From the Canadian Institutes of Health Research Group in Protein Structure and Function and the Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada
Received for publication, March 11, 2003 , and in revised form, April 24, 2003.
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ABSTRACT |
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INTRODUCTION |
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The structure of cardiac TnC, sharing sequence and structural similarities to the skeletal isoform, has been solved by both x-ray crystallography and NMR spectroscopy (7, 1519). Upon Ca2+ binding, there is an opening of the N-domain of the skeletal isoform, whereas the cardiac isoform remains closed, opening only upon binding to cTnI-(147163) (cSp) (16). The structure of the complete troponin complex is of critical importance in understanding molecular interactions during muscle contraction, yet only low resolution structures of the complex and several structures of TnC in complex with different TnI peptides are currently available (16, 18, 2124), giving insights into muscle contraction and regulation. The numbering of the residues of cTnI in this study are as presented previously (25), in contrast to the numbering of the wild type protein, which has an extra N-terminal Met residue (26).
TnI-TnC interactions have been studied extensively for over 3 decades. The functionality of domains of TnC was determined early by Head and Perry (27) by using various proteolytic/ cleavage techniques. These studies of rabbit skeletal muscle yielded a region of TnI that has been referred to as the "inhibitory region." Subsequent work by Talbot and Hodges (28) identified a refined inhibitory region, which has been mapped to a central region of its primary amino acid sequence, corresponding to residues Thr128Arg147 in the cardiac system (cIp). The inhibitory region contains the minimum sequence required to fully inhibit the myosin ATPase activity on the thin filament (29). Interactions of the cardiac TnI inhibitory region are of key importance in complete understanding of muscle regulation. It has been shown that the inhibitory region interacts with the protein actin on the thin filament during muscle relaxation and that movement of cIp off actin interacts with TnC upon Ca2+-induced muscle contraction. Previously, it has been shown that the inhibitory region of TnI binds to the C-domain of TnC and the central linker region of the N- and C-terminal domains of TnC (30). Additional interactions of TnC with other regions of TnI have been elucidated (31, 32), resulting in an intertwined system with multiple contacts between the subunits of the troponin complex.
Whereas elucidation of the structure of the inhibitory region of TnI in
complex with the troponin complex is of key importance in the understanding of
muscle contraction, the structure has remained elusive, and consequently many
models have been proposed. Early 1H NMR study of the skeletal TnI
isoform involving transferred NOEs by Campbell and co-workers
(33,
34) led to the proposal that
the inhibitory region, sTnI-(104115) (cTnI-(136147)), adopts a
short helix, distorted around two central proline residues, and this structure
was subsequently docked within the hydrophobic cleft on sCTnC
(35). The crystal structure of
sTnC in complex with the N-terminal regulatory peptide of sTnI-(147)
(Rp40) presented by Vassylyev et al.
(21) showed that Rp40 adopts a
helical structure and binds in the hydrophobic cleft of sCTnC, and these
workers modeled the inhibitory region as adopting a helical conformation away
from the hydrophobic cleft. The helical conformation of the inhibitory region
was directly challenged by Hernandez et al.
(36), who proposed an extended
conformation of the inhibitory region, with a two-stranded -hairpin away
from the hydrophobic cleft of the C-domain
(37).
Electron spin labeling work by Brown et al. (38) has shown that a section of the inhibitory region of the cardiac isoform (cTnI-(129137)) displays a helical profile, with the C-terminal residues 139145 displaying no discernible secondary structural characteristics. The inhibitory region possesses a large number of basic residues, and Tripet et al. (39) predicted that this highly basic region of cTnI makes numerous electrostatic interactions with the acidic TnC in the troponin complex. Recent work on the cardiac isoform using residual dipolar coupling by Dvoretsky et al. (19) determined the orientations of the domains of cTnC within a cTnI·cTnC complex, and a small angle scattering study by Heller et al. (40) has determined relative domain orientation within a cTnI·cTnC·cTnT-(198298) complex, which suggests that interactions between TnC and the TnI·TnT components differ significantly between the skeletal and cardiac isoforms. A preliminary crystallographic structure of a TnT·TnC·TnI ternary complex by Takeda et al. (41) indicated a coiled-coil region of cTnI·cTnT with multiple interactions with cTnC; however, the inhibitory region spanning cIp was not visualized within the structure.
We have been successful in elucidating the NMR solution structure of the cardiac isoform of the inhibitory region of TnI (cTnI-(128147)) in complex with the Ca2+-saturated C-terminal domain of TnC. The inhibitory region displays a helical secondary structure from residues Leu134Lys139, with several stabilizing electrostatic interactions with cCTnC. The structure correlates well with previous NMR chemical shift mapping of the interactions of the inhibitory region with TnC (22, 30, 42). The ability to isotopically label the inhibitory region has given us the unique ability to utilize 15N NMR relaxation to study dynamics of the bound inhibitory region. NMR relaxation indicates that the central core of cIp is rigid when bound to cCTnC, giving validation to the structure. This is the first high resolution structure determined for the inhibitory region of TnI in complex with TnC and provides a framework for understanding interactions within the troponin complex during heart contraction.
