Interaction of cardiac troponin C with calmodulin antagonist [corrected] W7 in the presence of three functional regions of cardiac troponin I

W7 is a well-known calmodulin (CaM) antagonist and has been implicated as an inhibitor of the troponin C-mediated Ca(2+) activation of cardiac muscle contraction. In this study, we use NMR spectroscopy to study binding of W7 to cardiac troponin C (cTnC) free or in complex with cardiac troponin I (cTnI) peptides. Titration of cTnC.3Ca(2+) with W7 shows that residues throughout the sequence, including the N- and C-domains of cTnC and the central linker, are affected. Analysis of the binding stoichiometry and the trajectories of chemical shift changes indicate that W7 binding occurs at multiple sites. To address the issue of whether multiple-site binding is relevant within the troponin complex, W7 is titrated to a cTnC-cTnI complex (cTnC.3Ca(2+).cTnI(34)(-)(71).cTnI(128)(-)(163)). In the presence of the N-terminal (residues approximately 34-71), inhibitory (residues approximately 128-147), and switch (residues approximately 147-163) regions of cTnI, W7 induces chemical shift changes only in the N-domain and not in the C-domain or the central linker of cTnC. The results indicate that in the presence of cTnI, W7 no longer binds to multiple sites of cTnC but instead binds specifically to the N-domain, and the binding (K(D) = 0.5 +/- 0.1 mM) can occur together with the switch region of cTnI. Hence, W7 may play a role in directly modulating the Ca(2+) sensitivity of the regulatory domain of cTnC and the interaction of the switch region of cTnI and cTnC.     

Phosphorylation-dependent conformational transition of the cardiac specific N-extension of troponin I in cardiac troponin

We present here the solution structure for the bisphosphorylated form of the cardiac N-extension of troponin I (cTnI(1-32)), a region for which there are no previous high-resolution data. Using this structure, the X-ray crystal structure of the cardiac troponin core, and uniform density models of the troponin components derived from neutron contrast variation data, we built atomic models for troponin that show the conformational transition in cardiac troponin induced by bisphosphorylation. In the absence of phosphorylation, our NMR data and sequence analyses indicate a less structured cardiac N-extension with a propensity for a helical region surrounding the phosphorylation motif, followed by a helical C-terminal region (residues 25-30). In this conformation, TnI(1-32) interacts with the N-lobe of cardiac troponin C (cTnC) and thus is positioned to modulate myofilament Ca2+-sensitivity. Bisphosphorylation at Ser23/24 extends the C-terminal helix (residues 21-30) which results in weakening interactions with the N-lobe of cTnC and a re-positioning of the acidic amino terminus of cTnI(1-32) for favorable interactions with basic regions, likely the inhibitory region of cTnI. An extended poly(L-proline)II helix between residues 11 and 19 serves as the rigid linker that aids in re-positioning the amino terminus of cTnI(1-32) upon bisphosphorylation at Ser23/24. We propose that it is these electrostatic interactions between the acidic amino terminus of cTnI(1-32) and the basic inhibitory region of troponin I that induces a bending of cTnI at the end that interacts with cTnC. This model provides a molecular mechanism for the observed changes in cross-bridge kinetics upon TnI phosphorylation.     

Solution structures of the C-terminal domain of cardiac troponin C free and bound to the N-terminal domain of cardiac troponin I.

The N-terminal domain of cardiac troponin I (cTnI) comprising residues 33-80 and lacking the cardiac-specific amino terminus forms a stable binary complex with the C-terminal domain of cardiac troponin C (cTnC) comprising residues 81-161. We have utilized heteronuclear multidimensional NMR to assign the backbone and side-chain resonances of Ca2+-saturated cTnC(81-161) both free and bound to cTnI(33-80). No significant differences were observed between secondary structural elements determined for free and cTnI(33-80)-bound cTnC(81-161). We have determined solution structures of Ca2+-saturated cTnC(81-161) free and bound to cTnI(33-80). While the tertiary structure of cTnC(81-161) is qualitatively similar to that observed free in solution, the binding of cTnI(33-80) results mainly in an opening of the structure and movement of the loop region between helices F and G. Together, these movements provide the binding site for the N-terminal domain of cTnI. The putative binding site for cTnI(33-80) was determined by mapping amide proton and nitrogen chemical shift changes, induced by the binding of cTnI(33-80), onto the C-terminal cTnC structure. The binding interface for cTnI(33-80), as suggested from chemical shift changes, involves predominantly hydrophobic interactions located in the expanded hydrophobic pocket. The largest chemical shift changes were observed in the loop region connecting helices F and G. Inspection of available TnC sequences reveals that these residues are highly conserved, suggesting a common binding motif for the Ca2+/Mg2+-dependent interaction site in the TnC/TnI complex.     

