Structure of Cardiac Muscle Troponin C Unexpectedly Reveals a Closed Regulatory Domain*

(Received for publication, March 5, 1997, and in revised form, May 8, 1997)

Samuel K. Sia Dagger , Monica X. Li Dagger , Leo Spyracopoulos Dagger , Stéphane M. Gagné Dagger , Wen Liu §, John A. Putkey § and Brian D. Sykes Dagger

From the Dagger  Department of Biochemistry, Medical Research Council Group in Protein Structure and Function, University of Alberta, Edmonton, Alberta T6G 2H7, Canada and the § Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston, Texas 77225

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

The regulation of cardiac muscle contraction must differ from that of skeletal muscles to effect different physiological and contractile properties. Cardiac troponin C (TnC), the key regulator of cardiac muscle contraction, possesses different functional and Ca2+-binding properties compared with skeletal TnC and features a Ca2+-binding site I, which is naturally inactive. The structure of cardiac TnC in the Ca2+-saturated state has been determined by nuclear magnetic resonance spectroscopy. The regulatory domain exists in a "closed" conformation even in the Ca2+-bound (the "on") state, in contrast to all predicted models and differing significantly from the calcium-induced structure observed in skeletal TnC. This structure in the Ca2+-bound state, and its subsequent interaction with troponin I (TnI), are crucial in determining the specific regulatory mechanism for cardiac muscle contraction. Further, it will allow for an understanding of the action of calcium-sensitizing drugs, which bind to cardiac TnC and are known to enhance the ability of cardiac TnC to activate cardiac muscle contraction.


INTRODUCTION

Transient increases in cytosolic Ca2+ levels in the cardiac muscle cell must be recognized by the thin filament to regulate cardiac muscle contraction. This critical function is accomplished by cardiac TnC1 (161 residues), a member of the EF-hand family of Ca2+-binding proteins, which relays the Ca2+ signal via a conformational change to the rest of the troponin-tropomyosin complex, and ultimately signals the activation of the myosin-actin ATPase reaction. Although the sequence of cardiac TnC is 70% identical to that of skeletal TnC, there are significant differences in the first 40 residues, the most crucial being the inactivation of Ca2+-binding site I due to an insertion (Val28) and substitutions of key ligands relative to skeletal TnC (Leu29 and Ala31 in cardiac TnC instead of Asp30 and Asp32 in skeletal TnC) (1). Despite the many functional, binding, and modeling studies performed on cardiac TnC (2), the absence of direct structural data makes the Ca2+-induced conformational change in cardiac TnC unclear. The structures of TnC in the skeletal system, on the other hand, have been solved both in the 2-Ca2+ (3, 4) and 4-Ca2+ states (5), showing TnC to be a dumbbell-shaped molecule with separate N- and C-terminal domains connected by a central linker. Upon Ca2+ binding, the regulatory N-domain of skeletal TnC switches from a "closed" to an "open" conformation, thereby exposing a patch of hydrophobic residues, which is thought to interact with skeletal TnI (6). In this report, we show that, in contrast to predicted models (7-9), the analogous conformational change does not occur in cardiac TnC, and that this is the direct structural consequence of inactivating Ca2+-binding site I. In addition, a structural understanding of cardiac TnC has potential therapeutic value in the understanding of the mechanism of cardiac TnC-binding drugs known as "calcium-sensitizing drugs" (8, 10).

For the purposes of this study, the two Cys residues at positions 35 and 84 of wild type cardiac TnC have been mutated to Ser residues. This prevents the formation of intra- and intermolecular disulfide bonds, which confer Ca2+-independent activity to cardiac TnC when assayed in skeletal muscle myofibrils (11). It has been shown that the conversion of these Cys residues to Ser residues has no effect on the ability of cardiac TnC to recover ATPase activity in TnC-extracted fast skeletal and cardiac myofibrils, and has little effect on Ca2+ binding to site II of cardiac TnC (11). Thus, it is unlikely that the introduction of these two conservative mutations would result in gross conformational changes in the secondary or tertiary structure of cardiac TnC.

