From the Department of Molecular Genetics, Biochemistry, and
Microbiology, University of Cincinnati, College of Medicine,
Cincinnati, Ohio 45267 and the Department of Physiology
and Biophysics, College of Medicine, University of Illinois,
Chicago, Illinois 60612
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ABSTRACT |
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Conformational exchange has been demonstrated
within the regulatory domain of calcium-saturated cardiac troponin C
when bound to the NH2-terminal domain of cardiac
troponin I-(1-80), and cardiac troponin I-(1-80)DD, having serine
residues 23 and 24 mutated to aspartate to mimic the phosphorylated
form of the protein. Binding of cardiac troponin I-(1-80) decreases
conformational exchange for residues 29, 32, and 34. Comparison of
average transverse cross correlation rates show that both the
NH2- and COOH-terminal domains of cardiac troponin C tumble
with similar correlation times when bound to cardiac troponin
I-(1-80). In contrast, the NH2- and COOH-terminal domains
in free cardiac troponin C and cardiac troponin C bound cardiac
troponin I-(1-80)DD tumble independently. These results suggest that
the nonphosphorylated cardiac specific NH2 terminus of
cardiac troponin I interacts with the NH2-terminal domain
of cardiac troponin C.
Troponin C (TnC)1 is the
Ca2+ binding component of the troponin complex that is
required to confer Ca2+ sensitivity on the actin-myosin
interaction. The troponin complex consists of three proteins: troponin
I (TnI), which inhibits the actomyosin Mg2+-ATPase; TnC,
which removes TnI inhibition triggering the Mg2+-ATPase;
and TnT, which makes primary protein-protein contacts with tropomyosin.
The cardiac isoforms of TnC and TnI differ significantly from skeletal
isoforms. In the cardiac isoform of TnC, Ca2+ binding site
I, comprising residues 28 through 40, is naturally inactive (1).
Residues 65 through 76 form the regulatory calcium binding site II. The
cardiac isoform of TnI is unique in that it contains an additional
NH2-terminal extension of approximately 32 residues. This
extension contains two adjacent serine residues that can be
phosphorylated by PKA. Phosphorylation has been demonstrated to
modulate myofilament sensitivity to Ca2+ by reducing
Ca2+ affinity for the NH2-terminal regulatory
site of cTnC (2). The reduction in the sensitivity of myofilament force
development to Ca2+ induced by phosphorylation of cTnI can
be mimicked by exchange of the native unphosphorylated cTnI with
cTnI-DD, in which Ser23 and Ser24 have been
mutated to Asp (3). Cardiac TnI-DD is also able to mimic effects of
phosphorylation on the steady-state and pre-steady-state binding of
cTnI to cTnC (4). Cardiac TnC and TnI are known to interact in an
antiparallel manner such that the COOH-terminal domain of TnC interacts
with the NH2-terminal domain of TnI (5).
Conformational changes induced by Ca2+ binding to the
NH2-terminal domain of skeletal TnC (sTnC) have been
followed by both x-ray (6) and solution NMR (7). For the free skeletal
TnC protein, Ca2+ binding results in a conformational
change from the "closed" form to an "open" form exposing a
patch of hydrophobic residues for interaction with TnI (7).
Surprisingly, the Ca2+-bound NH2-terminal
domain of cTnC was found to maintain a closed conformation (8). The
solution structure of the Ca2+ saturated regulatory domain
of human cTnC, cTnC-(1-91), in 9% trifluoroethanol, also reveals a
"closed" conformation with little exposed hydrophobic surface (9).
In addition, 15N relaxation studies on human cTnC-(1-91)
suggested conformational exchange for Val28,
Thr38, Lys39, Ile61, and
Val64. The authors suggest the origin of this
conformational exchange results from exchange between the closed form
and a low population of the open form. However, monomer-dimer exchange
could not unequivocally be ruled out.
To elucidate the role of phosphorylation of the cardiac-specific amino
terminus of cTnI on full-length cTnC and to unequivocally demonstrate
the presence of conformational exchange in the regulatory domain of
full-length cTnC, we have probed the dynamics of cTnC free and bound to
cTnI-(1-80) and cTnI-(1-80)DD. We show that residues within inactive
Ca2+ binding site I undergo chemical exchange consistent
with an equilibrium between closed and opened forms both in the
presence and absence of the NH2-terminal cTnI domain. Based
on chemical shift perturbation mapping and transverse relaxation rates,
the cardiac-specific NH2 terminus of cTnI-(1-80) appears
to make additional interactions with the regulatory domain of
cTnC, which results in slowing down the exchange rate in the defunct
Ca2+ binding site I as evidenced by the presence of
1H-15N cross-peaks representing two distinct
conformations for Leu29, Glu32, and
Gly34. These interactions were not observed in the free
protein or in the complex with cTnI-(1-80)DD.