The importance of the carboxyl-terminal domain of cardiac troponin C in Ca2+-sensitive muscle regulation

The interactions between troponin I and troponin C are central to the Ca(2+)-regulated control of striated muscle. Using isothermal titration microcalorimetry we have studied the binding of human cardiac troponin C (cTnC) and its isolated domains to human cardiac troponin I (cTnI). We provide the first binding data for these proteins while they are free in solution and unmodified by reporter groups. Our data reveal that the C-terminal domain of cTnC is responsible for most of the free energy change upon cTnC.cTnI binding. Importantly, the interaction between cTnI and the C-terminal domain of cTnC is 8-fold stronger in the presence of Ca(2+) than in the presence of Mg(2+), suggesting that the C-terminal domain of cTnC may play a modulatory role in cardiac muscle regulation. Changes in the affinity of cTnI for cTnC and its isolated C-terminal domain in response to ionic strength support this finding, with both following similar trends. At physiological ionic strength the affinity of cTnC for cTnI changed very little in response to Ca(2+), although the thermodynamic data show a clear distinction between binding in the presence of Ca(2+) and in the presence of Mg(2+).     

Characterization of the interaction between the N-terminal extension of human cardiac troponin I and troponin C

The N-terminal extension of cardiac troponin I (TnI) is bisphosphorylated by protein kinase A in response to beta-adrenergic stimulation. How this signal is transmitted between TnI and troponin C (TnC), resulting in accelerated Ca(2+) release, remains unclear. We recently proposed that the unphosphorylated extension interacts with the N-terminal domain of TnC stabilizing Ca(2+) binding and that phosphorylation prevents this interaction. We now use (1)H NMR to study the interactions between several N-terminal fragments of TnI, residues 1-18 (I1-18), residues 1-29 (I1-29), and residues 1-64 (I1-64), and TnC. The shorter fragments provide unambiguous information on the N-terminal regions of TnI that interact with TnC: I1-18 does not bind to TnC whereas the C-terminal region of unphosphorylated I1-29 does bind. Bisphosphorylation greatly weakens this interaction. I1-64 contains the phosphorylatable N-terminal extension and a region that anchors I1-64 to the C-terminal domain of TnC. I1-64 binding to TnC influences NMR signals arising from both domains of TnC, providing evidence that the N-terminal extension of TnI interacts with the N-terminal domain of TnC. TnC binding to I1-64 broadens NMR signals from the side chains of residues immediately C-terminal to the phosphorylation sites. Binding of TnC to bisphosphorylated I1-64 does not broaden these NMR signals to the same extent. Circular dichroism spectra of I1-64 indicate that bisphosphorylation does not produce major secondary structure changes in I1-64. We conclude that bisphosphorylation of cardiac TnI elicits its effects by weakening the interaction between the region of TnI immediately C-terminal to the phosphorylation sites and TnC either directly, due to electrostatic repulsion, or via localized conformational changes.     