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EXPERIMENTAL PROCEDURES |
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Preparation of cIp ProteincIp-s peptide acetyl-TQKIFDLRGKFKRPTLRRVR-amide was prepared as described for the sIp peptide in Tripet et al. (32). The engineering of the expression vector for expression and purification of cIp-r, 15N-cIp-r, and 13C/15N-cIp-r via a fusion protein approach has been described previously (42, 45). The procedure for purification of the GB-1-cIp-His fusion protein was modified as follows. Fusion proteins were eluted in 100 mM EDTA, 500 mM NaCl, 20 mM Tris-HCl, pH 7.9. Eluted proteins were then desalted by loading on a Sephadex G-25 medium (Amersham Biosciences) column in 10 mM NH4HCO3, pH 8.0, and lyophilized to dryness.
Titration of
15N-cCTnC·2Ca2+ with
cIpThe titration is as described previously
(42). Both one-dimensional
1H and two-dimensional {1H, 15N}-HSQC spectra
were acquired at every titration point. In order to check if the cIp-s and
cIp-r peptides bind the same way to cCTnC·2Ca2+,
8.1 mg of solid cIp-r was added to a 0.92 mM
15N-cCTnC·2Ca2+ in NMR buffer (NMR
buffer: solution containing 100 mM KCl, 10 mM imidazole,
90% H2O, 10% D2O, 0.01% NaN3. Buffer has been
treated with Chelex 100 to remove all residual metal ions prior to sample
preparation) with 5 µl of 1 M CaCl2 to generate a
complex cCTnC·2Ca2+·cIp-r, in which the
[cIp-r]total/[cCTnC·2Ca2+]total
is 5:1, corresponding to the final ratio for the
[cIp-s]total/[cCTnC·2Ca2+]total
complex. The two-dimensional {1H, 15N}-HSQC spectrum was
acquired and superimposed with that of the
cCTnC·2Ca2+·cIp complex.
Titration of 15N-cIp-r with cCTnC·2Ca2+5.11 mg of recombinant 15N-cIp-r was dissolved in 550 µl of NMR buffer, pH 6.7, containing 25 µl of 1 M CaCl2, and 500 µl was transferred to an NMR tube. Solid cCTnC was added in 1.2-mg additions, with thorough mixing after each addition. Both one-dimensional 1H and two-dimensional {1H, 15N}-HSQC spectra were acquired at every titration point. After every titration point, 1 µl of the resulting titrated solution was removed and used for amino acid analysis. Solid cCTnC was added until no further changes were observed in the HSQC spectra, ensuring complete cIp-r saturation. The change in cIp-r concentration due to changes in volume during the titration was taken into account for data analysis, and the change in pH for cCTnC addition was corrected by addition of NaOH during each addition. A small amount of white precipitate accumulated as increasing amounts of solid cCTnC was added.
Structural Studies on cCTnC·2Ca2+·cIp ComplexThree samples were prepared for structural studies on the complex, with subsequent structural determination carried out using the NMR experiments listed in Table I. 10.13 mg of 13C/15N-cCTnC was dissolved in 600 µl of NMR buffer, pH 6.7, containing 5 µlof1 M CaCl2 to which 6.23 mg of cIp-s was added, and 500 µl was added to an NMR tube. 3.34 mg of 13C/15N-cIp-r was dissolved in 600 µl of NMR buffer, pH 6.7, containing 5 µl of 1 M CaCl2 to which 25.5 mg of cCTnC was added to ensure complete cIp-r saturation, and 500 µl was added to an NMR tube. 5.06 mg of 13C/15N-cIp-r was dissolved with 16.44 mg of 13C/15N-cCTnC to ensure a 1:1 molar ratio in 575 µl of NMR buffer (100% D2O, pH 6.7) containing 5 µl of 1 M CaCl2, and 500 µl was added to an NMR tube.
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NMR SpectroscopyAll NMR data used in this study were acquired at 30 °C using Varian INOVA 500 MHz, Unity 600 MHz, and INOVA 800 MHz spectrometers. All three spectrometers are equipped with triple resonance probes and z axis pulsed field gradients (xyz gradients for the 800 MHz). For the cCTnC·cIp complex, the chemical shift assignments of the backbone and the side chain atoms and NOE interproton distance restraints were determined using the two-dimensional and three-dimensional NMR experiments described in Table I.
Data Processing and Peak CalibrationAll two-dimensional and three-dimensional NMR data were processed using NMRPipe (46), and all one-dimensional NMR data were processed using VNMR (Varian Associates). All spectra were analyzed using NMRView (47). For cCTnC and cIp in the complex, intramolecular distance restraints obtained from the NOESY experiments were calibrated according to Gagne et al. (48). Dihedral angle restraints were derived from data obtained from HNHA and NOESY-HSQC experiments according to Sia et al. (7).
