Structure and Dynamics of the C-domain of Human Cardiac Troponin C in Complex with the Inhibitory Region of Human Cardiac Troponin I*

Darrin A. Lindhout {ddagger} and Brian D. Sykes §

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.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cardiac troponin C is the Ca2+-dependent switch for heart muscle contraction. Troponin C is associated with various other proteins including troponin I and troponin T. The interaction between the subunits within the troponin complex is of critical importance in understanding contractility. Following a Ca2+ signal to begin contraction, the inhibitory region of troponin I comprising residues Thr128–Arg147 relocates from its binding surface on actin to troponin C, triggering movement of troponin-tropomyosin within the thin filament and thereby freeing actin-binding site(s) for interactions with the myosin ATPase of the thick filament to generate the power stroke. The structure of calcium-saturated cardiac troponin C (C-domain) in complex with the inhibitory region of troponin I was determined using multinuclear and multidimensional nuclear magnetic resonance spectroscopy. The structure of this complex reveals that the inhibitory region adopts a helical conformation spanning residues Leu134–Lys139, with a novel orientation between the E- and H-helices of troponin C, which is largely stabilized by electrostatic interactions. By using isotope labeling, we have studied the dynamics of the protein and peptide in the binary complex. The structure of this inhibited complex provides a framework for understanding into interactions within the troponin complex upon heart contraction.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The binding of Ca2+ to the troponin complex initiates cardiac muscle contraction (15). The troponin complex is composed of three subunits: troponin C (TnC),1 troponin I (TnI), and troponin T (TnT) (6). The three subunits of the troponin complex are necessary for Ca2+-induced regulation of cardiac muscle contractility. TnC, the Ca2+-sensitive component of the complex, is a member of the EF-hand family of Ca2+-binding proteins and contains two high affinity Ca2+/Mg2+-binding sites (sites III and IV) in the C-terminal domain and one low affinity Ca2+-binding site (site II) in the N-terminal domain (79). At physiological conditions during muscle relaxation, the two C-terminal Ca2+/Mg2+-binding sites are occupied, and the N-terminal Ca2+-binding site is unoccupied. During the onset of muscle contraction, a transient increase in cytosolic Ca2+ concentrations allows the low affinity N-terminal domain to bind Ca2+, resulting in the initiation of heart contraction (10). TnI is the subunit that in the presence of tropomyosin inhibits myosin Mg2+-ATPase activity. TnI inhibition is removed by the binding of Ca2+ to the N-terminal domain of the TnC subunit (11). TnT is the subunit that binds tropomyosin, TnC, and TnI, anchoring the troponin complex to the thin filament and aids in the propagation of Ca2+-induced conformational changes (12, 13).

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-(147–163) (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 Thr128–Arg147 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-(104–115) (cTnI-(136–147)), 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-(1–47) (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 {beta}-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-(129–137)) displays a helical profile, with the C-terminal residues 139–145 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-(198–298) 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-(128–147)) in complex with the Ca2+-saturated C-terminal domain of TnC. The inhibitory region displays a helical secondary structure from residues Leu134–Lys139, 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.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of cCTnC Protein—The engineering of the expression vector for the cCTnC-(90–161) protein was as described by Chandra et al. (43). The expression and purification of cCTnC, 15N-cCTnC, and 13C/15N-cCTnC proteins in Escherichia coli followed the procedure as described previously (44) for sNTnC. CCTnC-labeled proteins were further purified using a gravity flow Superdex 75 column (Amersham Biosciences) as described previously (42) for cCTnC.

Preparation of cIp Protein—cIp-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 cIp—The 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 Complex—Three 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.


View this table:
[in this window]
[in a new window]
 
TABLE I
NMR spectra acquired and experimental conditions used to obtain assignments and NOE restraints

 

NMR Spectroscopy—All 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 Calibration—All 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+·cIp—NOE 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, {varphi} 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.


View this table:
[in this window]
[in a new window]
 
TABLE II
Structural statistics of the family of the 30 structures calculated

 

View this table:
[in this window]
[in a new window]
 
TABLE III
Interhelical angles of various EF hands

 

Backbone Amide 15N Relaxation Measurements of cCTnC·2Ca2+·cIp Complex—Two 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).