The binding of W7, an inhibitor of striated muscle contraction, to cardiac troponin C

W7 is a well-characterized calmodulin antagonist. It decreases the maximal tension and rate of ATP hydrolysis in cardiac muscle fibers. Cardiac troponin C (cTnC) has been previously implicated as the mechanistically significant target for W7 in the myofilament. Two-dimensional NMR spectra ({1H,15N}- and {1H,13C}-HSQCs) were used to monitor the Ca2+-dependent binding of W7 to cTnC. Titration of cTnC x 3Ca2+ with W7 indicated binding to both domains of the protein. We examined the binding of W7 to the separated domains of cTnC to simplify the spectral analysis. In the titration of the C-terminal domain (cCTnC x 2Ca2+), the spectral peaks originating from a subset of residues changed nonuniformly, and could not be well-described as single-site binding. A global fit of the cCTnC x 2Ca2+ titration data to a two-site, sequential binding model (47 residues simultaneously fit) yielded a dissociation constant (Kd1) of 0.85-0.91 mM for the singly bound state, with the second dissociation constant fit to 3.40-3.65 mM (> or = 4 x Kd1). The titration data for the N-terminal domain (cNTnC x Ca2+) was globally fit to single-site binding model with a Kd of 0.15-0.30 mM (41 residues fit). The data are consistent with W7 binding to each domain's major hydrophobic pocket, coordinating side chains responsible for liganding cTnI. When in muscle fibers, W7 may compete with cTnI for target sites on cTnC.     

Interaction of cardiac troponin C with Ca(2+) sensitizer EMD 57033 and cardiac troponin I inhibitory peptide

The binding of Ca(2+) to cardiac troponin C (cTnC) triggers contraction in cardiac muscle. In diseased heart, the myocardium is often desensitized to Ca(2+), leading to weak cardiac contractility. Compounds that can sensitize cardiac muscle to Ca(2+) would have potential therapeutic value in treating heart failure. The thiadiazinone derivative EMD 57033 is an identified 'Ca(2+) sensitizer', and cTnC is a potential target of the drug. In this work, we used 2D ?(1)H, (15)N?-HSQC NMR spectroscopy to monitor the binding of EMD 57033 to cTnC in the Ca(2+)-saturated state. By mapping the chemical shift changes to the structure of cTnC, EMD 57033 is found to bind to the C-domain of cTnC. To test whether EMD 57033 competes with cardiac TnI (cTnI) for cTnC and interferes with the inhibitory function, we examined the interaction of cTnC with an inhibitory cTnI peptide (residues 128-147, cIp) in the absence and presence of EMD 57033, respectively. cTnC was also titrated with EMD 57033 in the presence of cIp. The results show that although both the drug and cIp interact with the C-domain of cTnC, they do not displace each other, suggesting noncompetitive binding sites for the two targets. Detailed chemical shift mapping of the binding sites reveals that the regions encompassing helix G-loop IV-helix H are more affected by EMD 57033, while residues located on helix E-loop III-helix F and the linker between sites III and IV are more affected by cIp. In both cases, the binding stoichiometry is 1:1. The binding affinities for the drug are 8.0 +/- 1.8 and 7.4 +/- 4.8 microM in the absence and presence of cIp, respectively, while those for the peptide are 78.2 +/- 10.3 and 99.2 +/- 30.0 microM in the absence and presence of EMD 57033, respectively. These findings suggest that EMD 57033 may exert its positive inotropic effect by not directly enhancing Ca(2+) binding to the Ca(2+) regulatory site of cTnC, but by binding to the structural domain of cTnC, modulating the interaction between cTnC and other thin filament proteins, and increasing the apparent Ca(2+) sensitivity of the contractile system.     

Investigation of cation-binding properties of cardiac troponin C peptides by circular-dichroism spectroscopy

Bovine cardiac troponin C was cleaved at residues cysteine-35 and cysteine-84. Three peptides, N-terminal (residues 1-34), central (residues 35-83) and C-terminal (residues 84-161), of cardiac troponin C were obtained in a homogeneous state. Saturation of troponin C or its C-terminal peptide with Ca2+ or Mg2+ is accompanied by an increase in the ellipticity at 222 nm in the c.d. spectrum. The half-maximal changes in the ellipticity of troponin C were observed at 32 nM-Ca2+ or 56 microM-Mg2+. The corresponding values for the C-terminal peptide are 7.1 nM for Ca2+ and 4.5 microM for Mg2+. The ellipticity of the central peptide (residues 35-83) containing the second cation-binding site was decreased on saturation with Ca2+. The half-maximal changes in the ellipticity occur at 80 microM-Ca2+. Study of the c.d. spectra suggests that the alpha-helices flanking the second cation-binding site of cardiac troponin C exist independently of Ca2+. Saturation of the third and fourth sites with these cations is associated with a considerable increase in the alpha-helix content, probably due to the formation of an alpha-helix flanking the third site on the N-terminus.     