Structural Calculations for Binary Complex
cCTnC·2Ca2+·cIpNOE
contacts between protein-protein, peptide-peptide, and protein-peptide were
derived from various three-dimensional 13C/15N-NOESY
experiments. The three-dimensional 13C-edited NOESY experiment
performed on 13C/15N-cCTnC complexed with
13C/15N-cIp-r was used to define protein-peptide
contacts with use of symmetrical peaks within the three-dimensional planes for
use in unambiguous NOE assignments. All protein-peptide NOEs were calibrated
to 4 ± 2 Å. An initial set of 100 structures of both cCTnC and
cIp was first generated separately using NOE distance restraints only. NOEs
with a distance violation of 0.2 Å or greater were closely examined
prior to further rounds of structure refinements. In the latest stages of
refinement, angles of cCTnC were added for residues located in well
defined regions, as determined using the program Procheck
(49). In the first round of
structure calculations of the cCTnC-cIp complex, an initial set of 100
structures was determined using pre-folded structures of cCTnC and cIp
respectively, with unambiguous protein-peptide NOEs being introduced. Further
refinement of structures of the complex using the initial set of 100
structures was performed using the program Procheck to inspect structures, as
well as closely examining NOE distance violations greater than 0.1 Å. To
ensure independent folding of the complex from pre-folded cCTnC and cIp
structures, a final set of 100 structures was derived from both cCTnC and cIp
starting in extended conformations. The default CNS
(50) built-in annealing
protocol was modified to allow the introduction of 12
Ca2+-distance restraints preceding the second cooling
step using Cartesian dynamics. The structural statistics of the family of the
30 calculated lowest energy structures is shown in
Table II. A comparison of
interhelical angles of the binary complex with previously solved EF-hand
structures is shown in Table
III.
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Backbone Amide 15N Relaxation Measurements of cCTnC·2Ca2+·cIp ComplexTwo samples of 15N-cCTnC were prepared, one of cCTnC·2Ca2+ free in solution and the other with binary complex cCTnC·2Ca2+·cIp-s. A first sample of 11.82 mg of 15N-cCTnC was dissolved in 600 µl of NMR buffer, pH 6.7, containing 5 µlof1 M CaCl2, and 500 µl was added to an NMR tube. A second sample of 12.49 mg of 15N-cCTnC was dissolved in 600 µl of NMR buffer, pH 6.7, containing 5 µl of 1 M CaCl2 to which 6.69 mg of cIp-s peptide was added to ensure complete cCTnC saturation, and 500 µl was added to an NMR tube. Two samples of 15N-cIp-r were prepared, one of cIp-r free in solution and the other with cIp-r·cCTnC·2Ca2+ in complex. 0.8 mg of recombinant 15N-cIp-r was dissolved in 550 µl of NMR buffer, pH 6.7, containing 5 µl of 1 M CaCl2, and 500 µl was added to an NMR tube. A second sample of 1.88 mg of recombinant 15N-cIp-r was dissolved 600 µl of NMR buffer, pH 6.7, containing 5 µl of 1 M CaCl2 to which 14.5 mg of 1H-cCTnC was dissolved to ensure complete cIp-r saturation, and 500 µl was added to an NMR tube.
All relaxation data were acquired on Varian INOVA 500 MHz and Unity 600 MHz spectrometers at 30 °C. Sensitivity enhanced pulse sequences developed by Farrow et al. (51) were used to measure 15N-T1, 15N-T2, and {1H}-15N NOE (where T1 is longitudinal (spin-lattice) and T2 is transverse (spin-spin) relaxation). By using two-dimensional spectroscopy a set of backbone 15N-T1, 15N-T2, and {1H}-15N NOE experiments were collected for each sample with parameters described in Table IV. The delay between repetitions of the pulse sequence was set to 3 s for both the T1 and T2 experiments. {1H}-15N NOE measurements were made in the absence (relaxation delay incorporation of 5 s between spectrometer pulses) and the presence or proton saturation (incorporation of 3 s of 1H saturation, with a delay of 2 s between spectrometer pulses). All relaxation data were processed using NMRPipe (46) and analyzed using NMRView (47).
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CoordinatesThe coordinates for the structure of cCTnC·2Ca2+·cIp have been deposited in the Protein Data Bank (code 10ZS).
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RESULTS |
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Resonances undergoing large backbone amide 1H and/or
15N chemical shifts were followed to monitor peptide-protein
binding. The normalized chemical shift data were fit to
Equation 1,
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cIp induces significant chemical shift perturbations of the amide resonances of 15N-cCTnC, with residues in the E-helix, H-helix, and the linker region showing the greatest changes. Superimposition of the {1H, 15N}-HSQC NMR spectra of cCTnC·2Ca2+·cIp-s and cCTnC·2Ca2+·cIp-r revealed that the spectra were identical, indicating that both recombinant and synthetic cIp peptides used in this study perturb cCTnC in an identical manner. Monitoring of the reverse titration of 15N-cIp-r with cCTnC also reveals large chemical shift perturbation, with the largest degree of perturbation in the central region of cIp-r peptide. Specified residues undergoing significant chemical shift perturbation are labeled on Fig. 1, A and B. Two species of hSer are observed in Fig. 1B, showing resonances of hSer and hSer-lactone species present on the C termini of the peptide cIp-r, neither of which undergo chemical shift perturbation, indicating that the C termini residues of cIp-r are not involved in binding of the complex.