View this table:
[in this window]
[in a new window]
 
TABLE IV
Backbone amide 15N relaxation measurements

 

Coordinates—The coordinates for the structure of cCTnC·2Ca2+·cIp have been deposited in the Protein Data Bank (code 10ZS).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Titration of cCTnC·2Ca2+ with cIp—The titration of 15N-cCTnC·2Ca2+ with cIp-s, as monitored by two-dimensional {1H, 15N}-HSQC NMR spectral changes, is shown in Fig. 1A.We also performed the reverse titration of 15N-cIp-r titrated with cCTnC to monitor chemical shift changes in cIp-r, as shown in Fig. 1B. The two-dimensional {1H, 15N}-HSQC NMR spectra of both cCTnC·2Ca2+ and cIp-r have been completely assigned and were used as starting points to monitor protein-peptide chemical shift changes. All of the chemical shift changes for both titrations fall into the fast exchange limit on the NMR time scale, so that each cross-peak corresponds to the weighted average of the bound and free chemical shifts. Linear movement of the cross-peaks indicates that only two species exist in solution; also the binding of cCTnC and cIp occurs in a 1:1 stoichiometry, as described previously (42).



View larger version (18K):
[in this window]
[in a new window]
 
FIG. 1.
Two-dimensional {1H, 15N}-HSQC NMR spectra arising from the backbone NH and side chain NH2 groups of 15N-cCTnC·2Ca2+ (A) and 15N-cIp (•) (B) at various concentrations of cIp (as adapted from Lindhout et al. (42)) (A) and cCTnC are superimposed ({circ}) (B), showing the progressive shifts of peaks. The direction of chemical shift changes for some residues undergoing large perturbations are indicated by an arrow. Conditions are described under "Experimental Procedures."

 

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,

(Eq. 1)
which yielded a dissociation constant (KD) of 31 ± 11 µM for 15N-cCTnC·2Ca2+ titrated with cIp-s, as reported previously (42), which is in the same order as the equivalent titration for the skeletal isoform (42 ± 7 µM) which our laboratory has reported previously (22). The titration of 15N-cIp-r with cCTnC is expected to produce the same KD; however, when solid cCTnC was added to the NMR tube containing 15N-cIp-r, some white precipitate was observed after the initial addition of cCTnC, and the level of precipitate increased as increasing levels of cCTnC were added; thus an accurate KD value was not obtained for this titration.

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 Complex—The 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-(95–155). 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 (ij >= 5) NOEs observed for cIp.



View larger version (56K):
[in this window]
[in a new window]
 
FIG. 2.
A, stereo ensemble of the 30 lowest energy structures of cCTnC·2Ca2+ (blue) in complex with the inhibitory region (red) of cardiac troponin I-(128–147) (cIp), superimposition of cCTnC (Glu95–Glu155) backbone. Residues Ile131–Lys139 are well ordered, whereas residues Arg140–Arg147 are flexible in the binary complex. B, ribbon diagram of the binary complex cCTnC·2Ca2+·cIp. The helical region of cIp encompasses residues Leu134–Lys139 (red) completing 1.5 turns of a helix is colored in red. N and C termini of each protein in the binary complex are as indicated, and all helices of cCTnC are defined. C, chemical shift surface map of cCTnC·2Ca2+·cIp as based upon the titration performed in Fig. 1A. Chemical shift changes are measured as {Delta}{delta}(ppm) = ({Delta}{delta}N2 + {Delta}{delta}H2)1/2, with areas of large chemical shift perturbation shown by the colored gradient. cIp peptide backbone is colored blue, with all heavy side chain atoms colored green. Stereo ensemble and ribbon diagram were generated using DINO (DINO: Visualizing Structural Biology (2002) www.dino3d.org), and the chemical shift surface map was generated using Insight II version 98 (Accelrys Inc.).

 

Structure of cCTnC in the Complex—The 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 E–H) and two well defined anti-parallel {beta}-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 92–105), 0.30 ± 0.07 Å for helix F (residues 114–123), 0.30 ± 0.13 Å for helix G (residues 130–140), and 0.27 ± 0.10 Å for helix H (residues 150–160). The two anti-parallel {beta}-sheets (residues 111–113 and 147–149) superimpose with a backbone r.m.s.d. value of 0.09 ± 0.04 Å. The N-terminal residues (residues 89–91) are not as well defined, yet unexpectedly the C-terminal residues (residues 159–161) 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 159–161.