Using lanthanide ions to align troponin complexes in solution: order of lanthanide occupancy in cardiac troponin C

The potential for using paramagnetic lanthanide ions to partially align troponin C in solution as a tool for the structure determination of bound troponin I peptides has been investigated. A prerequisite for these studies is an understanding of the order of lanthanide ion occupancy in the metal binding sites of the protein. Two-dimensional [(1)H, (15)N] HSQC NMR spectroscopy has been used to examine the binding order of Ce(3+), Tb(3+), and Yb(3+) to both apo- and holo-forms of human cardiac troponin C (cTnC) and of Ce(3+) to holo-chicken skeletal troponin C (sTnC). The disappearance of cross-peak resonances in the HSQC spectrum was used to determine the order of occupation of the binding sites in both cTnC and sTnC by each lanthanide. For the lanthanides tested, the binding order follows that of the net charge of the binding site residues from most to least negative; the N-domain calcium binding sites are the first to be filled followed by the C-domain sites. Given this binding order for lanthanide ions, it was demonstrated that it is possible to create a cTnC species with one lanthanide in the N-domain site and two Ca(2+) ions in the C-domain binding sites. By using the species cTnC.Yb(3+).2 Ca(2+) it was possible to confer partial alignment on a bound human cardiac troponin I (cTnI) peptide. Residual dipolar couplings (RDCs) were measured for the resonances in the bound (15)N-labeled cTnI(129-148) by using two-dimensional [(1)H, (15)N] inphase antiphase (IPAP) NMR spectroscopy.     

Ca(2+) induces an extended conformation of the inhibitory region of troponin I in cardiac muscle troponin.

The inhibitory region of troponin I (TnI) plays a central regulatory role in the contraction and relaxation cycle of skeletal and cardiac muscle through its Ca(2+)-dependent interaction with actin. Detailed structural information on the interface between TnC and this region of TnI has been long in dispute. We have used fluorescence resonance energy transfer (FRET) to investigate the global conformation of the inhibitory region of a full-length TnI mutant from cardiac muscle (cTnI) in the unbound state and in reconstituted complexes with the other cardiac troponin subunits. The mutant contained a single tryptophan residue at the position 129 which was used as an energy transfer donor, and a single cysteine residue at the position 152 labeled with IAEDANS as energy acceptor. The sequence between Trp129 and Cys152 in cTnI brackets the inhibitory region (residues 130-149), and the distance between the two sites was found to be 19.4 A in free cTnI. This distance was insensitive to reconstitution of cTnI with cardiac troponin T (cTnT), cTnC, or cTnC and cTnT in the absence of bound regulatory Ca(2+) in cTnC. An increase of 9 A in the Trp129-Cys152 separation was observed upon saturation of the Ca(2+) regulatory site of cTnC in the complexes. This large increase suggests an extended conformation of the inhibitory region in the interface between cTnC and cTnI in holo cardiac troponin. This extended conformation is different from a recent model of the Ca(2+)-saturated skeletal TnI-TnC complex in which the inhibitory region is modeled as a beta-turn. The observed Ca(2+)-induced conformational change may be a switch mechanism by which movement of the regulatory region of cTnI to the exposed hydrophobic patch of the open regulatory N-domain of cTnC pulls the inhibitory region away from actin upon Ca(2+) activation in cardiac muscle.     

An NMR and spin label study of the effects of binding calcium and troponin I inhibitory peptide to cardiac troponin C.