Overall Structure of the cCTnC·2Ca2+·cIp ComplexThe NMR experiments performed to obtain structural data for the TnC·TnI complex are summarized in Table I. A sample of 13C/15N-cCTnC in complex with cIp-s gave a two-dimensional {1H, 15N}-HSQC NMR spectra that was highly resolved (Fig. 1A), allowing the assignment of chemical shifts of the cCTnC backbone and side chain nuclei. Previously, our laboratory has obtained data for protein-peptide/ligand complexes using 13C/ 15N-labeled protein in complex with unlabeled peptides/ligands (1618), using two-dimensional 13C/15N-filtered DIPSI and NOESY experiments (52) to obtain chemical shift data of the bound peptide/ligand in the complex. Production of 13C/15N-cIp peptide was essential for this study as information obtained from standard filtered experiments produced very limited information, suggesting few close hydrophobic contacts, which would contribute to NOE cross-peaks, occur between the protein and the peptide in the binary complex (data not shown). Edited experiments of 13C/15N-cIp complexed with unlabeled cCTnC allowed for complete chemical shift assignment of cIp atoms in the bound binary complex. Three samples were used in this study to obtain NMR structural restraint data for the binary complex: 13C/15N-cCTnC·2Ca2+·cIp-s, cCTnC·2Ca2+·13C/ 15N-cIp-r, and 13C/15N-cCTnC·2Ca2+·13C/15N-cIp-r. Distance restraints for cCTnC and cIp-r in the complex were obtained by analyzing three-dimensional 15N-, 13C-, and/or 13C/15N-edited NOESY experiments. Dihedral angle restraints for cCTnC and cIp-r in the complex were obtained from three-dimensional HNHA experiments and 15N-edited NOESY experiments. Sample 13C/15N-cCTnC·2Ca2+·13C/15N-cIp-r was used for assignment of symmetrical peaks in the 13C-edited NOESY experiment for unambiguous protein-peptide side chain NOE cross-peak assignments.
A total of 1313 experimental distance restraints were obtained for the
complex and used to calculate the high resolution solution structure of the
complex of cCTnC·2Ca2+·cIp: 1046
intramolecular NOE distance restraints for cCTnC (15 restraints per
residue), 267 intramolecular NOE distance restraints for cIp (
13
restraints per residue), 23 intermolecular NOE distance restraints between
cCTnC and cIp, 29 dihedral restraints for cCTnC, and 12 cCTnC distance
restraints to Ca2+.
Fig. 2A depicts the
stereo view ensemble of the 30 lowest energy structures of the complex with
superimposition of the backbone heavy atoms of cCTnC-(95155). The
overall conformational energies and structural statistics for the ensemble are
provided in Table II. A ribbon
diagram of the lowest energy structure of the ensemble is provided in
Fig. 2B, and a surface
map depicting the perturbation of amide resonances of cCTnC upon cIp binding
is provided in Fig.
2C. The structures of cCTnC in the ensemble are of higher
quality than those of cIp, as a result of more restraints per residue for the
protein than for the peptide. The majority of assigned cIp intramolecular NOEs
in the complex are of intraresidue origin, with no long range (i
j
5) NOEs observed for cIp.
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Structure of cCTnC in the ComplexThe overall fold of cCTnC
in the complex resembles other Ca2+-bound domains in the
EF-hand family, such as the C-domain of skeletal TnC
(53). The secondary structure
elements of cCTnC in the complex are identical to unbound
cCTnC·2Ca2+
(7), displaying four well
defined helices (helices EH) and two well defined anti-parallel
-sheets. This helix-loop-helix structural motif is common in many
Ca2+-binding proteins (Ca2+ atoms
are not shown in Fig. 2). For
the 30 calculated lowest energy structures, the four helices superimpose with
individual backbone r.m.s.d. values of 0.34 ± 0.10 Å for helix E
(residues 92105), 0.30 ± 0.07 Å for helix F (residues
114123), 0.30 ± 0.13 Å for helix G (residues
130140), and 0.27 ± 0.10 Å for helix H (residues
150160). The two anti-parallel
-sheets (residues 111113
and 147149) superimpose with a backbone r.m.s.d. value of 0.09 ±
0.04 Å. The N-terminal residues (residues 8991) are not as well
defined, yet unexpectedly the C-terminal residues (residues 159161) are
well ordered when compared with the unbound structure
(7). This ordering of the
C-terminal residues is due to NOE contacts with cIp in the complex, with
contacts between cIp-Phe138 and cCTnC-Val160 minimizing
the flexibility of the C-terminal residues, yielding a more ordered H-helix
near residues 159161.
EF-hand structures can be described as "opened or closed," based upon the interhelical angles that the E-F and G-H-helices make with respect to one another. A comparison of interhelical angles of cCTnC in the binary complex with previously solved EF-hand structures is presented in Table III. The binding of cSp to calcium-saturated cNTnC reveals a conformational opening of over 20 degrees, yet binding of cIp to calcium-saturated cCTnC reveals only a slight conformational opening. Comparison with the x-ray structure of sTnC·2Ca2+·sTnI-(147) reveals identical interhelical angles of the G-H-helices and a slight closing of the E-F-helix. Thus it is shown that unlike the N-domain of cTnC, the C-domain is unable to undergo large conformational opening/closing upon target peptide binding.