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-(1–47) 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 Complex—The 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 Ile131–Asp133 making contacts with the hydrophobic cleft of cCTnC. Residues Ile131–Asp133 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 Leu134–Lys139 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 Leu134–Lys139 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 Leu134–Lys139. Residues Arg140–Arg147 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 147–163), 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 Lys90–Ser93 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{alpha} and 1H{alpha} 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{alpha} and an upfield shift observed for 1H{alpha} (thus {Delta}{delta}(ppm) = {delta}bound {delta}free). Incorporation of 13C label in cIp allows for measurement of both 13C{alpha} and 1H{alpha} chemical shifts of free and bound peptide. cIp undergoes 13C{alpha} chemical shift perturbation toward a more helical conformation (downfield shift ({delta}bound{delta}free) > 0) in the region of residues Leu134–Lys137, with significant shift (>1 ppm) for residue Arg135. Residues Ile131–Asp133 experience small upfield changes in 13C{alpha} chemical shift upon cCTnC binding, corresponding to the N-terminal region before the helical region of Leu134–Lys139. 13C{alpha} chemical shift changes for residues Arg140–Arg147 indicate that this region possesses no helical characteristics, corresponding to the flexibility of this region as shown in Fig. 2A. 1H{alpha} 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 Leu134–Lys139, residues Leu134 and Arg135 undergo an upfield shift, with all others undergoing minimal downfield shifts less than 0.05 ppm. Comparison with induced 1H{alpha} 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).



View larger version (32K):
[in this window]
[in a new window]
 
FIG. 3.
A, electrostatic surface map of binary complex cCTnC·2Ca2+·cIp with acidic regions colored red and basic regions colored blue. Ribbon diagram of domain orientation is as shown. Selected residues within the complex are as labeled. B, 90 °C rotation of complex about the y axis. Surface maps generated using the programs GRASP (67) and RASTER3D (68). Ribbon diagrams generated using the program Insight II version 98 (Accelrys Inc.).

 

Backbone Amide 15N Relaxation Studies of cCTnC—Backbone 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 Met90–Lys92 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.



View larger version (31K):
[in this window]
[in a new window]
 
FIG. 4.
15N NMR relaxation values of 15N-cCTnC for unbound (A) and 15N-cCTnC·2Ca2+·cIp binary complex (B) measured at 500 MHz ({circ}) and 600 MHz ({diamond}). T1 and T2 values are reported in milliseconds, and NOE values are a composite of the ratio of the NOE in the absence/presence of proton pre-saturation (see "Experimental Procedures").

 

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-cIp—Backbone 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.



View larger version (28K):
[in this window]
[in a new window]
 
FIG. 5.
15N NMR relaxation values of 15N-cIp unbound at 500 MHz ({square}), 15N-cIp unbound at 600 MHz ({blacktriangleup}), 15N-cIp bound to cCTnC·2Ca2+ at 500 MHz ({diamond}), and 15N-cIp bound to cCTnC·2Ca2+ at 600 MHz ({blacktriangledown}). T1 and T2 values are reported in milliseconds, and NOE values are a composite of the ratio of the NOE in the absence/ presence of proton pre-saturation (see "Experimental Procedures").

 

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 ({tau}C), assuming isotropic tumbling. Predicted {tau}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.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The general mechanism of contraction involving temporal interactions of the thin and thick filaments in muscle fibers upon a Ca2+ signaling cascade is now quite well understood (15). However, no high resolution structures of the troponin complex are available to date. In the last few years, successful determination of various structures of both cardiac and skeletal TnC isoforms in various states and in complex with various TnI segments have become available (7, 1619, 21, 24, 41, 53), yielding insights into the molecular interactions of muscle contraction. Coupled with these studies has been the advent of backbone and side chain NMR relaxation data (59, 62, 63), which have provided details into the dynamics and energetics of peptide and Ca2+ binding to TnC. Several research groups (21, 32, 37, 64) have proposed structural models of the TnC·TnI·TnT ternary complex, involving specific interactions of the domains during muscle contraction.