For NMR analysis, the protein was uniformly labeled with 13C and/or 15N by expression in Escherichia coli. Triple-resonance NMR experiments were used for assigning the resonances and subsequently to derive distance and dihedral angle restraints. 35 structures were then calculated using the simulated annealing protocol (12). Structural statistics for the 30 lowest energy structures (Table I) show that the N- and C-domains are very well defined separately, with the central linker shown to be flexible by relaxation measurements.2

Table I. Structural statistics


N-domain (2-89) C-domain (90-161)

r.m.s.d. from the average   structure (Å)a
  Backbone atoms 0.54  ± 0.09 0.46  ± 0.07
  All heavy atoms 0.97  ± 0.09 0.94  ± 0.10
NOE restraints
  Total 1239 1080
  Intra-residue  477  421
  Sequential (|i - j| = 1)  285  267
  Medium-range (2 <=  |i - j| <=  4)  315  222
  Long-range (|i - j| >=  5)  162  170
Distance restraints to Ca2+ -ionb    6   12
Dihedral restraints
  Total  104   87
  phi   49   41
  psi   37   26
  chi 1   18   20
Energiesc
  Etotal 105  ± 4 84  ± 2
  ENOE 2  ± 1 4  ± 1
  Edihedral 0.03  ± 0.03 0.03  ± 0.05
r.m.s.d. from idealized geometry
  bond lengths (Å) 0.0012  ± 0.0001 0.0011  ± 0.0001
  bond angle (°) 0.47  ± 0.01 0.44  ± 0.01
  impropers (°) 0.35  ± 0.01 0.33  ± 0.01
Restraint violations
  distance > 0.1 Å but < 0.2 Åd 52 (1.7/structure) 50 (1.7/structure)
  dihedral > 1°  0 (0/structure)  0 (0/structure)
 phi , psi  in core or allowed regions   98%   99%

a 30 structures were calculated with the method of simulated annealing (12), using the program X-PLOR (35). Because the central linker (85-94) is unstructured, the structures of the N- and C-domains were calculated separately. Root-mean-squared deviations (r.m.s.d.) are for residues 5-29, 34-48, 56-65, and 69-84 for the N-domain, and residues 97-124 and 130-158 for the C-domain.
b Note that calcium ions are not directly observed by NMR spectroscopy. No restraints involving the calcium ions were used in the initial stages of structure calculations, and were added only in the final stages of refinement (see "Materials and Methods").
c The final force constants were KNOE = 50 kcal mol-1 and Kdihedral = 200 kcal mol-1rad-2. phi , psi  core and allowed regions were as determined by the program PROCHECK (36).
d There are no distance violations over 0.2 Å for the N-domain, and there is 1 distance violation over 0.2 Å for 30 structures for the C-domain.


MATERIALS AND METHODS

Sample Preparation

To produce high level expression of chicken cardiac TnC with the mutations C35S and C84S (denoted cTnC(A-Cys)), the expression plasmid pTnC(A-Cys)PL (11), which uses the PL promoter, was digested with NcoI and HindIII, and the small fragment containing the full amino acid coding region was ligated into the NcoI/HindIII sites of the plasmid pET-23d (Novagen), which has a T7 promoter. Plasmids were maintained, and cTnC(A-Cys) was expressed in the BL21(DE3)pLysS host strain E. coli after induction with isopropyl-1-thio-beta -D-galactopyranoside. Isotope enrichment media consisted of M9 minimal media, in which the NH4Cl and/or the D-glucose was replaced with one of the following: 1) 15NH4Cl (1 g/liter) for uniform 15N labeling; 2) D-glucose-U-13C6 (2 g/liter) for uniform 13C labeling; 3) 15NH4Cl (1 g/liter) and D-glucose-U-13C6 (2 g/liter) for uniform 13C and 15N labeling; or 4) D-glucose-U-13C6 (0.5 g/liter) and D-glucose (1.5 g/liter) for partial 13C labeling. All isotopes were purchased from Cambridge Isotope Laboratories. After induction, bacteria were collected and lysed, and a soluble protein fraction was prepared as described previously (13). This was applied directly to a Macro-Q (Bio-Rad) anion exchange column and eluted with a 0-500 mM KCl gradient containing 0.5 mM EGTA. Appropriate fractions were pooled, made 3 M in (NH4)2SO4, and centrifuged to remove precipitates, after which the soluble fraction was dialyzed against 50 mM MOPS, 0.5 mM CaCl2 at pH 7.0. The dialyzed sample was then applied to a semiprep HPLC DEAE 5-PW anion exchange column (Millipore) and eluted with a 0-500 mM KCl gradient with 0.5 mM CaCl2. Purified cTnC(A-Cys) was pooled and dialyzed against 10 mM (NH4)HCO3 at pH 8.0, and lyophilized.