Structure of cIp in the ComplexThe structure of cCTnC· 2Ca2+·cIp is presented in Fig. 2. The structure of cIp in the binary complex displays a region of helical content in the central region of the peptide, flanked by a non-helical structured region on the N termini and an unstructured random coil region on the C termini. The overall fold of cIp in complex with cCTnC is novel, possessing no structural similarities to known Ca2+-binding protein/peptide interactions. cIp runs anti-parallel to cCTnC in the complex, with residues Ile131Asp133 making contacts with the hydrophobic cleft of cCTnC. Residues Ile131Asp133 possess no definitive helical secondary structure, yet superimposition of the 30 lowest energy (Fig. 2A) structures reveals a structured region. This structured region makes a near 90° turn along the hydrophobic cleft of cCTnC to begin the helical region beginning at residue Leu134. This abrupt turn in the sequence is evidenced by multiple side chain inter-peptide NOE contacts of Ile131 to Leu134 and Arg135. Multiple hydrophobic NOE contacts were observed within this region, with cIp-Phe132 making contacts with cCTnC-Thr127/Ile128/Ile133 (linker region of cCTnC). Residues Leu134Lys139 adopt a helical secondary structure, with NOE contacts of cIp-Phe138 making hydrophobic contacts with cCTnC-Val160/Leu100 (E- and H-helices), with E-helix contacts closely matching those predicted by Cachia et al. (54) for the inhibitory region. Checking of the helical region spanning residues Leu134Lys139 by the program Procheck (49) for the 30 calculated lowest energy structures reveals various violations of the backbone dihedral angles in comparison with accepted Ramachandran values, indicating a non-ideal helix. The structure of the inhibitory region could change in the troponin complex containing full-length TnC, TnI, and TnT, wherein additional stabilizing protein-protein interactions may be present. For the 30 calculated lowest energy structures, the central helix of cIp superimposes with individual backbone r.m.s.d. values of 0.45 ± 0.12 Å for residues Leu134Lys139. Residues Arg140Arg147 comprising the C-terminal region of cIp-r display no evidence of a well ordered secondary structure. Superimposition of the 30 calculated lowest energy structures of the binary complex (Fig. 2A) reveals this region as very labile in solution. No long range peptide-peptide NOE contacts were observed for this region, with only i, i + 1 contacts observed between residues, and no protein-peptide NOE contacts were observed for this region. Arg147 begins the segment of the "switch peptide" (cSp, residues 147163), which has been shown to start a helical secondary structure when complexed with cNTnC·Ca2+ (16, 18, 24). Arg147 makes multiple contacts with cNTnC and thus is not expected to make interactions with cCTnC in the complex.
Fig. 2C displays the chemical shift surface map of cCTnC upon cIp binding, which we have reported previously (42). Changes in the two-dimensional {1H, 15N}-HSQC spectra of 15N-cCTnC were monitored during a titration of cIp (Fig. 1A) and were mapped on the surface of the binary complex of cCTnC·2Ca2+·cIp. No chemical shifts were measured for Lys90Ser93 due to exchange with water and are thus reported as no change in chemical shift and are colored white. The regions of greatest chemical shift change are shown in red, with the largest chemical shift perturbations of cCTnC upon the binding surface area of cIp, within the linker region and the E- and H-helices. A large chemical shift was observed for the linker region, as cIp-Phe132 is within close proximity to the linker region, with aromatic ring effects predicted to be responsible for the large shifts observed.
Chemical shift values for 13C and
1H
nuclei are important indicators for predicting
secondary structure
(5557).
NMR studies have shown that upon the initiation of helical secondary structure
formation, there is a downfield shift observed for
13C
and an upfield shift observed for
1H
(thus
(ppm) =
bound
free). Incorporation of
13C label in cIp allows for measurement of both
13C
and 1H
chemical
shifts of free and bound peptide. cIp undergoes
13C
chemical shift perturbation toward a more
helical conformation (downfield shift (
bound
free) > 0) in the region of residues
Leu134Lys137, with significant shift (>1 ppm)
for residue Arg135. Residues
Ile131Asp133 experience small upfield changes in
13C
chemical shift upon cCTnC binding,
corresponding to the N-terminal region before the helical region of
Leu134Lys139. 13C
chemical shift changes for residues Arg140Arg147
indicate that this region possesses no helical characteristics, corresponding
to the flexibility of this region as shown in
Fig. 2A.
1H
perturbation measurements show minimal changes
for all residues for cIp-r upon cCTnC binding, with only residues
Gln129, Asp133, Arg135, Arg145,
and Arg147 undergoing changes greater than 0.05 ppm. For the
helical region Leu134Lys139, residues
Leu134 and Arg135 undergo an upfield shift, with all
others undergoing minimal downfield shifts less than 0.05 ppm. Comparison with
induced 1H
chemical shift changes of the skeletal
inhibitory peptide isoform (sIp) upon binding to sTnC as reported by Hernandez
et al. (36) yields
similar results of no changes greater than 0.1 ppm for the majority of
residues, with Arg135 undergoing the largest upfield chemical shift
for both the cardiac and skeletal isoforms upon TnC binding. cIp and sIp share
a conserved sequence with only a mutation of Thr142 in cIp to that
of a Pro residue in sIp, thus it is predicted that both cIp and sIp bind to
TnC in a similar manner.
The electrostatic surface map of the binary complex cCTnC· 2Ca2+·cIp and its components are shown in Fig. 3. The highly basic cIp peptide has the potential for many favorable electrostatic interactions with the highly acidic cCTnC. At physiological conditions, pH 6.7, cIp (pI = 11.0) will have an overall charge of 6.9, whereas cCTnC (pI = 4.1) will possess an overall charge of 14.8 (58). It is supposed that the highly basic C-terminal end of cIp (Arg140, Arg144, Arg145, and Arg147) will make beneficial interactions with the acidic E-helix of cCTnC (Glu94, Glu95, and Glu96), thereby stabilizing the binary complex (54). As we have shown previously, mutation of R145G of cIp results in a 4-fold reduction of binding affinity with cCTnC when compared with wild type cIp. The resulting mutation resulted in a change of a basic residue for one that is uncharged, thereby effectively reducing the charge on cIp-R145G and thus affecting binding. Similar results were seen for phosphorylation of Thr142 of cIp (42).