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-(1–47), 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-(128–147) (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-(104–115)) 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 {beta}-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 Leu134–Lys139 adopt a helical arrangement within the binary complex, with residues Arg140–Arg147 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 {beta}-sheet secondary structure present, as is indicated by the presence of traditional helical NOEs (i.e. d{alpha}{beta}(i, i + 3) and d{alpha}N(i, i + 3)) and by chemical shift data for 1H{alpha} and 13C{alpha} atoms in the protein backbone. No long range NOE (i j >= 5) contacts are observed, which would be predicted if the inhibitory region possessed {beta}-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-(104–115), 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 Gln129–Lys137, with residues Phe138–Arg145 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 Lys130–Arg135 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-(1–47) 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-(32–79) and sTnI-(1–47)), 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-(128–139) 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 Thr128–Arg135, of which Brown et al. (38) have shown Lys130–Arg135 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-(34–160)·cTnT-(181–288) revealed a coiled-coil of TnT with Thr128–Lys137 of cIp, with an undefined region spanning Phe138–Arg147, implying this region as unstructured or flexible.

It is possible that Thr128–Lys139 of cIp makes a coiled-coil interaction with cTnT, with Arg135–Lys139 of cIp making contacts with the E- and H-helices of cCTnC up to Lys139 with an unstructured region of Arg140–Arg147, followed by cSp which has been shown to have interactions with cNTnC. The region Arg140–Arg147 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 Thr128–Arg135 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 (Leu134–Lys139) 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.


    FOOTNOTES
 
The atomic coordinates and structure factors (code 1OZS) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* 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. Back

{ddagger} Supported by an Alberta Heritage Foundation for Medical Research Studentship. Back

§ 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 128–147); cIp-r, recombinant cIp peptide produced from fusion protein construct (residues 128–147); hSer, homoserine/homoserine-lactone residue found on C termini of cIp-r after cyanogen bromide cleavage; Rp40, skeletal TnI regulatory peptide residues 1–47; cTnC, recombinant cardiac troponin C residues 1–161; cCTnC, recombinant C-domain cardiac troponin C residues 90–161; cNTnC, recombinant N-domain cardiac troponin C residues 1–89; sTnC, recombinant skeletal troponin C residues 1–162; sCTnC, recombinant C-domain skeletal troponin C residues 89–162; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy; HSQC, heteronuclear single quantum coherence; r.m.s.d., root mean squared deviation. Back


    ACKNOWLEDGMENTS
 
We gratefully acknowledge Dr. G. Marius Clore and co-workers (National Institutes of Health, Bethesda) for the kind gift of the GEV-1 vector and for helpful discussions. We are indebted to Drs. John Solaro and Murali Chandra (University of Illinois, Chicago) for providing the cCTnC construct and David Corson and Angela Thiessen for assistance with protein expression. We thank Paul Semchuck and Lorne Burke for assistance with high pressure liquid chromatographic purification and matrix-assisted laser desorption ionization mass spectroscopy measurements. We also thank Gerry McQuaid for spectrometer maintenance and Pascal Mercier, Leo Spyracopoulos, and Jason Maynes for helpful discussions and software troubleshooting. We are grateful to Dr. Monica Li, Xu Wang, and Dean Schieve for helpful discussions.