The paramagnetic relaxation reagent, 4-hydroxy-2,2,6,6-tetramethylpiperidinyl-1-oxy (HyTEMPO), was used to probe the surface exposure of methionine residues of recombinant cardiac troponin C (cTnC) in the absence and presence of Ca2+ at the regulatory site (site II), as well as in the presence of the troponin I inhibitory peptide (cTnIp). Methyl resonances of the 10 Met residues of cTnC were chosen as spectral probes because they are thought to play a role in both formation of the N-terminal hydrophobic pocket and in the binding of cTnIp. Proton longitudinal relaxation rates (R1's) of the [13C-methyl] groups in [13C-methyl]Met-labeled cTnC(C35S) were determined using a T1 two-dimensional heteronuclear single- and multiple-quantum coherence pulse sequence. Solvent-exposed Met residues exhibit increased relaxation rates from the paramagnetic effect of HyTEMPO. Relaxation rates in 2Ca(2+)-loaded and Ca(2+)-saturated cTnC, both in the presence and absence of HyTEMPO, permitted the topological mapping of the conformational changes induced by the binding of Ca2+ to site II, the site responsible for triggering muscle contraction. Calcium binding at site II resulted in an increased exposure of Met residues 45 and 81 to the soluble spin label HyTEMPO. This result is consistent with an opening of the hydrophobic pocket in the N-terminal domain of cTnC upon binding Ca2+ at site II. The binding of the inhibitory peptide cTnIp, corresponding to Asn 129 through Ile 149 of cTnI, to both 2Ca(2+)-loaded and Ca(2+)-saturated cTnC was shown to protect Met residues 120 and 157 from HyTEMPO as determined by a decrease in their measured R1 values. These results suggest that in both the 2Ca(2+)-loaded and Ca(2+)-saturated forms of cTnC, cTnIp binds primarily to the C-terminal domain of cTnC.     

Comparative NMR studies on cardiac troponin C and a mutant incapable of binding calcium at site II

One- and two-dimensional NMR techniques were used to study both the influence of mutations on the structure of recombinant normal cardiac troponin C (cTnC3) and the conformational changes induced by Ca2+ binding to site II, the site responsible for triggering muscle contraction. Spin systems of the nine Phe and three Tyr residues were elucidated from DQF-COSY and NOESY spectra. Comparison of the pattern of NOE connectivities obtained from a NOESY spectrum of cTnC3 with a model of cTnC based on the crystal structure of skeletal TnC permitted sequence-specific assignment of all three Tyr residues, as well as Phe-101 and Phe-153. NOESY spectra and calcium titrations of cTnC3 monitoring the aromatic region of the 1H NMR spectrum permitted localization of six of the nine Phe residues to either the N- or C-terminal domain of cTnC3. Analysis of the downfield-shifted C alpha H resonances permitted sequence-specific assignment of those residues involved in the beta-strand structures which are part of the Ca(2+)-binding loops in both the N- and C-terminal domains of cTnC3. The short beta-strands in the N-terminal domain of cTnC3 were found to be present and in close proximity even in the absence of Ca2+ bound at site II. Using these assignments, we have examined the effects of mutating Asp-65 to Ala, CBM-IIA, a functionally inactive mutant which is incapable of binding Ca2+ at site II [Putkey, J.A., Sweeney, H. L., & Campbell, S. T. (1989) J. Biol. Chem. 264, 12370]. Comparison of the apo, Mg(2+)-, and Ca(2+)-bound forms of cTnC3 and CBM-IIA demonstrates that the inability of CBM-IIA to trigger muscle contraction is not due to global structural changes in the mutant protein but is a consequence of the inability of CBM-IIA to bind Ca2+ at site II. The pattern of NOEs between aromatic residues in the C-terminal domain is nearly identical in cTnC3 and CBM-IIA. Similar interresidue NOEs were also observed between Phe residues assigned to the N-terminal domain in the Ca(2+)-saturated forms of both cTnC3 and CBM-IIA. However, chemical shift changes were observed for the N-terminal Phe residues in CBM-IIA. This suggests that binding of Ca2+ to site II alters the chemical environment of the residues in the N-terminal hydrophobic cluster without disrupting the spatial relationship between the Phe residues located in helices A and D.     