Structure Determination

All spectra were collected at 30 °C on a Varian Unity Plus 500-MHz spectrometer or a Varian Unity Plus 600-MHz spectrometer, both equipped with pulsed field gradient accessories. The NMR samples contained 2-4 mM cTnC(A-Cys) in 16 mM CaCl2, 100 mM KCl, 10 mM imidazole at pH 6.7, in either 90% H2O, 10% D2O or 99.99% D2O. The high affinities of cardiac TnC for calcium (with dissociation constants on the order of 10-6 M for the N-domain and 10-8 M for the C-domain) ensure that the protein is Ca2+-saturated at 16 mM CaCl2 (11).

Assignment of the main-chain NH, N, Calpha , and Cbeta resonances were made based on the three-dimensional experiments HNCACB and CBCA(CO)NH (14). Three-dimensional 15N-edited total correlation spectroscopy (TOCSY) (15) and three-dimensional HCCH-TOCSY (16) experiments were used to assign the chemical shifts for the side-chain atoms. A high resolution two-dimensional 1H-1H nuclear Overhauser enhancement spectroscopy (NOESY) experiment and assignments made by a previous study (17) were used to confirm the assignments for resonances of aromatic protons. Interproton distances were derived from three-dimensional 15N- and 15N/13C-edited NOESY experiments (18), both collected at 50 ms of mixing time. In converting NOE intensities into distance restraints, the NOE intensities were calibrated for each residue using its intraresidue dNalpha (i, i) and sequential dalpha N(i - 1, i) NOEs as reference intensities (6). In cases where neither NOE was present, the upper bound distance was given the calibration factor corresponding to the loosest restraint. In all cases, a 40% error on NOE intensities was used. Based on the analysis of the crystal structures of skeletal TnC (19), 18 distance restraints to the Ca2+ ion (6 at each of site II, III, and IV) of 2.0-2.8 Å were assigned.

A three-dimensional HNHA experiment (20) was used to derive 3JHNHalpha coupling constants. phi  restraints were imposed if the 3JHNHalpha values were greater than 8 Hz or less than 5.5 Hz. These restraints were given different errors depending on the region on the Karplus curve to which they corresponded, with a minimum error of ±20°. psi  restraints were assigned for the (i - 1) residue if the dNalpha (i, i) to dalpha N(i - 1, i) NOE intensity ratio was greater than 1.2 or less than 0.83, which were given loose restraints of -30 ± 110° and 110 ± 110°, respectively (28). Stereospecific assignments of the methyl protons of valines and leucines (20 out of 21 possible) were made based on the presence of singlets (pro-S) or doublets (pro-R) in a two-dimensional 13C-1H HSQC spectrum with the protein being expressed in 25% [13C]glucose (21). Hbeta -methylene protons were stereospecifically assigned (32 out of 115 possible) by analyzing 3JHalpha Hbeta values from the three-dimensional HACAHB experiment (22), 3JNHbeta values from the three-dimensional HNHB experiment (23), and intraresidue NOE intensities. chi 1 restraints of +60°, 180°, or -60° (±60°) were also given if all of the above data were consistent with one chi 1 conformation. All spectra were processed with NMRPipe (24) and peak-picked with the program PIPP (25).