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Adelstein, R. S., and Eisenberg, E. (1980) Annu. Rev. Biochem. 49, 921–956[CrossRef][Medline] [Order article via Infotrieve]
  2. Leavis, P. C., and Gergely, J. (1984) CRC Crit. Rev. Biochem. 16, 235–305[Medline] [Order article via Infotrieve]
  3. Zot, A. S., and Potter, J. D. (1987) Annu. Rev. Biophys. Biophys. Chem. 16, 535–559[CrossRef][Medline] [Order article via Infotrieve]
  4. Tobacman, L. S. (1996) Annu. Rev. Physiol. 58, 447–481[CrossRef][Medline] [Order article via Infotrieve]
  5. Farah, C. S., and Reinach, F. C. (1995) FASEB J. 9, 755–767[Abstract/Free Full Text]
  6. Greaser, M. L., and Gergely, J. (1973) J. Biol. Chem. 248, 2125–2133[Abstract/Free Full Text]
  7. Sia, S. K., Li, M. X., Spyracopoulos, L., Gagné, S. M., Liu, W., Putkey, J. A., and Sykes, B. D. (1997) J. Biol. Chem. 272, 18216–18221[Abstract/Free Full Text]
  8. Herzberg, O., Moult, J., and James, M. N. G. (1986) J. Biol. Chem. 261, 2638–2644[Abstract/Free Full Text]
  9. Strynadka, N. C. J., Cherney, M., Sielecki, A. R., Li, M. X., Smillie, L. B., and James, M. N. G. (1997) J. Mol. Biol. 273, 238–255[CrossRef][Medline] [Order article via Infotrieve]
  10. Johnson, J. D., Charlton, S. C., and Potter, J. D. (1979) J. Biol. Chem. 254, 3497–3502[Medline] [Order article via Infotrieve]
  11. Farah, C. S., Miyamoto, C. A., Ramos, C. H. I., da Silva, A. C. R., Quaggio, R. B., Fujimori, K., Smillie, L. B., and Reinach, F. C. (1994) J. Biol. Chem. 269, 5230–5240[Abstract/Free Full Text]
  12. Mak, A. S., and Smillie, L. B. (1981) J. Mol. Biol. 149, 541–550[Medline] [Order article via Infotrieve]
  13. Potter, J. D., Sheng, Z., Pan, B. S., and Zhao, J. (1995) J. Biol. Chem. 270, 2557–2562[Abstract/Free Full Text]
  14. Pascal, S. M., Muhandiram, D. R., Yamazaki, T., Forman-Kay, J. D., and Kay, L. E. (1994) J. Magn. Res. 103, 197–201[CrossRef]
  15. Li, Y., Love, M. L., Putkey, J. A., and Cohen, C. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 5140–5145[Abstract/Free Full Text]
  16. Li, M. X., Spyracopoulos, L., and Sykes, B. D. (1999) Biochemistry 38, 8289–8298[CrossRef][Medline] [Order article via Infotrieve]
  17. Wang, X., Li, M. X., Spyracopoulos, L., Beier, N., Chandra, M., Solaro, R. J., and Sykes, B. D. (2001) J. Biol. Chem. 276, 25456–25466[Abstract/Free Full Text]
  18. Wang, X., Li, M. X., and Sykes, B. D. (2002) J. Biol. Chem. 277, 31124–31133[Abstract/Free Full Text]
  19. Dvoretsky, A., Abusamhadneh, E. M., Howarth, J. W., and Rosevear, P. R. (2002) J. Biol. Chem. 277, 38565–38570[Abstract/Free Full Text]
  20. Kay, L. E., Xu, G. Y., Singer, A. U., Muhandiram, D. R., and Forman-Kay, J. D. (1993) J. Magn. Res. 101, 333–337
  21. Vassylyev, D. G., Takeda, S., Wakatsuki, S., Maeda, K., and Maeda, Y. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 4847–4852[Abstract/Free Full Text]
  22. Mercier, P., Li, M. X., and Sykes, B. D. (2000) Biochemistry 39, 2902–2911[CrossRef][Medline] [Order article via Infotrieve]
  23. McKay, R. T., Pearlstone, J. R., Corson, D. C., Gagne, S. M., Smillie, L. B., and Sykes, B. D. (1998) Biochemistry 37, 12419–12430[CrossRef][Medline] [Order article via Infotrieve]
  24. Mercier, P., Ferguson, R. E., Irving, M., Corrie, J. E. T., Trentham, D. R., and Sykes, B. D. (2003) Biochemistry 42, 4333–4348[CrossRef][Medline] [Order article via Infotrieve]