Proteins--
The single cysteine form of cTnC (cTnC35S) was
used in the study (10, 11). 15N and 2H labeling
was accomplished using minimal medium containing 90% 2H2O and 1 g/liter
15NH4Cl. Cardiac troponin C was purified as
described previously (5). Cardiac troponin I-(1-80) and cTnI-(1-80)DD
were expressed as inclusion bodies purified in 8 M
urea.2 All proteins were
judged to be homogeneous by SDS-polyacrylamide gel electrophoresis and
staining with Coomassie Brilliant Blue.
Complex formation for cTnC·cTnI-(1-80) and cTnC·cTnI-(1-80)DD was
carried out as described previously (5). Samples of 1.0 mM
concentration were prepared in 10% 2H2O, 20 mM Tris-d11 buffer (pH = 6.8), 150 mM potassium chloride, 10 mM Ca 2+,
10 mM NMR Spectroscopy--
All experiments were carried out on Varian
Inova 600 or 800 MHz spectrometers. Amide 1H and
15N resonances of cTnC were obtained by means of
heteronuclear triple resonance NMR experiments.3 To confirm
the assignments for cTnC bound to cTnI-(1-80) and cTnI-(1-80)DD,
1H-15N NOESY-HSQC experiments with mixing times
of 85 and 200 ms were performed at 800 MHz. The 15N
R2 and dipolar CSA interference experiments were
performed on Ca2+-saturated
[2H,15N]cTnC free and bound to either
cTnI-(1-80) or cTnI-(1-80)DD. For all experiments, the spectral
widths in t1 and t2 dimensions were 2.00 and 15.62 kHz, respectively.
The 1H carrier was set to the frequency of the water
resonance at 4.77 ppm. The number of transients used in
R2 and Iauto transverse cross-correlation experiments was 16, while Icross
transverse cross-correlation experiments were performed with 48 transients. 15N dipolar CSA interference experiments were
performed as described by Kroenke et al. (12). Relaxation
delays used for the transverse cross-correlation rate
( Data Processing--
NMR data was processed using Felix 97.2 (MSI). Free induction decays were apodized with a skewed sine bell
90° function. Generally, peak heights were measured using Felix
routines. For the transverse cross-correlation experiments
Icross/Iauto intensity ratios were normalized
for the number of transients used in each experiment. Curve-fits for
transverse relaxation and cross-correlation rates were performed using
the CURVEFIT software (13).
To compare interactions of cTnI-(1-80) and cTnI-(1-80)DD with
cTnC, 1H and 15N chemical shift differences of
bound cTnC were analyzed. Chemical shift differences for amide
1H and 15N resonances observed between the two
complexes are presented in Fig. 1. These
chemical shift differences can be used to map to binding site of
cTnI-(1-80) on the regulatory domain of cTnC. In addition, analysis of
chemical shift differences between free cTnC and cTnC bound to
cTnC·cTnI-(1-80) or cTnC·cTnI-(1-80)DD reveals that both proteins
bind predominantly to the COOH-terminal domain of cTnC causing a
conformational change, as was observed for cTnI-(33-80) binding to
cTnC-(81-161).4 Taken together, these results allow us to
propose that both nonphosphorylated and phosphorylated forms of cTnI
bind to the COOH-terminal domain of cTnC with the nonphosphorylated
form making limited contacts with the NH2-terminal domain
of cTnC. It is also apparent that binding of the nonphosphorylated
NH2-terminal domain of cTnI causes only minor structure
perturbations in the NH2-terminal domain of cTnC.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol, and 10 mM
dithiothreitol. All samples contained 0.2 mM leupeptin and
0.4 mM pefablock to prevent protein degradation.