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Backbone Amide 15N Relaxation Studies of cCTnCBackbone amide 15N NMR relaxation data for 15N-cCTnC·2Ca2+ and 15N-cCTnC·2Ca2+·cIp were obtained at 500 and 600 MHz (Fig. 4). Backbone resonances for Met90Lys92 were not observed due to rapid amide proton exchange with water for both samples. Resonances Met131, Lys137, and Arg147 were not included in 15N-cCTnC·2Ca2+·cIp data, and resonances Ala99 and Arg147 were not included in 15N-cCTnC·2Ca2+, due to partial peak overlap in the {1H, 15N}-HSQC spectra.
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The T1, T2, and NOE values of 15N-cCTnC·2Ca2+ are shown in Fig. 4A. The measured values at 500 MHz for 15N-cCTnC·2Ca2+ show a decrease in T1, a decrease in T2, and an increase in NOE values for the first 5 N-terminal residues on the E-helix when compared with calcium-saturated skeletal C-domain isoform (sCTnC), indicating that the N-terminal residues on the E-helix of cCTnC are more ordered when compared with the skeletal isoform (59), as well as small differences in calcium-binding sites III and IV. Residues whose internal motions affect their measured relaxation values were excluded from the calculation of the averages, as determined using NOE criteria (NOE500 > 0.6 and NOE600 > 0.65). As expected, the average T1600/T1500 ratio is >1; the ratio of T2600/ T2500 is approximately equal to 1, and the average NOE600/ NOE500 ratio is approximately equal to 1 for the majority of the residues studied. These ratios are within accepted theoretical calculations, taking into account effects from dipole-dipole relaxation and chemical shift anisotropy at the two magnetic field strengths studied.
The relaxation values for the 8.2-kDa domain of 15N-cCTnC·2Ca2+ upon the addition of the 2.5-kDa cIp peptide (Fig. 4B) are consistent with calculated values for a 10.7-kDa binary complex. There is an increase for T1 values and a decrease for T2 values, with NOE values remaining largely unchanged when compared with cCTnC·2Ca2+. The linker region and the H-helix of cCTnC display the largest changes in relaxation values upon cIp binding, indicating that this region is becoming more ordered upon cIp binding. These results correspond well with the structure of the binary complex of 15N-cCTnC· 2Ca2+·cIp, as multiple NOE contacts are observed between cIp and cCTnC·2Ca2+ in the linker region and the H-helix.
15N NMR Relaxation Studies of 15N-cIpBackbone amide 15N NMR relaxation data for 15N-cIp-r and cCTnC·2Ca2+·15N-cIp-r were obtained at 500 and 600 MHz (Fig. 5). Backbone resonances for Thr128 and Gln129 were not observed due to rapid amide proton exchange with water for both samples. Residue Pro141 was not observed due to the absence of a residual amide proton. The T1, T2, and NOE values observed at the two frequencies are typical for an unstructured 2.5-kDa domain in solution. Addition of cCTnC·2Ca2+ produces large effects on the relaxation rates of all residues present on cIp. As expected for an increase in total molecular mass to 10.7 kDa (assuming a 1:1 ratio), decreases of both T1 and T2 values are observed at both magnetic field strengths, along with a large increase in the recorded values for NOE. Interpretation of the data reveals that cIp is becoming increasingly rigid upon cCTnC binding.
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Comparison of the total molecular weights of both
15N-cCTnC·2Ca2+·cIp and
cCTnC·2Ca2+·15N-cIp-r indicates
that both systems should produce equivalent experimental correlation times
(C), assuming isotropic tumbling. Predicted
C values for a 1:1 binary complex of
15N-cCTnC·2Ca2+·cIp and
cCTnC·2Ca2+·15N-cIp-r are 5.5
and 5.5 ns, respectively, whereas experimentally measured values of 5.8 and
5.7 ns are observed, enforcing that the binary complex of cCTnC and cIp is
binding in a 1:1 ratio. The measured T2 values of
15N-cCTnC· 2Ca2+·cIp mirror the
values measured for
cCTnC·2Ca2+·15N-cIp-r (
120
ms) also supporting a 1:1 binary complex, as well implying that cIp is
becoming increasingly rigid upon cCTnC binding even though the structure of
cIp in the binary complex is not as well defined as cCTnC.