  25. Dargis, R., Pearlstone, J. R., Barrette-Ng, I., Edwards, H., and Smillie, L. B. (2002) J. Biol. Chem. 277, 34662–34665[Abstract/Free Full Text]
  26. Vallins, W. J., Brand, N. J., Dabhade, N., Butler-Browne, G., Yacoub, M. H., and Barton, P. J. (1990) FEBS Lett. 270, 57–61[CrossRef][Medline] [Order article via Infotrieve]
  27. Head, J. F., and Perry, S. V. (1974) Biochem. J. 137, 145–154[Medline] [Order article via Infotrieve]
  28. Talbot, J. A., and Hodges, R. S. (1979) J. Biol. Chem. 254, 3720–3723[Abstract]
  29. Syska, H., Wilkinson, J. M., Grand, R. J., and Perry, S. V. (1976) Biochem. J. 153, 375–387[Medline] [Order article via Infotrieve]
  30. Li, M. X., Spyracopoulos, L., Beier, N., Putkey, J. A., and Sykes, B. D. (2000) Biochemistry 39, 8782–8790[CrossRef][Medline] [Order article via Infotrieve]
  31. Ngai, S.-M., and Hodges, R. S. (1992) J. Biol. Chem. 267, 15715–15720[Abstract/Free Full Text]
  32. Tripet, B., Van Eyk, J. E., and Hodges, R. S. (1997) J. Mol. Biol. 271, 728–750[CrossRef][Medline] [Order article via Infotrieve]
  33. Campbell, A. P., and Sykes, B. D. (1991) J. Mol. Biol. 222, 405–421[Medline] [Order article via Infotrieve]
  34. Campbell, A. P., Van Eyk, J. E., Hodges, R. S., and Sykes, B. D. (1992) Biochim. Biophys. Acta 1160, 35–54[Medline] [Order article via Infotrieve]
  35. Ngai, S.-M., Sönnichsen, F. D., and Hodges, R. S. (1994) J. Biol. Chem. 269, 2165–2172[Abstract/Free Full Text]
  36. Hernandez, G., Blumenthal, D. K., Kennedy, M. A., Unkefer, C. J., and Trewhella, J. (1999) Biochemistry 38, 6911–6917[CrossRef][Medline] [Order article via Infotrieve]
  37. Tung, C. S., Wall, M. E., Gallagher, S. C., and Trewhella, J. (2000) Protein Sci. 9, 1312–1326[Abstract]
  38. Brown, L. J., Sale, K. L., Hills, R., Rouviere, C., Song, L., Zhang, X., and Fajer, P. G. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 12765–12770[Abstract/Free Full Text]
  39. Tripet, B., De Crescenzo, G., Grothe, S., O'Connor-McCourt, M., and Hodges, R. (2002) J. Mol. Biol. 323, 345–362[CrossRef][Medline] [Order article via Infotrieve]
  40. Heller, W. T., Abusamhadneh, E., Finley, N., Rosevear, P. R., and Trewhella, J. (2002) Biochemistry 41, 15654–15663[CrossRef][Medline] [Order article via Infotrieve]
  41. Takeda, S., Yamashita, A., Maeda, K., and Maeda, Y. (2002) Biophys. J. 82, 170A
  42. Lindhout, D. A., Li, M. X., Schieve, D., and Sykes, B. D. (2002) Biochemistry 41, 7267–7274[CrossRef][Medline] [Order article via Infotrieve]
  43. Chandra, M., Dong, W. J., Pan, B. S., Cheung, H. C., and Solaro, R. J. (1997) Biochemistry 36, 13305–13311[CrossRef][Medline] [Order article via Infotrieve]
  44. Li, M. X., Gagné, S. M., Tsuda, S., Kay, C. M., Smillie, L. B., and Sykes, B. D. (1995) Biochemistry 34, 8330–8340[Medline] [Order article via Infotrieve]
  45. Huth, J. R., Bewley, C. A., Jackson, B. M., Hinnebusch, A. G., Clore, G. M., and Gronenborn, A. M. (1997) Protein Sci. 6, 2359–2364[Abstract/Free Full Text]
  46. Delaglio, F., Grzesiek, S., Vuister, G. W., Zhu, G., Pfeifer, J., and Bax, A. (1995) J. Biomol. NMR 6, 277–293[Medline] [Order article via Infotrieve]
  47. Johnson, A. J., and Blevins, R. A. (1994) J. Biomol. NMR 4, 603–614
  48. Gagné, S. M., Li, M. X., and Sykes, B. D. (1997) Biochemistry 36, 4386–4392[CrossRef][Medline] [Order article via Infotrieve]