xy) measurements were 0.0320, 0.0534, 0.0748, 0.0961, and 0.1068 s. The R2 experiments were performed with recovery delays of 0.000, 0.032, 0.064, 0.096, 0.128, 0.160, 0.192, and 0.224 s as described previously (14). Double points were
collected with recovery delays of 0.032, 0.128, and 0.192 s to estimate
the uncertainties.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Absolute value amide nitrogen
(A) and amide proton (B) chemical
shift differences between Ca2+-saturated
cTnC·cTnI-(1-80) and cTnC·cTn-(1-80)DD. The horizontal
lines in A and B represent the average
chemical shift differences plus 1 S.D., 0.02 ± 0.02 ppm and
0.18 ± 0.17 ppm, respectively. Filled diamonds mark
residues for which resonance assignments in
1H-15N correlation spectrum could be not
confirmed due to a lack of sequential NOEs in NOESY-HSQC spectra. These
residues are 6, 8, 10, 15, 17, 20, 24, 41, 44, 47, 48, 51, 58, 62, 64, 65, 75, 83, 85, 95, 96, 97, 101, 103, 118, 126, 132, 136, 150, and 154. Residues 52 and 54 are Pro. Cross-peaks for residues 28, 36, 37, 38, 39, and 40 are broadened beyond detection in
1H-15N correlation spectra of
cTnC·cTnI-(1-80), and residues 38 and 39 are broadened beyond
detection in 1H-15N correlation spectra of
cTnC·cTnI-(1-80)DD. For residues 29, 32, and 34, chemical shifts of
the highest intensity peaks representing the predominant conformation
of cTnC bound to cTnI-(1-80) were chosen.
1H-15N correlation NMR spectroscopy was used to
study the interactions of cTnC with TnI-(1-80) and TnI-(1-80)DD.
Residues excluded from the analysis due to peak overlap, even at 800 MHz, in the 1H-15N correlation spectra of cTnC
bound to cTnI-(1-80) or cTnI-(1-80)DD are listed in Fig.
2. In the cTnC·cTnI-(1-80)DD complex,
cross-peaks for cTnC residues 38 and 39 were broadened beyond
detection. Cross-peaks for residues 24, 28, 36, 37, 38, 39, and 40 were
broadened beyond detection in the cTnC·cTnI-(1-80) complex.
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To further investigate interactions between the regulatory domain of cTnC and the NH2-terminal domain of cTnI, 15N transverse relaxation and 1H-15N diploar/15N CSA cross-correlation rates were measured at 800 MHz on [2H,15N]cTnC free and bound to cTnI-(1-80) and cTnI-(1-80)DD. Perdeuteration offered an opportunity to obtain precise relaxation parameters and enhanced spectral resolution. Data analysis was performed as described under "Materials and Methods." The experimental values for R2 obtained at 800 MHz on cTnC·cTnI-(1-80), cTnC·cTnI-(1-80)DD, and free cTnC are depicted in Fig. 2. Not surprisingly, low R2 values for flexible NH2 and COOH termini of cTnC free and bound are observed. It is also apparent that Met85 through Thr93 in the linker region of cTnC free and cTnI bound exhibit reduced R2 values. In addition, Ala30 through Ser35 in the inactive calcium binding site I of cTnC bound to cTnI-(1-80) or cTnI-(1-80)DD exhibit reduced R2 values. This fact is indicative of increased mobility in these regions.
Elevated R2 relaxation rates are found for Val28, Ile36, and Ser37 in both free cTnC and cTnC bound to cTnI-(1-80)DD (Fig. 2B). R2 relaxation rates could not be obtained for these residues in the cTnC·cTnI-(1-80) complex due to extreme broadening of 1H-15N cross-peaks in HSQC spectra recorded for the R2 measurements. This broadening may best be explained by the possibility that these residues are engaged in slow conformational exchange.
To prove the existence of conformational exchange in the inactive
Ca2+ binding site I of cTnC, measurements of
1H-15N dipolar/15N CSA transverse
(xy) cross-correlation rates were carried out. These
cross-correlation rates do not depend on chemical exchange effects and
are directly proportional to the generalized order parameters (15).
