prolines. Other missing data points are
due to overlaps or extreme broadening beyond observation. B,
transverse relaxation rate (R2) ( ) and
cross-correlation rate ( ) ( ) versus residue.
|
|
The heteronuclear Overhauser enhancement was nearly constant and close
to the maximum value (+0.82) (28) for the helices and the
calcium-binding site but decreased for a few residues at the N and C
termini. Heteronuclear NOEs could not be determined reliably for
residues Ser-37 and Thr-38 due to the line broadening.
The principle components of the inertia tensor computed for the cNTnC
structure were (0.81:0.85:1), which implies that the overall rotational
diffusion can be regarded approximately isotropic. Furthermore, the
ratio of R2/R1 for the
helices did not vary significantly, which also implies an approximately
isotropic rotational diffusion characterized by
m = 7.7 ns.
Therefore we considered the analysis of relaxation data in terms of the
commonly used Lipari-Szabo model-free spectral density (26) reasonable.
First the simple model was used with the order parameter
(S2) as the only free parameter. Subsequently,
the time constant
e for the fast internal motion was allowed
to vary as well. In this way a statistically good fit was obtained for
the majority of the residues. For the residues in the helices and in
the calcium binding site S2 is approximately
0.9. The extended model in which the internal motion is divided into
two components (31) resulted for the residues at the N and C termini
(2-8 and 86-91) in a statistically good fit. At the N and C termini
S2 drops rapidly (Fig.
4). For the remaining residues 20, 23, 25, 28, 30, 37-40, 48, 60, and 63-64, the conformational exchange contribution (Rex) was included in the analysis.
For the residues 28, 37-40, 61, and 64, reliable values for
S2 were not obtained due to the fast loss of
transverse coherence.

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Fig. 4.
Generalized order parameter
S2 ( ) versus residue. The
order parameter at the Lipari-Szabo model-free spectral density was
computed from longitudinal transverse relaxation and from heteronuclear
NOE measurements.
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 |
DISCUSSION |
For comparisons to skeletal troponin C, the angle between helices
A and B in cNTnC (135°) is approximately the same as that of
apo-sNTnC (138°) but very different from that of
2·Ca2+-sNTnC (81°) (17). The angle between the C and D
helices (130°) is more similar to the corresponding angle in
apo-sNTnC (145°) than to the angle in 2·Ca2+-sNTnC
(78°). Owing to the comparatively large distance bounds of the few
NOEs in the calcium binding site the angle between the C and D helices
is subject to uncertainty which does not allow us to prove
unambiguously differences between cNTnC and sNTnC. The relative
orientations of the N, A, and D helices of cNTnC are similar to the
corresponding orientations in both apo- and 2·Ca2+-sNTnC.
In this NAD frame of reference the orientation of the B and C helices
of cNTnC resemble more those of the apo-form than the two-calcium form
of sNTnC. This is obvious from the DDMs computed from the families of
structures in order to obtain unbiased comparisons (Fig.
5). In cNTnC the B helix is closer to
(approximately 10 Å) the N and D helices than in
2·Ca2+-sNTnC. On the other hand the differences between
cNTnC and apo-sNTnC are small; however, the cNTnC form appears slightly
more compact (Fig. 5B). The residue Val-28 not in the sNTnC
sequence was excluded from the comparisons. For the sake of
completeness a DDM was computed between apo- and
2·Ca2+-sNTnC (Fig. 5C). The result is quite
similar to the DDM between cNTnC and 2·Ca2+-sNTnC. These
comparisons imply only minor conformational changes to be expected upon
the calcium binding as already shown by Spyracopoulos et al.
(18). This may indeed be a fundamental property of cTnC, which enables
the fast switching between apo and holo states necessary for the heart
function. On the other hand, so far no high resolution structure has
been obtained from a complex comprising the key parts of cardiac TnC
and troponin I. The association of cTnC with troponin I may affect the
degree of opening of cTnC upon calcium binding (18).

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Fig. 5.
Difference distance matrices computed between
the cNTnC and the two-calcium form (A) and apo form
(B) of the skeletal troponin C. C, for the
completeness the DDM is computed between the apo and two-calcium forms
of the sNTnC. Distances are coded according to the color code bar on
top.