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DISCUSSION |
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Within the cardiac system, the calcium-binding sites in the C-domain of TnC are believed to always be occupied by Ca2+ (65), and the C-domain is thus designated the structural domain. The N-domain of TnC is believed to be the regulatory domain, undergoing large structural changes upon the onset of Ca2+-induced contraction. In the resting state the Ca2+-binding site in the N-domain of TnC is unoccupied. The ability of the N-domain to sense the Ca2+ signal in order to propagate muscle contraction has led to the widely accepted designation as the molecular switch for muscle contraction. The classification of the N and C domains of TnC as the regulatory and structural domains are, however, somewhat arbitrary as physiological roles of the two domains may vary within intact muscle fibers. The C domain of TnC has been shown to interact with specific regions of both TnT and TnI, with a high resolution structure presented by Vassylyev et al. (21), showing interactions of sCTnC with an N-terminal fragment of TnI-(147), revealing a helical structure of the TnI fragment that interacts with the hydrophobic cleft of the C-domain. Recent functional studies (32) have required researchers to re-evaluate the role of the C domain as one that actively participates in the muscle contraction signaling process. Following Ca2+ binding to NTnC, the inhibitory region of TnI-(128147) (cardiac isoform) is believed to relocate from its binding partner actin to TnC on the thin filament. This reorganization of the inhibitory region allows for myosin binding to actin, which allows for completion of the power stroke during muscle contraction. The binding of cIp to cTnC has been shown to have interactions with the C domain, as well as with the central linker region between the D- and E-helices (16).
Early NMR work by Campbell and co-workers
(33,
34) using transferred NOEs in
the skeletal system (sTnI-(104115)) predicted the inhibitory region to
comprise two turns of a helix, surrounding two central proline residues, and
subsequent modeling of a binary complex docked the bound inhibitory region
within the hydrophobic core of sCTnC
(35). The structure of a
N-terminal regulatory peptide Rp40 in complex with sTnC presented by Vassylyev
et al. (21)
conflicted with this model of the inhibitory region, as Rp40 was shown to bind
to the hydrophobic core of sCTnC. Vassylyev et al.
(21) have proposed a model of
the troponin complex with the inhibitory region having a helical secondary
structure, away from the hydrophobic cleft of sCTnC that binds Rp40. NMR
chemical shift mapping data presented by Mercier et al.
(22) agreed with results by
Vassylyev et al. (21)
that Rp40 binds within the hydrophobic cleft of sCTnC, yet predicted that the
inhibitory region may bind across the top of the hydrophobic patch within the
ternary troponin complex. Hernandez et al.
(36) challenged the idea of a
helical inhibitory region and suggested that the inhibitory region adopts an
extended conformation in the binary complex, with a structural model presented
by Tung et al. (37)
predicting the inhibitory region to possess a -hairpin secondary
structure, away from the hydrophobic cleft of sCTnC.
The purpose of this study was to explore and solve the NMR solution structure of cIp when bound to the troponin complex. We have shown previously that cIp binds to the C-domain of cTnC, with large chemical shift perturbations in the {1H, 15N}-HSQC spectrum (30, 42). Considering these data, and to minimize the total molecular weight of the complex for ease of assignment in the NMR spectrum, it was decided to pursue the structure of cIp in complex with cCTnC·2Ca2+. By using isotope labeling strategies of recombinant peptide production as a cost-effective alternative to synthetic peptide labeling (see under "Experimental Procedures"), we have been successful in elucidating the structure of the binary complex of cCTnC·2Ca2+·cIp, which is presented in Fig. 2. In the binary complex, cIp adopts a helical conformation, making NOE contacts with the linker region of cCTnC, as well as with both the E- and H-helices. Residues Leu134Lys139 adopt a helical arrangement within the binary complex, with residues Arg140Arg147 adopting an extended conformation, void of any secondary structure elements.
Reflection on the structure of the binary complex reveals that all previous
predicted models are in part incorrect. The inhibitory region has no
-sheet secondary structure present, as is indicated by the presence of
traditional helical NOEs (i.e. d
(i, i +
3) and d
N(i, i + 3)) and by chemical shift
data for 1H
and 13C
atoms in the protein backbone. No long range NOE (i
j
5) contacts are observed, which would be predicted if the
inhibitory region possessed
-sheet characteristics. The model predicted
by Vassylyev et al.
(21) correctly predicted a
helical region of cIp, yet positioning of the helix in reference to cTnC was
incorrect. The model by Campbell et al.
(33) predicted a helical
orientation of sTnI-(104115), which we have shown to be unstructured in
the cardiac isoform. Our results correlate well with recent results published
by Brown et al. (38)
in which the structure of the inhibitory region was proposed by spin labeling
EPR. Within this study it was proposed that the inhibitory region in a ternary
troponin complex possesses a helical region from residues
Gln129Lys137, with residues
Phe138Arg145 showing no secondary structure
elements, which is in good agreement with the preliminary ternary troponin
structure by Takeda et al.
(41). As well, it was
predicted that residues Lys130Arg135 are
important in making contacts with TnT, which are immediately N-terminal to the
region which comprises the 1.5 turns of the helix presented in
Fig. 2.
It is observed that numerous stabilizing electrostatic interactions occur
between the acidic C domain and the highly basic inhibitory region, as there
is only 23 cCTnC·cIp NOEs in the binary complex. Electrostatic NOE
contacts are traditionally difficult to measure by NMR, due to proton exchange
and NOE distance limitations (<5 Å). The probability of stabilizing
electrostatic interactions within the complex is high, as inspection of the
structure yields close association of basic/acidic residue overlap, with the
potential of the unstructured region containing residues Arg140,
Arg144, Arg145, and Arg147 in cIp to interact
with the acidic residues Glu94, Glu95, and
Glu96 present on the E-helix
(Fig. 3). Recent work by Tripet
et al. (39) has shown
a decrease in the affinity of cIp to cTnC with increasing concentrations of
KCl, suggesting that electrostatics play a part in binding. Previous work from
our laboratory has shown that mutation R145G and phosphorylation of
Thr142 diminishes the binding affinity of cIp for cCTnC by 4-
and
14-fold (42),
respectively. The mutation and phosphorylation events of cIp effectively
change the charge of the residue side chain by 1, which diminishes
electrostatic interactions when compared with the wild type inhibitory region.