Therefore, the
xy values are sensitive only to internal
and overall motions (12, 16). The transverse cross-correlation rates
are also presented in Fig. 2. In general, the transverse cross-correlation rates repeat the same trend as
R2 values. However, for residues
Val28, Ile36, and Ser37 the
cross-correlation values are not increased like the
R2 relaxation rates in this region. Thus, the
results of these experiments prove that residues adjacent to the
calcium binding site I undergo conformational exchange in free cTnC and
when bound to cTnI-(1-80)DD. Conformational exchange in
Val28, Ile36, and Ser37 is slower
when cTnC is bound to cTnI-(1-80), since these residues have broadened
beyond detection. In support of this, multiple resonances for
Leu29, Glu32, and Gly34 of cTnC
were observed in the complex with cTnI-(1-80) (Fig. 3). Careful
analysis of NOESY-HSQC spectra revealed that both cross-peaks for each
residue have NOEs to the same amide protons. In the case of
Leu29 and Gly34, mutual sequential NOEs are
observed to Gly30 (Fig. 3A) and
Asp33 (Fig. 3B), respectively. These results
allowed us to suggest that the new cross-peaks represent a different
conformation for the defunct Ca2+ binding site I of cTnC.
The resonance for Glu32 also appears to consist of multiple
peaks, but HN-HN NOEs for Glu32
could not be observed in the recorded NOESY-HSQC spectra. Thus, we are
unable to unequivocally assign both cross-peaks to Glu32.
Cross-peaks for Leu29, Glu32, and
Gly34 are well resolved, and it is unlikely that the new
peaks belong to some other unassigned amide protons experiencing
extremely large chemical shift perturbations and having identical
i to i + 1 NOEs. It seems possible that
cTnI-(1-80) binds to the NH2-terminal domain of cTnC and
slows down chemical exchange in defunct Ca2+ binding site
I, presumably changing the dynamic equilibrium between open and closed
conformations.
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On average, the R2 values for the COOH-terminal
domain of the free protein are smaller than those values for the
NH2-terminal domain (Fig. 2C). This can be
attributed to the fact that the NH2-terminal domain of cTnC
is slightly larger than the COOH-terminal domain. The average
R2 value for the COOH-terminal domain,
comprising residues 104 through 135, was 11.0 ± 1.9 s1, while the average R2 value for
the NH2 terminus, comprising residues 14 through 23 and
residues 41 through 80, was 14.6 ± 1.6 s
1. The
linker region, the NH2 and COOH termini, and inactive
calcium binding site I were excluded from this analysis on the basis of either increased mobility or possible conformational exchange in these regions.
When cTnC is bound to cTnI-(1-80)DD the average
R2 value (20.0 ± 1.9 s1) for
the COOH-terminal domain is larger than the average
R2 value for the NH2-terminal domain
(16.0 ± 1.8 s
1) as shown in Fig. 2B.
This difference could be explained by the fact that the NH2
terminus of cTnI binds to the COOH terminus of cTnC (5). However, for
the cTnC·cTnI-(1-80) complex both the NH2- and
COOH-terminal domains have similar average R2
values, (30.3 ± 2.9 s
1) and (31.6 ± 2.1 s
1), respectively (Fig. 2A). A possible
explanation of this phenomenon is that the NH2 terminus of
the nonphosphorylated form of cTnI also makes contacts with the
NH2-terminal domain of cTnC.
The two domains of cTnC which are connected by a flexible linker tumble
independently, with different rotational correlation times, in both the
free protein and in a complex with cTnI-(1-80)DD. This results in
different 15N R2 values for the two
domains of cTnC. Binding of cTnI-(1-80) to both the NH2-
and COOH-terminal domains of cTnC reduces the possibility of
independent tumbling of the two domains, which results in more uniform
rotational correlation times across the molecule and subsequently in
similar 15N transverse relaxation rates. Interaction of
cTnI-(1-80) with both domains of Ca2+-saturated cTnC does
not preclude flexibility in the linker region as is observed in the
complex (Fig. 2A).
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DISCUSSION |
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The solution structure for cardiac troponin C unexpectedly revealed a closed conformation for the regulatory domain of cardiac TnC (8). In this regard, the Ca2+-saturated cardiac isoform of TnC differs from the Ca2+-saturated skeletal isoform, which is in the open state (7). The authors suggest that the functional form of Ca2+- saturated cTnC could be the closed form. However, based on relaxation studies performed on the isolated NH2-terminal domain of human cTnC, it has been suggested that the regulatory domain exists in two conformations: open and closed (9).