  49. Laskowski, R. A., MacArthur, M. W., Moss, D. S., and Thorton, J. M. (1993) J. Appl. Crystallogr. 26, 283–290[CrossRef]
  50. Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T., and Warren, G. L. (1998) Acta Crystallogr. Sect. D. Biol. Crystallogr. 54, 905–921[CrossRef][Medline] [Order article via Infotrieve]
  51. Farrow, N. A., Muhandiram, R., Singer, A. U., Pascal, S. M., Kay, C. M., Gish, G., Shoelson, S. E., Pawson, T., Forman-Kay, J. D., and Kay, L. E. (1994) Biochemistry 33, 5984–6003[Medline] [Order article via Infotrieve]
  52. Ogura, K., Terasawa, H., and Inagaki, F. (1996) J. Biomol. NMR 8, 492–498
  53. Herzberg, O., and James, M. N. G. (1985) Nature 313, 653–659[Medline] [Order article via Infotrieve]
  54. Cachia, P. J., Sykes, B. D., and Hodges, R. S. (1983) Biochemistry 22, 4145–4152[Medline] [Order article via Infotrieve]
  55. Wishart, D. S., and Sykes, B. D. (1994) Methods Enzymol. 239, 363–392[Medline] [Order article via Infotrieve]
  56. Wishart, D. S., and Sykes, B. D. (1994) J. Biomol. NMR 4, 171–180[Medline] [Order article via Infotrieve]
  57. Wishart, D. S., Sykes, B. D., and Richards, F. M. (1991) J. Mol. Biol. 222, 311–333[Medline] [Order article via Infotrieve]
  58. Wishart, D. S., Boyko, R. F., Willard, L., Richards, F. M., and Sykes, B. D. (1994) Comput. Appl. Biosci. 10, 121–132[Abstract]
  59. Mercier, P., Spyracopoulos, L., and Sykes, B. D. (2001) Biochemistry 40, 10063–10077[CrossRef][Medline] [Order article via Infotrieve]
  60. Slupsky, C. M., and Sykes, B. D. (1995) Biochemistry 34, 15953–15964[Medline] [Order article via Infotrieve]
  61. Spyracopoulos, L., Li, M. X., Sia, S. K., Gagné, S. M., Chandra, M., Solaro, R. J., and Sykes, B. D. (1997) Biochemistry 36, 12138–12146[CrossRef][Medline] [Order article via Infotrieve]
  62. Spyracopoulos, L., Gagné, S. M., Li, M. X., and Sykes, B. D. (1998) Biochemistry 37, 18032–18044[CrossRef][Medline] [Order article via Infotrieve]
  63. Gagne, S. M., Tsuda, S., Spyracopoulos, L., Kay, L. E., and Sykes, B. D. (1998) J. Mol. Biol. 278, 667–686[CrossRef][Medline] [Order article via Infotrieve]
  64. Luo, Y., Wu, J. L., Li, B., Langsetmo, K., Gergely, J., and Tao, T. (2000) J. Mol. Biol. 296, 899–910[CrossRef][Medline] [Order article via Infotrieve]
  65. Finley, N., Dvoretsky, A., and Rosevear, P. R. (2000) J. Mol. Cell. Cardiol. 32, 1439–1446[CrossRef][Medline] [Order article via Infotrieve]
  66. Pearlstone, J. R., and Smillie, L. B. (1985) Can. J. Biochem. Cell Biol. 63, 212–218[Medline] [Order article via Infotrieve]
  67. Nicholls, A., Sharp, K., and Honig, B. (1991) Proteins Struct. Funct. Genet. 11, 281–296[Medline] [Order article via Infotrieve]
  68. Merritt, E. A., and Bacon, D. J. (1997) Methods Enzymol. 277, 505–524
  69. Neri, D., Szyperski, T., Ottig, G., Senn, H., and Wuthrich, K. (1989) Biochemistry 28, 7510–7516[Medline] [Order article via Infotrieve]
  70. Kay, L. E., Keifer, P., and Saarinen, T. (1992) J. Am. Chem. Soc. 114, 10663–10665
  71. Zhang, O., Kay, L. E., Olivier, J. P., and Forman-Kay, J. D. (1994) J. Biomol. NMR 4, 845–858[Medline] [Order article via Infotrieve]
  72. Kuboniwa, H., Grzesiek, S., Delaglio, F., and Bax, A. (1994) J. Biomol. NMR 4, 871–878[Medline] [Order article via Infotrieve]
  73. Archer, S. J., Ikura, M. I., Torchia, D. A., and Bax, A. (1991) J. Magn. Res. 95, 636–641
  74. Muhandiram, D. R., and Kay, L. E. (1994) J. Magn. Res. 103, 203–216[CrossRef]