In this regard, it is not surprising that the phosphorylation of
Thr142 has a large reduction of binding when compared with R145G,
as Thr142 is in much closer proximity to cCTnC in the presented
structure.
The structure of the inhibitory region may be altered within the full-length TnC·TnT·TnI troponin complex when compared with the presented binary complex of cCTnC·cIp, wherein additional interacting subunits may play a factor in ternary assembly. Comparison of the structure of cCTnC·2Ca2+·cIp with the structure of sCTnC·2Ca2+ in complex with the N-terminal domain of sTnI-(147) presented by Vassylyev et al. (21) reveals that there would be steric clashes between the two cTnI subunits when bound to the C-domain of TnC. Specifically, there is steric overlap of the N-terminal residues of cIp with Rp40 in the hydrophobic cleft of the C-domain. Previously, it has been shown that the regulatory peptide Rp40 will displace sIp in a titration with sCTnC·2Ca2+ (22), implying that the Rp40 is always bound to sCTnC·2Ca2+ during both muscle relaxation and during contraction. Thus a rationale must be presented for validation of the presented structure. There are notable differences in primary sequence between the cardiac and skeletal isoforms, specifically large differences in the regulatory region of TnI (cTnI-(3279) and sTnI-(147)), thus the regulatory peptide might have altered binding in the cardiac system. As well, EPR results from Brown et al. (38) have shown cIp to be in contact with cTnT, as well as cTnC. The observed binary structure (Fig. 2) may of course be altered somewhat in the context of the intact troponin complex. In particular, a section of cTnI-(128139) has been predicted to be involved in a coiled-coil with TnT (66). The residues of steric clash of superimposition of sTnC·Rp40 and cCTnC·cIp are those of Thr128Arg135, of which Brown et al. (38) have shown Lys130Arg135 to be in contact with TnT. As well, preliminary data from the 2.6 Å x-ray structure by Takeda et al. (41) of the complex of cTnC·cTnI-(34160)·cTnT-(181288) revealed a coiled-coil of TnT with Thr128Lys137 of cIp, with an undefined region spanning Phe138Arg147, implying this region as unstructured or flexible.
It is possible that Thr128Lys139 of cIp makes a coiled-coil interaction with cTnT, with Arg135Lys139 of cIp making contacts with the E- and H-helices of cCTnC up to Lys139 with an unstructured region of Arg140Arg147, followed by cSp which has been shown to have interactions with cNTnC. The region Arg140Arg147 makes stabilizing electrostatic interactions with the acidic E-helix and the linker region of cTnC, where cSp begins. As well the coiled-coil interaction of cTnT to Thr128Arg135 of cIp will bring the N-terminal residues of cIp out of the hydrophobic cleft of cCTnC, allowing the regulatory peptide Rp40 to bind. Supporting this, in the binary complex presented (Fig. 2) Leu134 was found to possess no NOE contacts to cCTnC. In this model, modulation of the interaction between cSp and cNTnC during intracellular calcium efflux is sufficient to excise cIp from its binding site on actin to that of cCTnC, thereby releasing the inhibition of heart contraction.
We have been successful in elucidating the NMR solution structure of the binary complex of cCTnC·2Ca2+·cIp, which reveals a helical conformation of the inhibitory region. The helical region of cIp (Leu134Lys139) binds to cCTnC in a novel orientation, with contacts to regions of the E- and H-helices and the linker region of cCTnC. The structure further reveals the potentiality of stabilizing electrostatic interactions in the binary complex. Coupled with 15N NMR relaxation data, we have shown that the binary complex forms a 1:1 complex in solution. Further studies on the ternary complex are encouraged as the interactions predicted between TnC·TnI·TnT might currently be too simplistic.
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FOOTNOTES |
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* This work was supported in part by the Canadian Institutes of Health
Research and the Heart and Stroke Foundation of Canada. The costs of
publication of this article were defrayed in part by the payment of page
charges. This 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.: 780-492-5460; Fax:
780-492-0886; E-mail:
Brian.Sykes{at}ualberta.ca.
1 The abbreviations used are: TnC, troponin C; TnI, troponin I; TnT, troponin
T; cIp-s, synthetic cIp peptide acetyl-TQKIFDLRGKFKRPTLRRVR-amide (residues
128147); cIp-r, recombinant cIp peptide
produced from fusion protein construct (residues 128147); hSer,
homoserine/homoserine-lactone residue found on C termini of cIp-r after
cyanogen bromide cleavage; Rp40, skeletal TnI regulatory peptide residues
147; cTnC, recombinant cardiac troponin C residues 1161; cCTnC,
recombinant C-domain cardiac troponin C residues 90161; cNTnC,
recombinant N-domain cardiac troponin C residues 189; sTnC, recombinant
skeletal troponin C residues 1162; sCTnC, recombinant C-domain skeletal
troponin C residues 89162; NOE, nuclear Overhauser effect; NOESY, NOE
spectroscopy; HSQC, heteronuclear single quantum coherence; r.m.s.d., root
mean squared deviation.
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ACKNOWLEDGMENTS |
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REFERENCES |
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