The results of our experiments demonstrate that chemical exchange does
occur in the defunct Ca2+ binding site I in both intact
free cTnC and cTnC bound to the NH2-terminal domain of
cTnI. Thus, the regulatory domain of cTnC may be in equilibrium between
a closed and open form both free in solution and when it is bound to
the phosphorylated and nonphosphorylated forms of the
NH2-terminal domain of cTnI. This argument is supported by
the presence of conformational exchange as evidenced by differences in
the R2 values and transverse cross-correlation
rates for Val28, Ile36, and Ser37
in the inactive Ca2+ binding site I. Taken together, our
studies suggest that when cTnI is phosphorylated, the regulatory domain
of cTnC is destabilized, resulting in a decrease in Ca2+
affinity. This would explain the enhanced relaxation observed when cTnI
is phosphorylated by PKA in response to -adrenergic stimulation of
the heart (17). Binding of the nonphosphorylated form of cTnI does not
prevent chemical exchange in inactive calcium binding site I of cTnC.
We propose, based on chemical shift mapping, R2
values, and the presence of multiple cross-peaks for certain residues
in defunct Ca2+ binding site I that nonphosphorylated cTnI
interacts with both domains of cTnC and decreases chemical exchange
presumably changing the dynamic equilibrium between open and closed
conformations (8, 18). The fact that the transverse relaxation rates
for the NH2 terminus of cTnC are not increased upon binding
cTnI-(1-80)DD provides strong evidence that the phosphorylated mimetic
of cTnI binds only to the COOH terminus of cTnC. This interpretation is in agreement with the fact that there are no significant amide proton
and amide nitrogen chemical shift differences in the
NH2-terminal domain of cTnC upon binding of cTnI-(1-80)DD.
The crystal structure of sTnC complexed with a fragment of sTnI that
corresponds to cardiac TnI-(33-80) clearly shows that sTnI interacts
with both the NH2 and COOH termini of TnC (19).
Specifically, the A-helix of sTnC makes several contacts with the
NH2-terminal portion of sTnI-(1-47) (19). We have shown
recently that the solution structure of cTnC-(81-161) bound to
cTnI-(33-80) is similar to that of sTnC bound to sTnI-(1-47). General
agreement between sTnC and cTnC bound TnI structures suggest a common
binding motif for the
Ca2+/Mg2+-dependent interaction
site in the TnI·TnC complex. It now appears that in the absence of
phosphorylation, the
Ca2+/Mg2+-dependent interaction
site of cTnI makes additional contacts with the regulatory domain of
cTnC similar to those interactions observed in the crystal structure
for sTnC·sTnI-(1-47) (19). Data presented here are all consistent
with this hypothesis. It is intriguing to speculate that loss of these
interactions are responsible for a decrease in calcium affinity in
Ca2+ binding site II of cTnC when cTnI is phosphorylated.
However, the exact sequence of events in this process is not understood and requires further investigation.
This study provides the first structural and dynamic insight on the
mechanism by which PKA phosphorylation of cTnI, in response to
-adrenergic stimulation of the myocyte, produces a decrease in the
calcium affinity of the regulatory site in cTnC. The proposed mechanism
utilizes the unique cardiac isoform differences found in both TnC and
TnI. Knowledge of this mechanism provides the basis for molecular
approaches aimed at modifying cardiac muscle contraction.
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ACKNOWLEDGEMENTS |
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We thank Prof. Art Palmer (Columbia University) for providing CURVEFIT Software and Chris Kroenke for helpful discussions.
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FOOTNOTES |
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* This work supported by Grants AR 44324 (to P. R. R.), HL 49934 (to R. J. S.), and GM 40089 (to M. R.) from 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.
§ Authors (M. R. or P. R. R.) to whom correspondence should be addressed. E-mail: rance{at}rabi.med.uc.edu (for M. R.) or rosevear{at}proto.med.uc.edu (for P. R. R.).
2 M. B. Abbott, unpublished data.
3 N. Finley, unpublished data.
4 Gasmi-Seabrook, G. M. C., Howarth, J. W., Finley, N., Abusamhadneh, E., Gaponenko, V., Brito, R. M. M., Solaro, R. J., and Rosevear, P. R. (1999) Biochemistry, in press.
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ABBREVIATIONS |
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The abbreviations used are: Tn, troponin; cTnC, recombinant cardiac troponin C (desMet1-Ala2, C35S); sTnC, skeletal TnC; cTnI, cardiac troponin I; PKA, cyclic AMP-dependent protein kinase A; NOESY-HSQC, nuclear Overhauser effect spectroscopy-heteronuclear single quantum coherence; CSA, chemical shift anisotropy.
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