Function of the N-terminal calcium-binding sites in cardiac/slow troponin C assessed in fast skeletal muscle fibers

Fast skeletal troponin C (sTnC) has two low affinity Ca(2+)-binding sites (sites I and II), whereas in cardiac troponin C (cTnC) site I is inactive. By modifying the Ca2+ binding properties of sites I and II in cTnC it was demonstrated that binding of Ca2+ to an activated site I alone is not sufficient for triggering contraction in slow skeletal muscle fibers (Sweeney, H.L., Brito, R. M.M., Rosevear, P.R., and Putkey, J.A. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 9538-9542). However, a similar study using sTnC showed that Ca2+ binding to site I alone could partially activate force production in fast skeletal muscle fibers (Sheng, Z., Strauss, W.L., Francois, J.M., and Potter, J.D. (1990) J. Biol. Chem. 265, 21554-21560). The purpose of the current study was to examine the functional characteristics of modified cTnC derivatives in fast skeletal muscle fibers to assess whether or not either low affinity site can mediate force production when coupled to fast skeletal isoforms of troponin (Tn) I and TnT. Normal cTnC and sTnC were compared with engineered derivatives of cTnC having either both sites I and II active, or only site I active. In contrast to what is seen in slow muscle, binding of Ca2+ to site I alone recovered about 15-20% of the normal calcium-activated force and ATPase activity in skinned fast skeletal muscle fibers and myofibrils, respectively. This is most likely due to structural differences between TnI and/or TnT isoforms that allow for partial recognition and translation of the signal represented by binding Ca2+ to site I of TnC when associated with fast skeletal but not slow skeletal muscle.     

Mutation of the high affinity calcium binding sites in cardiac troponin C.

Fast skeletal and cardiac troponin C (TnC) contain two high affinity Ca2+/Mg2+ binding sites within the C-terminal domain that are thought to be important for association of TnC with the troponin complex of the thin filament. To test directly the function of these high affinity sites in cardiac TnC they were systematically altered by mutagenesis to generate proteins with a single inactive site III or IV (CBM-III and CBM-IV, respectively), or with both sites III and IV inactive (CBM-III-IV). Equilibrium dialysis indicated that the mutated sites did not bind Ca2+ at pCa 4. Both CBM-III and CBM-IV were similar to the wild type protein in their ability to regulate Ca(2+)-dependent contraction in slow skeletal muscle fibers, and Ca(2+)-dependent ATPase activity in fast skeletal and cardiac muscle myofibrils. The mutant CBM-III-IV is capable of regulating contraction in permeabilized slow muscle fibers but only if the fibers are maintained in a contraction solution containing a high concentration of the mutant protein. CBM-III-IV also regulates myofibril ATPase activity in fast skeletal and cardiac myofibrils but only at concentrations 10-100-fold greater than the normal protein. The pCa50 and Hill coefficient values for Ca(2+)-dependent activation of fast skeletal muscle myofibril ATPase activity by the normal protein and all three mutants are essentially the same. Competition between active and inactive forms of cardiac and slow TnC in a functional assay demonstrates that mutation of both sites III and IV greatly reduces the affinity of cardiac and slow TnC for its functionally relevant binding site in the myofibrils. The data indicate that although neither high affinity site is absolutely essential for regulation of muscle contraction in vitro, at least one active C-terminal site is required for tight association of cardiac troponin C with myofibrils. This requirement can be satisfied by either site III or IV.     

Site-directed spin labeling electron paramagnetic resonance study of the calcium-induced structural transition in the N-domain of human cardiac troponin C complexed with troponin I