RESULTS

Fig. 1 shows the solution structures of the N- and C-terminal domains, with the structural statistics provided in Table I. The overall solution structure of Ca2+-saturated cardiac TnC, like the solution structures of Ca2+-saturated skeletal TnC (5) and calmodulin-target peptide complex (26), resembles a dumbbell in shape, consisting of two separate domains connected by a flexible central linker (residues 86-94 in cardiac TnC). However, despite the general structural similarities to homologous Ca2+-binding proteins, the regulatory N-domain of Ca2+-saturated cardiac TnC is significantly more compact than the N-domain of Ca2+-saturated skeletal TnC (5, 6), exposing approximately 800 Å2 less total accessible surface area (residues 5-84) than its skeletal counterpart (residues 7-85). In particular, the B-helix of defunct site I exists in the "closed" conformation, exhibiting an A-B interhelical angle of 142° (with the A- and B-helices corresponding to the two helices of the helix-loop-helix motif in Ca2+-binding proteins; Table II). The closed conformation is evidenced by 21 NOE connectives observed between the A- and B-helices (Fig. 2), most of which would not be observed if the B-helix were in an "open" conformation as in skeletal TnC (Fig. 3A). A compact regulatory domain is also consistent with a previous cysteine-reactivity study on wild type cardiac TnC (27).


Fig. 1. Structure of Ca2+-saturated cardiac TnC as determined in this study (sequence starts at Ala2 and ends at Val161). The alpha -helices are approximately as follows: N-helix (residues 6-10), A-helix (14-28), B-helix (38-48), C-helix (54-64), D-helix (74-83), E-helix (95-103), F-helix (114-123), G-helix (130-140), H-helix (150-158). The antiparallel beta -sheets connect residues 35-37 and 71-73 in the N-domain, and residues 111-113 and 147-149 in the C-domain. All regions of the molecule are well defined, with the exceptions of the following regions: the N- and C-terminal residues (2-4 and 159-161), residues 30-33 of defunct site I, residues 49-55 of the B-C linker, residues 66-68 of site II, the central linker (86-94), and residues 125-129 of the F-G linker. Stereo views of the superposition of 30 structures for the regulatory N-domain (residues 5-84, A) and the structural C-domain (95-158, B) are shown. Positions of the Ca2+ ions are indicated by gray spheres. This figure was prepared with the program RASTER3D (37).
[View Larger Version of this Image (51K GIF file)]

Table II. Interhelical angles of various EF-hands


Calcium-binding proteina Interhelical angles (°)b
A-B C-D E-F G-H

Cardiac TnC(1 Ca2+/2 Ca2+) 138  ± 3 108  ± 4 115  ± 4 121  ± 4
Skeletal TnC(2 Ca2+/2 Ca2+) 81  ± 5 78  ± 7 89  ± 6 104  ± 7
Skeletal TnC(apo/2 Ca2+) 138 145 105 111
Calmodulin(2 Ca2+/2 Ca2+)  88  92  98  97

a The parentheses indicate first the state of the N-domain (i.e., A-B and C-D helix-loop-helices), followed by the state of the C-domain (i.e., E-F and G-H helix-loop-helices). Note that in cardiac TnC(1 Ca2+/2 Ca2+) as determined in the present study, defunct site I (i.e. A-B helix-loop-helix) is free of Ca2+, while sites II, III, and IV are Ca2+-bound. Protein Data Bank accession codes are: 1TNW for the NMR structure of skeletal TnC(2 Ca2+/2 Ca2+), 5TNC for the crystal structure of skeletal TnC(apo/2 Ca2+), and 4CLN for the crystal structure of calmodulin(2 Ca2+/2 Ca2+).
b A large angle defines a "closed" conformation, whereas a small angle defines an "open" conformation. The axis for an alpha -helix is defined by two points, the two points being the average coordinates of the first and last 11 backbone atoms of the alpha -helix.