Ca2+-induced conformational transition in the inhibitory and regulatory regions of cardiac troponin I

Cardiac muscle activation is initiated by the binding of Ca(2+) to the single N-domain regulatory site of cardiac muscle troponin C (cTnC). Ca(2+) binding causes structural changes between cTnC and two critical regions of cardiac muscle troponin I (cTnI): the regulatory region (cTnI-R, residues 150-165) and the inhibitory region (cTnI-I, residues130-149). These changes are associated with a decreased cTnI affinity for actin and a heightened affinity for cTnC. Using F?rster resonance energy transfer, we have measured three intra-cTnI distances in the deactivated (Mg(2+)-saturated) and Ca(2+)-activated (Ca(2+)-saturated) states in reconstituted binary (cTnC-cTnI) and ternary (cTnC-cTnI-cTnT) troponin complexes. Distance A (spanning cTnI-R) was unaltered by Ca(2+). Distances B (spanning both cTnI-R and cTnI-I) and C (from a residue flanking cTnI-I to a residue in the center of cTnI-R) exhibited Ca(2+)-induced increases of >8 A. These results compliment our previous determination of the distance between residues flanking cTnI-I alone. Together, the data suggest that Ca(2+) activation causes residues within cTnI-I to switch from a beta-turn/coil to an extended quasi-alpha-helical conformation as the actin-contacts are broken, whereas cTnI-R remains alpha-helical in both Mg(2+)- and Ca(2+)-saturated states. We have used the data to construct a structural model of the cTnI inhibitory and regulatory regions in the Mg(2+)- and Ca(2+)-saturated states.     

Kinetic studies of calcium and cardiac troponin I peptide binding to human cardiac troponin C using NMR spectroscopy

Ca2+ and human cardiac troponin I (cTnI) peptide binding to human cardiac troponin C (cTnC) have been investigated with the use of 2D [1H,15N] HSQC NMR spectroscopy. The spectral intensity, chemical shift, and line-shape changes were analyzed to obtain the dissociation ( K(D)) and off-rate ( k(off)) constants at 30 degrees C. The results show that sites III and IV exhibit 100-fold higher Ca2+ affinity than site II ( K(D(III,IV)) approximately 0.2 microM, K(D(II)) approximately 20 microM), but site II is partially occupied before sites III and IV are saturated. The addition of the first two equivalents of Ca2+ saturates 90% of sites III and IV and 20% of site II. This suggests that the Ca2+ occupancy of all three sites may contribute to the Ca2+-dependent regulation in muscle contraction. We have determined a k(off) of 5000 s(-1) for site II Ca2+ dissociation at 30 degrees C. Such a rapid off-rate had not been previously measured. Three cTnI peptides, cTnI(34-71), cTnI(128-147), and cTnI(147-163), were titrated to Ca2+-saturated cTnC. In each case, the binding occurs with a 1:1 stoichiometry. The determined K(D) and k(off) values are 1 microM and 5 s(-1) for cTnI(34-71), 78+/-10 microM and 5000 s(-1) for cTnI(128-147), and 150+/-10 microM and 5000 s(-1) for cTnI(147-163), respectively. Thus, the dissociation of Ca2+ from site II and cTnI(128-147) and cTnI(147-163) from cTnC are rapid enough to be involved in the contraction/relaxation cycle of cardiac muscle, while that of cTnI(34-71) from cTnC may be too slow for this process.     

Phosphorylation and mutation of human cardiac troponin I deferentially destabilize the interaction of the functional regions of troponin I with troponin C