Calcium-induced structural transition in the amino-terminal domain of troponin C (TnC) triggers skeletal and cardiac muscle contraction. The salient feature of this structural transition is the movement of the B and C helices, which is termed the "opening" of the N-domain. This movement exposes a hydrophobic region, allowing interaction with the regulatory domain of troponin I (TnI) as can be seen in the crystal structure of the troponin ternary complex [Takeda, S., Yamashita, A., Maeda, K., and Maeda, Y. (2003) Nature 424, 35-41]. In contrast to skeletal TnC, Ca(2+)-binding site I (an EF-hand motif that consists of an A helix-loop-B helix motif) is inactive in cardiac TnC. The question arising from comparisons with skeletal TnC is how both helices move according to Ca(2+) binding or interact with TnI in cardiac TnC. In this study, we examined the Ca(2+)-induced movement of the B and C helices relative to the D helix in a cardiac TnC monomer state and TnC-TnI binary complex by means of site-directed spin labeling electron paramagnetic resonance (EPR). Doubly spin-labeled TnC mutants were prepared, and the spin-spin distances were estimated by analyzing dipolar interactions with the Fourier deconvolution method. An interspin distance of 18.4 A was estimated for mutants spin labeled at G42C on the B helix and C84 on the D helix in a Mg(2+)-saturated monomer state. The interspin distance between Q58C on the C helix and C84 on the D helix was estimated to be 18.3 A under the same conditions. Distance changes were observed by the addition of Ca(2+) ions and the formation of a complex with TnI. Our data indicated that the C helix moved away from the D helix in a distinct Ca(2+)-dependent manner, while the B helix did not. A movement of the B helix by interaction with TnI was observed. Both Ca(2+) and TnI were also shown to be essential for the full opening of the N-domain in cardiac TnC.     

The dilated cardiomyopathy G159D mutation in cardiac troponin C weakens the anchoring interaction with troponin I

NMR spectroscopy has been employed to elucidate the molecular consequences of the DCM G159D mutation on the structure and dynamics of troponin C, and its interaction with troponin I (TnI). Since the molecular effects of human mutations are often subtle, all NMR experiments were conducted as direct side-by-side comparisons of the wild-type C-domain of troponin C (cCTnC) and the mutant protein, G159D. With the mutation, the affinity toward the anchoring region of cTnI (cTnI 34-71) was reduced ( K D = 3.0 +/- 0.6 microM) compared to that of the wild type ( K D < 1 microM). Overall, the structure and dynamics of the G159D.cTnI 34-71 complex were very similar to those of the cCTnC.cTnI 34-71 complex. There were, however, significant changes in the (1)H, (13)C, and (15)N NMR chemical shifts, especially for the residues in direct contact with cTnI 34-71, and the changes in NOE connectivity patterns between the G159D.cTnI 34-71 and cCTnC.cTnI 34-71 complexes. Thus, the most parsimonious hypothesis is that the development of disease results from the poor anchoring of cTnI to cCTnC, with the resulting increase in the level of acto-myosin inhibition in agreement with physiological data. Another possibility is that long-range electrostatic interactions affect the binding of the inhibitory and switch regions of cTnI (cTnI 128-147 and cTnI 147-163) and/or the cardiac specific N-terminus of cTnI (cTnI 1-29) to the N-domain of cTnC. These important interactions are all spatially close in the X-ray structure of the cardiac TnC core.     

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.     

Conformational changes in the metal-binding sites of cardiac troponin C induced by calcium binding.

Isotope labeling of recombinant normal cardiac troponin C (cTnC3) with 15N-enriched amino acids and multidimensional NMR were used to assign the downfield-shifted amide protons of Gly residues at position 6 in Ca(2+)-binding loops II, III, and IV, as well as tightly hydrogen-bonded amides within the short antiparallel beta-sheets between pairs of Ca(2+)-binding loops. The amide protons of Gly70, Gly110, and Gly146 were found to be shifted significantly downfield from the remaining amide proton resonances in Ca(2+)-saturated cTnC3. No downfield-shifted Gly resonance was observed from the naturally inactive site I. Comparison of downfield-shifted amide protons in the Ca(2+)-saturated forms of cTnC3 and CBM-IIA, a mutant having Asp65 replaced by Ala, demonstrated that Gly70 is hydrogen bonded to the carboxylate side chain of Asp65. Thus, the hydrogen bond between Gly and Asp in positions 6 and 1, respectively, of the Ca(2+)-binding loop appears crucial for maintaining the integrity of the helix-loop-helix Ca(2+)-binding sites. In the apo- form of cTnC3, only Gly70 was found to be shifted significantly downfield with respect to the remaining amide proton resonances. Thus, even in the absence of Ca2+ at binding site II, the amide proton of Gly70 is strongly hydrogen bonded to the side-chain carboxylate of Asp65. The amide protons of Ile112 and Ile148 in the C-terminal domain and Ile36 in the N-terminal domain data-sheets exhibit chemical shifts consistent with hydrogen-bond formation between the pair of Ca(2+)-binding loops in each domain of Ca(2+)-saturated cTnC3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distance measurements in cardiac troponin C