Fig. 2. Evidence for the closed conformation for the regulatory domain. A, three-dimensional 15N/13C-edited NOESY showing 4 of the 21 NOE contacts between the A- and B-helices. Here, the Hbeta protons of Ala23 show NOE connectives to the Hdelta 1 and Hdelta 2 protons of Leu48 and to the Hgamma 1 and Hgamma 2 protons of Val44. Negative (folded) peaks are indicated by dotted lines. B, structure of the A-helix-loop-B-helix (residues 14-49) of the regulatory domain. The four dotted lines correspond to the four NOEs shown in A. This figure was prepared with the software INSIGHT II (Biosym).
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Fig. 3. A, comparison of the regulatory N-domains of TnC in various states. Shown here are 2Ca·skeletal TnC (green, left panel; neither site occupied by Ca2+), 3Ca·cardiac TnC (red, middle panel; site II occupied by Ca2+ and site I unable to bind Ca2+), and 4Ca·skeletal TnC (blue, right panel; both sites I and II occupied by Ca2+). The B- and C-helices of all three structures are shown in gray. The residue E40 is labeled by an arrow. B, comparison of various structural C-domains of TnC, all in the Ca2+-saturated state (both sites III and IV occupied by Ca2+). Shown here are 2Ca·skeletal TnC (green, left panel), 3Ca·cardiac TnC (red, middle panel), and 4Ca·skeletal TnC (blue, right panel). This figure was prepared with the program RASTER3D (37).
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The difference in the conformation of the B-helix is reflected most clearly in the main chain conformation of residue Glu40 in cardiac TnC (equivalent to Glu41 in skeletal TnC). Glu41 of skeletal TnC has been proposed to be pivotal in the mechanism of the coupling between Ca2+ binding and the Ca2+-induced conformational change in Ca2+-binding proteins (28, 29). Indeed, the apo form of skeletal TnC (3) features a kink in the B-helix at Glu41 which straightens out upon Ca2+ binding to sites I and II (28). In cardiac TnC, however, there exists a kink in the B-helix at Glu40, even in the Ca2+-bound state (Ca2+ ions bound at sites II, III, and IV). The non-helical nature at Glu40 is supported by a 3JHNHalpha value of 7.8 Hz, an absence of an upfield-shift of its Halpha resonance (4.37 ppm), and an absence of a downfield shift in its Calpha resonance (56.8 ppm), all of which indicate non-helical conformations (30). On the other hand, both adjacent residues Lys39 and Leu41 exhibit 3JHNHalpha values of less than 5.5 Hz, as well as appropriate shifts in their Halpha and Calpha resonances which indicate an alpha -helical conformation.

The above structural differences between cardiac and skeletal TnC can be explained by what is in fact the most striking functional difference between the two proteins: namely that site I in cardiac TnC is inactive due to an insertion and key substitutions of key ligands. In cardiac TnC, there is no Ca2+ at site I to pull the Glu40 side-chain carboxylate group over to contribute to the enthalpy necessary to overcome the entropic loss associated with exposing buried residues (which occurs in the "opening" of the B- and C-helices relative to helices N, A, and D). Thus, the B-helix remains closed due to favorable packing forces with the A-helix and D-helix (Figs. 1A and 3A; Table II, A-B interhelical angle). On the other hand, with Ca2+ bound at site II, the C-helix is in fact in an open conformation, but does not open up to the extent seen in skeletal TnC (Figs. 1A and 3A; Table II, C-D interhelical angle). These observations demonstrate that the inability of Glu40 to coordinate Ca2+ results in a more compact conformation for the B-helix, and possibly the C-helix, than is observed in skeletal TnC (Fig. 3A); in effect, Ca2+ binding to sites I and II of skeletal TnC locks open the whole regulatory domain, whereas Ca2+ binding to site II of cardiac TnC only partially opens up the regulatory domain. (This discussion assumes that the structure of the apo form of the regulatory domain of cardiac TnC is similar to that of skeletal TnC, as has been recently demonstrated.3) The model of Glu40 acting as a pivot for the N-domain is further supported by a recent structural study of a skeletal TnC mutant in which Glu41 is replaced by Ala41, such that residue 41 can no longer coordinate the Ca2+ ion present at site I (29). In the Ca2+-saturated state of this protein, the single substitution results in a kink at Ala41 and a closed conformation for the B-helix, similar to what is seen in Ca2+-saturated cardiac TnC.

The structure of defunct site I shows that Leu29, which comes just after the insertion at Val28, forms an extra half-turn at the end of the A-helix, as evidenced by dalpha N(i, i + 3) and dalpha beta (i, i + 3) NOE connectives from Ile26 to Leu29. Site I, being Ca2+-free, is not as well defined as the rest of the molecule (root mean square deviation of 0.78 Å for backbone atoms of residues 30-33), and is shown to be more flexible than the rest of the regulatory domain by relaxation measurements.