We have utilized 2D [(1)H,(15)N]HSQC NMR spectroscopy to elucidate the binding of three segments of cTnI in native, phosphorylated, and mutated states to cTnC. The near N-terminal region (cRp; residues 34-71) contains the protein kinase C (PKC) phosphorylation sites S41 and S43, the inhibitory region (cIp; residues 128-147) contains another PKC site T142 and a familial hypertrophic cardiomyopathy (FHC) mutation R144G, and the switch region (cSp; residues 147-163) contains the novel p21-activated kinase (PAK) site S149 and another FHC mutation R161W. While S41/S43 phosphorylation of cRp had minimal disruption in the interaction of cRp and cTnC.3Ca(2+), T142 phosphorylation reduced the affinity of cIp for cCTnC.2Ca(2+) by approximately 14-fold and S149 phosphorylation reduced the affinity of cSp for cNTnC.Ca(2+) by approximately 10-fold. The mutation R144G caused an approximately 6-fold affinity decrease of cIp for cCTnC.2Ca(2+) and mutation R161W destabilized the interaction of cSp and cNTnC.Ca(2+) by approximately 1.4-fold. When cIp was both T142 phosphorylated and R144G mutated, its affinity for cCTnC.2Ca(2+) was reduced approximately 19-fold, and when cSp was both S149 phosphorylated and R161W mutated, its affinity for cNTnC.Ca(2+) was reduced approximately 4-fold. Thus, while the FHC mutation R144G enhances the effect of T142 phosphorylation on the interaction of cIp and cCTnC.2Ca(2+), the FHC mutation R161W suppresses the effect of S149 phosphorylation on the interaction of cSp and cNTnC.Ca(2+), demonstrating linkages between the FHC mutation and phosphorylation of cTnI. The observed alterations corroborate well with structural data. These results suggest that while the modifications in the cRp region have minimal influence, those in the key functional cIp-cSp region have a pronounced effect on the interaction of cTnI and cTnC, which may correlate with the altered myofilament function and cardiac muscle contraction under pathophysiological conditions.     

Drug binding to cardiac troponin C.

Compounds that sensitize cardiac muscle to Ca(2+) by intervening at the level of regulatory thin filament proteins would have potential therapeutic benefit in the treatment of myocardial infarctions. Two putative Ca(2+) sensitizers, EMD 57033 and levosimendan, are reported to bind to cardiac troponin C (cTnC). In this study, we use heteronuclear NMR techniques to study drug binding to [methyl-(13)C]methionine-labeled cTnC when free or when complexed with cardiac troponin I (cTnI). In the absence of Ca(2+), neither drug interacted with cTnC. In the presence of Ca(2+), one molecule of EMD 57033 bound specifically to the C-terminal domain of free cTnC. NMR and equilibrium dialysis failed to demonstrate binding of levosimendan to free cTnC, and the presence of levosimendan had no apparent effect on the Ca(2+) binding affinity of cTnC. Changes in the N-terminal methionine methyl chemical shifts in cTnC upon association with cTnI suggest that cTnI associates with the A-B helical interface and the N terminus of the central helix in cTnC. NMR experiments failed to show evidence of binding of levosimendan to the cTnC.cTnI complex. However, levosimendan covalently bound to a small percentage of free cTnC after prolonged incubation with the protein. These findings suggest that levosimendan exerts its positive inotropic effect by mechanisms that do not involve binding to cTnC.     

Ca2+-dependent interaction of the inhibitory region of troponin I with acidic residues in the N-terminal domain of troponin C

Ca2+ regulation of vertebrate striated muscle contraction is initiated by conformational changes in the N-terminal, regulatory domain of the Ca2+-binding protein troponin C (TnC), altering the interaction of TnC with the other subunits of troponin complex, TnI and TnT. We have investigated the role of acidic amino acid residues in the N-terminal, regulatory domain of TnC in binding to the inhibitory region (residues 96-116) of TnI. We constructed three double mutants of TnC (E53A/E54A, E60A/E61A and E85A/D86A), in which pairs of acidic amino acid residues were replaced by neutral alanines, and measured their affinities for synthetic inhibitory peptides. These peptides had the same amino acid sequence as TnI segments 95-116, 95-119 or 95-124, except that the natural Phe-100 of TnI was replaced by a tryptophan residue. Significant Ca2+-dependent increases in the affinities of the two longer peptides, but not the shortest one, to TnC could be detected by changes in Trp fluorescence. In the presence of Ca2+, all the mutant TnCs showed about the same affinity as wild-type TnC for the inhibitory peptides. In the presence of Mg2+ and EGTA, the N-terminal, regulatory Ca2+-binding sites of TnC are unoccupied. Under these conditions, the affinity of TnC(E85A/D86A) for inhibitory peptides was about half that of wild-type TnC, while the other two mutants had about the same affinity. These results imply a Ca2+-dependent change in the interaction of TnC Glu-85 and/or Asp-86 with residues (117-124) on the C-terminal side of the inhibitory region of TnI. Since Glu-85 and/or Asp-86 of TnC have also been demonstrated to be involved in Ca2+-dependent regulation through interaction with TnT, this region of TnC must be critical for troponin function.     