Intramolecular distance measurements were made in cardiac troponin C (cTnC) by fluorescence energy transfer using Eu3+ or Tb3+ as energy donors and Nd3+ or an organic chromophore as acceptors. The laser-induced luminescence of bound Eu3+ is quenched in Eu1Nd1cTnC with a lifetime of 0.328 ms, compared with 0.43 ms for Eu2cTnC. The enhanced decay corresponds to an energy transfer efficiency of 0.25, or a distance of 1.1 nm between the two high affinity sites. We have also labeled cTnC with 4-dimethylaminophenylazophenyl-4'-maleimide (DAB-Mal) at the two cysteine residues (Cys-35 and Cys-84). Energy transfer measurements were carried out between Tb3+ bound to the high affinity sites and the labels attached to the domain containing the low affinity site. Upon uv irradiation at pH 6.7, Tb1cTnCDAB emits tyrosine-sensitized Tb3+ luminescence that decays bioexponentially with lifetimes of 1.29 and 0.76 ms. The shorter lifetime is ascribed to energy transfer from Tb3+ to the DAB labels, yielding an average distance of 3.4 nm between the donor and the acceptors. At pH 5.0, however, the luminescence decays exclusively with a single lifetime of 1.31 ms, suggesting that under these conditions all Tb3+ ions are more than 5.2 nm away from the label. Thus cTnC, like skeletal TnC, undergoes a pH-dependent conformational transition which converts an elongated structure at lower pH's to a rather compact conformation in a more physiological medium.     

Solution structure of calcium-saturated cardiac troponin C bound to cardiac troponin I

Cardiac troponin C (TnC) is composed of two globular domains connected by a flexible linker. In solution, linker flexibility results in an ill defined orientation of the two globular domains relative to one another. We have previously shown a decrease in linker flexibility in response to cardiac troponin I (cTnI) binding. To investigate the relative orientation of calcium-saturated TnC domains when bound to cTnI, (1)H-(15)N residual dipolar couplings were measured in two different alignment media. Similarity in alignment tensor orientation for the two TnC domains supports restriction of domain motion in the presence of cTnI. The relative spatial orientation of TnC domains bound to TnI was calculated from measured residual dipolar couplings and long-range distance restraints utilizing a rigid body molecular dynamics protocol. The relative domain orientation is such that hydrophobic pockets face each other, forming a latch to constrain separate helical segments of TnI. We have utilized this structure to successfully explain the observed functional consequences of linker region deletion mutants. Together, these studies suggest that, although linker plasticity is important, the ability of TnC to function in muscle contraction can be correlated with a preferred domain orientation and interdomain distance.     

Identification of sites phosphorylated in bovine cardiac troponin I and troponin T by protein kinase C and comparative substrate activity of synthetic peptides containing the phosphorylation sites

As an extension of our previous reports that cardiac and skeletal muscle troponin I (Tn-I) and troponin T (Tn-T) are excellent substrates for protein kinase C (PKC) (Katoh, N., Wise, B. C., and Kuo, J. F. (1983) Biochem. J. 209, 189-195; Mazzei, G. J., and Kuo, J. F. (1984) Biochem. J. 218, 361-369), we have now determined that PKC phosphorylated serine 43 (and/or serine 45), serine 78, and threonine 144 in the free Tn-I subunit and threonine 190, threonine 199, and threonine 280 in the free Tn-T subunit of bovine cardiac troponin. PKC appeared to phosphorylate the same sites of the subunits present in the form of the troponin complex, as indicated by the similarity in the two-dimensional phosphopeptide maps. Although some of the phosphorylation sites were shared by other classes of protein kinases, PKC exhibited a distinct substrate specificity. It was also noted that phosphorylated serine and threonine residues in Tn-I and Tn-T had neighboring basic amino acid residues separated by 1 or 2 other residues both at the amino and carboxyl termini, in agreement with the conclusion of House et al. (House, C., Wettenhall, R. E. H., and Kemp, B. E. (1987) J. Biol. Chem. 262, 772-777) based upon their studies on other substrate proteins. Several peptides having sequences around the phosphorylating sites have been synthesized. The phosphorylation experiments indicated that these peptides were substrates for PKC, and their relative substrate activity (determined by the ratios of Vmax/Km) compared with other proteins, in descending order, was Tn-I = Tn-I(134-154) greater than Tn-T much greater than histone H1 greater than Tn-I(33-35) approximately Tn-T(268-284) greater than Tn-T(179-198) approximately Tn-T(191-209). It is suggested that PKC phosphorylation of Tn-I and Tn-T could be biologically significant in terms of possible modifications in interactions among the individual contractile protein components as well as the Ca2+ sensitivity and activity of actomyosin ATPase.     