The structural C-domain of cardiac TnC (Figs. 1B and 3B) is predictably similar to those in skeletal TnC and calmodulin, although the interhelical angles of the two EF-hands in the C-domain indicate that this domain is in fact slightly more compact in cardiac TnC than in its counterparts (10-20° more closed in the E-F and G-H interhelical angles; see Table II). Overall, the backbone atoms of residues 95-157 of cardiac TnC superimpose within 1.9 Å with their equivalent residues (96-158) in the NMR structure of skeletal TnC with Ca2+-saturated N-domain, and 1.3 Å with the same region in the crystal structure of skeletal TnC with apo N-domain.


DISCUSSION

We have shown for the first time the three-dimensional structure of Ca2+-saturated cardiac TnC, which reveals an unexpected compact regulatory domain as a direct consequence of an inactive Ca2+-binding site I. These results provide a structural precedent for a Ca2+-binding regulatory protein in which one of the two sites in the paired set of EF-hands is inactive (for example, some invertebrate TnCs also have this feature; Ref. 31)). This unique structural feature sets cardiac TnC apart from other "calcium sensor" EF-hand Ca2+-binding proteins such as skeletal TnC and calmodulin, as well as "calcium buffer" EF-hand proteins such as parvalbumin and calbindin. The compact regulatory domain is a surprising result because it violates the general rule with Ca2+-binding proteins that a small conformational change accompanies Ca2+ binding in buffering proteins, and that a large conformational change accompanies Ca2+ binding in regulatory proteins such as cardiac TnC (32). In particular, it is believed that in general the action of Ca2+ binding in calcium sensor proteins is to induce an exposure of a large hydrophobic surface, allowing the protein to interact with targets to accomplish regulatory functions, whereas the capture of Ca2+ ions by calcium buffer proteins is accompanied by only minor conformational changes. Thus, it has long been believed that the mechanism for the activation of cardiac TnC involves the exposure of a large hydrophobic patch upon Ca2+ binding as observed for other calcium sensors. In fact, cardiac TnC models based on the conformational changes observed in skeletal TnC have been widely used to interpret the functional, Ca2+-binding and drug-binding properties of cardiac TnC (7-9), despite the unique inactive Ca2+-binding site I in cardiac TnC. The present results show that the hydrophobic exposure in the Ca2+-saturated regulatory domain of cardiac TnC (Fig. 4B) is dramatically reduced compared with that of skeletal TnC (Fig. 4A) as well as a previous widely used model of cardiac TnC (9) (Fig. 4C).


Fig. 4. Comparison of the surface structures of the regulatory N-domains of 4Ca·skeletal TnC (NMR) (A), 3Ca·cardiac TnC (NMR) (B), and 3Ca·cardiac TnC (model, Ref. 9) (C), displayed in the same orientation as Fig. 1A. Side chains of hydrophobic residues (Ala, Ile, Leu, Met, Pro, Phe, Tyr, and Val) are shown in yellow, negatively charged residues (Asp and Glu) in red, positively charged residues (Arg and Lys) in blue, and all other residues in gray. The major hydrophobic pocket of 3Ca·cardiac TnC involves residues Phe20, Phe24, Leu48, Phe74, Phe77, Leu78, Met81, and Met85, and residues Ile36, Leu41, Met45, Leu57, Met60, Ile61, Val64, Val72, and Met80. Other hydrophobic contacts are also observed from Phe20, Ala23, and Phe27 of the A-helix to Val44, Met47, and Leu48 of the B-helix, and from Ala8, Val9, and Leu12 of the N-helix to Leu78, Val79, and Val82 of the D-helix. This figure was generated using the program GRASP (38).
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The substantially reduced hydrophobic surface of Ca2+-saturated cardiac TnC has important implications for the association of cardiac TnI with cardiac TnC. In particular, given that in both the cardiac and skeletal systems the Ca2+-dependent binding of TnI involves the N-domain of TnC (33), and that residues 5-84 of Ca2+-saturated cardiac TnC expose less total and hydrophobic surface area than residues 7-85 of Ca2+-saturated skeletal TnC (Fig. 4), it is possible that the mode of interaction between TnI and TnC in cardiac muscle is in fact different from that in skeletal muscle. A smaller surface of interaction between 3Ca·cardiac TnC-cardiac TnI as compared with 4Ca·skeletal TnC-skeletal TnI would explain the finding that the free energy (Delta G) of Ca2+ binding to the TnC-TnI complex is 4 times smaller in cardiac than it is in skeletal muscle (34). It may also be that the hydrophobic or electrostatic force dominates more in one isoform than in the other in the binding of TnI to TnC. If indeed the interaction between cardiac TnC and cardiac TnI involves less surface contact than that between skeletal TnC and skeletal TnI, cardiac TnC would be a more dynamic calcium sensor than its skeletal counterpart. In fact, a recent study has shown that the Ca2+ off rate measured for site II in cardiac TnC is about 3-fold faster than observed for the N-terminal signaling domain of calmodulin or skeletal troponin C.4 On the other hand, as an alternative to the above proposal, it is possible that the Ca2+-dependent binding of TnI forces open the regulatory domain of cardiac TnC, with the end result being that the cardiac TnI-TnC complex binds in a similar fashion to skeletal TnI-TnC (here cardiac TnC may adopt a structure similar to that in Fig. 4C). Such a model would be consistent with the finding that the chemical environment of Met81, which is mostly buried in this structure (accessible surface area of 14 Å2), changes upon the binding of cardiac TnI (33). This may also imply that cardiac TnC opens up to different degrees in response to events of muscle contraction such as TnI phosphorylation. At present, there is no compelling evidence to either favor or discount either model for cardiac TnI-TnC binding.