Various properties of cardiac troponin C tryptic peptides

Using chromatography and preparative polyacrylamide gel electrophoresis, tryptic peptides TP 1 (residues 47-83), TP 2 (residues 84-118) and TP 3 (residues 119-161) were isolated in a highly homogeneous state from cardiac troponin C. Peptides TP 1, TP 2 and TP 3 were found to contain isolated cation-binding sites II, III and IV of cardiac troponin C. The interaction of these peptides with troponins I and T was studied. It was found that only peptide TP 2 could interact with troponin I. Neither of the peptides isolated interacted with troponin T. The cation-binding properties and structural peculiarities of peptide TP 1 were investigated. It was shown that despite its small size (37 amino acid residues), peptide TP 1 retained its ability to bind Ca2+ which caused conformational changes in the peptide structure. This was accompanied by changes in the electrophoretic mobility and absorption of TP 1 on phenyl-Sepharose.     

NMR and mutagenesis studies on the phosphorylation region of human cardiac troponin I

Phosphorylation of the cardiac troponin complex by PKA at S22 and S23 of troponin I (TnI) accelerates Ca(2+) release from troponin C (TnC). The region of TnI around the bisphosphorylation site binds to, and stabilizes, the Ca(2+) bound N-terminal domain of TnC. Phosphorylation interferes with this interaction between TnI and TnC resulting in weaker Ca(2+) binding. In this study, we used (1)H-(15)N-HSQC NMR to investigate at the atomic level the interaction between an N-terminal fragment of TnI consisting of residues 1-64 of TnI (I1-64) and TnC. We produced several mutants of I1-64, TnI, and TnC to test the contribution of certain residues to the transmission of the phosphorylation signal in both NMR experiments and functional assays. We also investigated how phosphorylation of the PKC sites in I1-64 (S41 and S43) affects the interaction of I1-64 with TnC. We found that phosphorylation of S22 and S23 produced only localized effects in the structure of I1-64 between residues 24 and 34. Residues 1-17 of I1-64 did not bind to TnC, and residues 38-64 bound tightly to the C-terminal domain of TnC regardless of phosphorylation. Residues 22-34 bound weakly to TnC in a phosphorylation sensitive manner. Bisphosphorylation prevented this phosphorylation switch region from interacting with TnC. Systematic mutation of residues in the phosphorylation switch did not prevent PKA phosphorylation from accelerating Ca(2+) release from troponin. We conclude that the phosphorylation switch binds to TnC via an extended interaction site spanning residues R19 to A34.     

Chymotryptic subfragments of troponin T from rabbit skeletal muscle. Interaction with tropomyosin, troponin I and troponin C

The binding of the chymotryptic troponin T subfragments to tropomyosin, troponin I, and troponin C was semiquantitatively examined by using affinity chromatography, and also by co-sedimentation with F-actin and polyacrylamide gel electrophoresis in 14 mM Tris/90 mM glycine. Circular dichroism spectra of the subfragments were measured to confirm that the subfragments retained their conformational structures. Based on these results, the binding sites of tropomyosin, troponin I, and troponin C on the troponin T sequence were elucidated. Tropomyosin bound mainly to the region of troponin T1 (residues 1-158) with the same binding strength as to the original troponin T. The C-terminal region of troponin T (residues 243-259) was the second binding site to tropomyosin under physiological conditions. The binding site of troponin I was concluded to be the region including residues 223-227. The binding of troponin C was dependent on Ca2+ ion concentration. The C-terminal region of troponin T2 (residues 159-259) was indicated to be the Ca2+-independent troponin C-binding site and the N-terminal side of troponin T2 to be the Ca2+-dependent site.