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Qualitative comparisons with the structures of the avian (17) and human
(18) regulatory domains of cardiac TnC, reported just recently, reveal
a high degree of similarity. Apparently the C35S and C84S mutations,
used to prevent possible disulfide formation, in the avian cTnC study
are immaterial for the overall structure. Nevertheless, we notice that
NOEs between Ala-23 and Val-44 from the A and B helices, respectively,
reported for the avian cTnC are absent in our NOE spectra. This could
be explained by a small difference in the twist of the B helix with
respect to the A helix between the human and avian cTnC. Furthermore, we notice some differences in the chemical shifts of certain NHs between our data and the data by Spyracopoulos et al. (18). This is partly due to the slightly longer (1-91 amino acids) sequence used in the present study compared with the protein (1-89 amino acids)
studied by Spyracopoulos et al. (18) (Fig. 1 caption). In
addition NH chemical shifts depend on the concentration of TFE but we
do not observe noticeable structural consequences. According to an
earlier study on sTnC (33) the use of 15% TFE should prevent the
dimerization involving the N-terminal domain. The lack of apparent
variation in R2/R1 for
the approximately globular tertiary structure of the cNTnC excludes the
presence of specific dimers, but it may be that 9% of TFE used in the
present study does not completely prevent a transient unspecified
dimerization. Indeed our estimate of the overall correlation time
(
m = 7.7 ns) based on
R2/R1 (28) appears
somewhat high in comparison with values obtained for proteins of
similar sizes. This leaves the possibility for the monomer-dimer
exchange, which leads to an erroneous estimate for the
m and
consequently also to a small systematic error in the values of
S2 assuming the isotropic model. The fast
dynamics itself is unlikely to be affected by the dimerization (34).
The monomer-dimer exchange is indistinguishable from a conformational
exchange (34). In the present study it appears more likely that the
comparatively fast loss of the transverse coherence for residues 28, 37-40, 61, and 64 is caused by the conformational exchange rather than by the monomer-dimer exchange. These residues do not appear to form a
contact surface.
Evenäs et al. (35) have studied a mutated C-terminal
half of calmodulin, E140Q, and observed a conformational equilibrium between the open and closed forms with a population ratio close to 1:1.
They observed the most pronounced effects on resonances from residues
in the calcium binding loops. The strongest line broadening observed in
the present work is for residues in the defunct calcium binding loop
37-40 and hydrophobic residues Val-28, Ile-61, and Val-64.
Coincidentally, Val-64 is also the "hinge" about which the end of
the C helix rotates relative to the NAD unit in sTnC (36) as well as
Glu-40 is the "hinge" about which the B helix reorients with
respect to the NAD unit in cTnC (18). Therefore, it is interesting to
consider the possibility of an exchange between the closed form and a
low population of the open state as an explanation for the line
broadening. In particular because prior to the results by Sia et
al. (17) a large conformational change similar to the one observed
for sTnC was generally expected for cTnC. It was shown, for example,
that upon a calcium titration the chemical shift of the methyl of
Val-28 changes much (
CH3 = 0.16). In retrospect this
significant change is probably explained by small movements of the
nearby phenyls, because the structures of apo- and
Ca2+-cNTnC are so similar (18). In the case of the mutant
calmodulin two conformers were distinguished from a double set of
mutually excluding NOEs. We find no clear evidence for a similar
situation, which at least implies that the determined closed
conformation of cNTnC has a population above 80-90%. A small
population of the minor conformation could still cause the rapid
transverse relaxation provided that the chemical shifts for the two
conformations would differ significantly for instance due to ring
current effects. A study of the structures of the apo and holo states
of cNTnC in the presence of the binding peptide from TnI, now in
progress, should settle if the presently known calcium-loaded closed
conformation is truly the biologically relevant conformation of the
regulatory domain of cardiac troponin C.
The time-limiting step in muscle relaxation is most likely the release
of calcium from the N-terminal half of troponin C. For free troponin C
and calmodulin the off-rate of the Ca2+ ions from the
regulatory sites is about 500 s
1 (3). However, in the
presence of a binding peptide or drug this rate is dramatically
reduced. For skeletal muscle with sTnC and smooth muscle with
calmodulin, even a 100-fold reduction is acceptable, but not so for
heart muscle. For the heart muscle to relax to about 90% in half a
second the calcium off-rate has to be about 7 s
1. Maybe
the cardiac TnC has developed to its present sequence with the defunct
site to cope with the strict timing requirements, which can be more
easily met with a somewhat weaker calcium binding.
We thank Dr. L. E. Kay for providing
pulse sequences for heteronuclear spectroscopy, Dr. A. Palmer for
providing programs for dynamics calculations, and Dr. P. J. Fleming for making available the program for the difference distance
matrix calculations.