Cardiac TnC is a potential target in therapy for patients with acute myocardial infarctions and subsequently congestive heart failure, where the diseased myocardium is "desensitized" to increases in cytosolic Ca2+ levels. A novel group of positive inotropic agents known as "calcium sensitizers" (10) is known to increase the affinity of cardiac TnC for Ca2+, possibly by binding to a hydrophobic patch in the N-domain of cardiac TnC (8). The exposed hydrophobic patches in Ca2+-saturated cardiac TnC can now be identified (Fig. 4B). Although several residues (e.g. Phe77, Met81, and Met85) have been implicated in earlier studies as possible binding sites for these drugs (8), the proposed modes of binding must now be re-evaluated since most of these residues lie on the side of the D-helix facing the B-helix, and therefore are more buried by the B-helix than previously suspected (B-D interhelical distance of 12 Å for cardiac TnC versus 18 Å for skeletal TnC). In addition, significant surface topology differences between the cardiac TnC model and the solution structure (Fig. 4) warrants for a re-interpretation of most of the previous drug binding studies performed based on the now-disproved model (8). Thus, the solution structure of cardiac TnC presented here will allow for the accurate modeling of the binding of calcium-sensitizing drugs to cardiac TnC, in addition to revealing the structural basis for the regulation of cardiac versus skeletal muscle contraction.


FOOTNOTES

*   This work was supported by grants from the Medical Research Council of Canada, the Heart and Stroke Foundation of Canada, the National Institutes of Health, the Robert Welch Foundation, and the Alberta Heritage Foundation for Medical Research (to S. K. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and structure factors (code 1AJ4) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY. Coordinates of the 30 calculated structures for the N-domain (code 2CTN) and the C-domain (code 3CTN) have also been deposited.


   To whom correspondence should be addressed. E-mail: brian.sykes{at}ualberta.ca.
1   The abbreviations used are: TnC, troponin C; TnI, troponin I; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser enhancement spectroscopy; TOCSY, total correlation spectroscopy; MOPS, 4-morpholinepropanesulfonic acid.
2   S. K. Sia, M. X. Li, L. Spyracopoulos, S. M. Gagné, W. Liu, J. A. Putkey, and B. D. Sykes, unpublished data.
3   L. Spyracopoulos, M. X. Li, S. K. Sia, S. M. Gagné, M. Chandra, R. J. Solaro, and B. D. Sykes, unpublished data.
4   A. L. Hazard, N. L. Stricker, J. A. Putkey, and J. J. Falke, unpublished data.

ACKNOWLEDGEMENTS

We thank G. McQuaid for upkeep of the spectrometers; L. Willard, T. Jellard, and R. Boyko for computer assistance; and L. Kay for generously providing the pulse sequences. We also acknowledge the Protein Engineering Network Center of Excellence for the use of their Varian Unity 600-MHz spectrometer.


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