人心肌肌钙蛋白C两种突变转基因小鼠模型建立与对比分析

目的：为建立心肌组织特异性表达人cTnCD145E和cTnCG159D突变基因转基因小鼠,为对比分析两种不同心肌病的发生发展建立模型。方法利用定点突变技术分别制备人cTnC基因的cTnCD145E和cTnCG159D两个突变体,随后插入心肌特异性表达启动子α-MHC下游构建人cTnCD145E和cTnCG159D基因转基因载体。通过显微注射法建立转基因C57BL/6小鼠。利用心脏超声和病理观察对比分析不同年龄转基因小鼠心脏的结构与功能。结果建立了心肌组织高表达人cTnCD145E和cTnCG159D突变基因转基因小鼠,cTnCD145E和cTnCG159D转基因小鼠随年龄增加,有分别向HCM和DCM发展的趋势,12月龄时,cTnCD145E转基因小鼠收缩末期和舒张末期左室容积（ left ventricle end-diastolic volume and end-systolic volume,EDV and ESV）与同窝阴性小鼠相比下降,射血分数（ejection fraction, EF）和收缩末期左心室后壁厚度（left ventricle end-systolic posterior wall thickness ,ESPWT）增加,而cTnCG159D转基因小鼠EDV和ESV与同窝阴性小鼠相比上升,EF和ESPWT减少。结论心肌组织特异性表达人cTnCD145E突变基因转基因小鼠表现肥厚型心肌病病理表型,而心肌组织特异性表达人cTnCG159D突变基因转基因小鼠表现扩张型心肌病病理表型,二者可作为对比研究由不同发病机制导致的心肌病模型。     

Interaction of bepridil with the cardiac troponin C/troponin I complex

Mammalian cells are characterized by an endomembrane system. Nevertheless, some cells lose these membranes during their terminal differentiation, e.g. red blood cells and lens fiber cells of the eye. 15-Lipoxygenase is believed to be critical for this membrane degradation. Here we use cultivated rabbit reticulocytes in the presence or absence of a lipoxygenase inhibitor to provide further evidence for the importance of 15-lipoxygenase for the in vivo degradation of mitochondria. We find that inhibitor treatment retarded mitochondrial degradation, as shown by persistence of marker proteins and by direct visualization of mitochondria by electron microscopy.     

The Ca/Mg sites of troponin C modulate crossbridge-mediated thin filament activation in cardiac myofibrils

The Ca²⁺/Mg²⁺ sites (III and IV) located in the C-terminal domain of cardiac troponin C (cTnC) have been generally considered to play a purely structural role in keeping the cTnC bound to the thin filament. However, several lines of evidence, including the discovery of cardiomyopathy-associated mutations in the C-domain, have raised the possibility that these sites may have a more complex role in contractile regulation. To explore this possibility, the ATPase activity of rat cardiac myofibrils was assayed under conditions in which no Ca²⁺ was bound to the N-terminal regulatory Ca²⁺-binding site (site II). Myosin-S1 was treated with N-ethylmaleimide to create strong-binding myosin heads (NEM-S1), which could activate the cardiac thin filament in the absence of Ca²⁺. NEM-S1 activation was assayed at pCa 8.0 to 6.5 and in the presence of either 1 mM or 30 μM free Mg²⁺. ATPase activity was maximal when sites III and IV were occupied by Mg²⁺ and it steadily declined as Ca²⁺ displaced Mg²⁺. The data suggest that in the absence of Ca²⁺ at site II strong-binding myosin crossbridges cause the opening of more active sites on the thin filament if the C-domain is occupied by Mg²⁺ rather than Ca²⁺. This finding could be relevant to the contraction–relaxation kinetics of cardiac muscle. As Ca²⁺ dissociates from site II of cTnC during the early relaxing phase of the cardiac cycle, residual Ca²⁺ bound at sites III and IV might facilitate the switching off of the thin filament and the detachment of crossbridges from actin.     

Calcium binding to cardiac troponin C

The binding of Ca²⁺ to cardiac troponin C was studied by determining changes in the fluorescence and circular dichroism of the protein and by following changes in the free Ca²⁺ concentration by means of a Ca²⁺-specific electrode. Cardiac troponin C contains three Ca²⁺-binding sites which fall into two classes —two sites with a higher affinity and one with a lower affinity. The higher-affinity sites also bind Mg²⁺ which competes with the Ca²⁺.     

Conformation of the regulatory domain of cardiac muscle troponin C in its complex with cardiac troponin I.

Calcium activation of fast striated muscle results from an opening of the regulatory N-terminal domain of fast skeletal troponin C (fsTnC), and a substantial exposure of a hydrophobic patch, essential for Ca(2+)-dependent interaction with fast skeletal troponin I (fsTnI). This interaction is obligatory to relieve the inhibition of strong, force-generating actin-myosin interactions. We have determined intersite distances in the N-terminal domain of cardiac TnC (cTnC) by fluorescence resonance energy transfer measurements and found negligible increases in these distances when the single regulatory site is saturated with Ca(2+). However, in the presence of bound cardiac TnI (cTnI), activator Ca(2+) induces significant increases in the distances and a substantial opening of the N-domain. This open conformation within the cTnC.cTnI complex has properties favorable for the Ca(2+)-induced interaction with an additional segment of cTnI. Thus, the binding of cTnI to cTnC is a prerequisite to achieve a Ca(2+)-induced open N-domain similar to that previously observed in fsTnC with no bound fsTnI. This role of cardiac TnI has not been previously recognized. Our results also indicate that structural information derived from a single protein may not be sufficient for inference of a structure/function relationship.     

Additional PKA phosphorylation sites in human cardiac troponin I.

We used mass spectrometry to monitor cAMP-dependent protein kinase catalysed phosphorylation of human cardiac troponin I in vitro. Phosphorylation of isolated troponin I by cAMP-dependent protein kinase resulted in the covalent incorporation of phosphate on at least five different sites on troponin I, and a S22/23A troponin I mutant incorporated phosphates on at least three sites. In addition to the established phosphorylation sites (S22 and S23) we found that S38 and S165 were the other two main sites of phosphorylation. These 'overphosphorylation' sites were not phosphorylated sufficiently slower than S22 and S23 that we could isolate pure S22/23 bisphosphorylated troponin I. Overphosphorylation of troponin I reduced its affinity for troponin C, as measured by isothermal titration microcalorimetry. Phosphorylation of S22/23A also decreased its affinity for troponin C indicating that phosphorylation of S38 and/or S165 impedes binding of troponin I to troponin C. Formation of a troponin I/troponin C complex prior to cAMP-dependent protein kinase treatment did not prevent overphosphorylation. When whole troponin was phosphorylated by cAMP-dependent protein kinase, however, [(32)P]phosphate was incorporated only into troponin I and only at S22 and S23. Mass spectrometry confirmed that overphosphorylation is abolished in the ternary complex. Troponin I bisphosphorylated exclusively at S22 and S23 troponin I showed reduced affinity for troponin C but the effect was diminished with respect to overphosphorylated troponin I. These results show that care should be exercised when interpreting data obtained with troponin I phosphorylated in vitro.     

Resolution and calcium-binding properties of the two major isoforms of troponin C from crayfish

Crayfish tail muscle troponin C (TnC) has been fractionated into its five components and the Ca2+-binding properties of the two major isoforms (alpha and gamma) determined by equilibrium dialysis. alpha-TnC contains one Ca2+-binding site with a binding constant of 1 x 10(6) M-1 and one Ca2+ site with a binding constant of 1 x 10(4) M-1. In the complex of alpha-TnC with troponin I (TnI) or with TnI and troponin T (TnT), both sites bind Ca2+ with a single affinity constant of 2-4 x 10(6) M-1. gamma-TnC contains two Ca2+-binding sites with a binding constant of 2 x 10(4) M-1. In the gamma-TnC.TnI and gamma-TnC.TnI.TnT complexes, the binding constant of one of the sites is increased to 4-5 x 10(6) M-1, while Ca2+ binding to the second site is hardly affected (KCa = 4-7 x 10(4) M-1). In the presence of 10 mM MgCl2, the two Ca2+-binding sites of both TnC isoforms exhibit a 2-3-fold lower affinity. Assuming competition between Ca2+ and Mg2+ for these sites, their binding constants for Mg2+ were 120-230 M-1. In the absence of Ca2+, however, alpha-TnC and gamma-TnC bind 4-5 mol of Mg2+/mol with a binding constant of 1 x 10(3) M-1. These results suggest that the effect of Mg2+ on Ca2+ binding at the two Ca2+ sites is noncompetitive, i.e. Mg2+ does not bind directly to these sites (Ca2+-specific sites). Since the formation of the complex of crayfish TnI with alpha-TnC or gamma-TnC increases significantly the affinity of one of their two Ca2+-specific sites, I conclude that the binding of Ca2+ to only one site (regulatory Ca2+-specific site) controls the Ca2+-dependent interaction between crayfish TnCs and TnI.     

Introduction of negative charge mimicking protein kinase C phosphorylation of cardiac troponin I. Effects on cardiac troponin C

Protein kinase C phosphorylation of cardiac troponin, the Ca(2+)-sensing switch in muscle contraction, is capable of modulating the response of cardiac muscle to a Ca(2+) ion concentration. The N-domain of cardiac troponin I contains two protein kinase C phosphorylation sites. Although the physiological consequences of phosphorylation at Ser(43)/Ser(45) are known, the molecular mechanisms responsible for these functional changes have yet to be established. In this work, NMR was used to identify conformational and dynamic changes in cardiac troponin C upon binding a phosphomimetic troponin I, having Ser(43)/Ser(45) mutated to Asp. Chemical shift perturbation mapping indicated that residues in helix G were most affected. Smaller chemical shift changes were observed in residues located in the Ca(2+)/Mg(2+)-binding loops. Amide hydrogen/deuterium exchange rates in the C-lobe of troponin C were compared in complexes containing either the wild-type or phosphomimetic N-domain of troponin I. In the presence of a phosphomimetic domain, exchange rates in helix G increased, whereas a decrease in exchange rates for residues mapping to Ca(2+)/Mg(2+)-binding loops III and IV was observed. Increased exchange rates are consistent with destabilization of the Thr(129)-Asp(132) helix capping box previously characterized in helix G. The perturbation of helix G and metal binding loops III and IV suggests that phosphorylation alters metal ion affinity and inter-subunit interactions. Our studies support a novel mechanism for protein kinase C signal transduction, emphasizing the importance of C-lobe Ca(2+)/Mg(2+)-dependent troponin interactions.     

Effects of T142 phosphorylation and mutation R145G on the interaction of the inhibitory region of human cardiac troponin I with the C-domain of human cardiac troponin C

Cardiac troponin I (cTnI) is the inhibitory component of the troponin complex, and its interaction with cardiac troponin C (cTnC) plays a critical role in transmitting the Ca(2+) signal to the other myofilament proteins in heart muscle contraction. The switch between contraction and relaxation involves a movement of the inhibitory region of cTnI (cIp) from cTnC to actin-tropomyosin. This region of cTnI is prone to missense mutations in heart disease, and a specific mutation, R145G, has been associated with familial hypertrophic cardiomyopathy. It also contains the unique cardiac PKC phosphorylation site at residue T142. To determine the structural consequences of the mutation R145G and the T142 phosphorylation on the interaction of cIp with cTnC, we have utilized 2D [(1)H, (15)N]-HSQC NMR spectroscopy to monitor the binding of native cIp, cIp-R (R145G), and cIp-P (phosphorylated T142), respectively, to the Ca(2+)-saturated C-domain of cTnC (cCTnC.2Ca(2+)). We also report a strategy for cloning, expression, and purification of cTnI peptide, and both synthetic and recombinant peptides are used in this study. NMR chemical shift mapping indicates that the binding epitope of cIp on cCTnC.2Ca(2+) is not greatly affected, but the affinity is reduced by approximately 14-fold by the T142 phosphorylation and approximately 4-fold by the mutation R145G, respectively. This suggests that these modifications of cIp have an adverse effect on the binding of cIp to cCTnC.2Ca(2+). These perturbations may correlate with the impairment or loss of cTnI function in heart muscle contraction.     

Cardiac troponin I inhibitory peptide: location of interaction sites on troponin C

Cardiac troponin I(129-149) binds to the calcium saturated cardiac troponin C/troponin I(1-80) complex at two distinct sites. Binding of the first equivalent of troponin I(129-149) was found to primarily affect amide proton chemical shifts in the regulatory domain, while the second equivalent perturbed amide proton chemical shifts within the D/E linker region. Nitrogen-15 transverse relaxation rates showed that binding the first equivalent of inhibitory peptide to the regulatory domain decreased conformational exchange in defunct calcium binding site I and that addition of the second equivalent of inhibitory peptide decreased flexibility in the D/E linker region. No interactions between the inhibitory peptide and the C-domain of cardiac troponin C were detected by these methods demonstrating that the inhibitory peptide cannot displace cTnI(1-80) from the C-domain.     

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.