Ca2+ binding to skeletal muscle troponin C in skeletal and cardiac myofibrils

Ca2+ binding to skeletal muscle troponin C in skeletal or cardiac myofibrils was measured by the centrifugation method using 45Ca. The specific Ca2+ binding to troponin C was obtained by subtracting the amount of Ca2+ bound to the CDTA-treated myofibrils (troponin C-depleted myofibrils) from that to the myofibrils reconstituted with troponin C. Results of Ca2+ binding measurement at various Ca2+ concentrations showed that skeletal troponin C had two classes of binding sites with different affinity for Ca2+. The Ca2+ binding of low-affinity sites in cardiac myofibrils was about eight times lower than that in skeletal myofibrils, while the high-affinity sites of troponin C in skeletal or cardiac myofibrils showed almost the same affinity for Ca2+. The Ca2+ sensitivity of the ATPase activity of skeletal troponin C-reconstituted cardiac myofibrils was also about eight times lower than that of skeletal myofibrils reconstituted with troponin C. These findings indicated that the difference in the sensitivity to Ca2+ of the ATPase activity between skeletal and cardiac CDTA-treated myofibrils reconstituted with skeletal troponin C was mostly due to the change in the affinity for Ca2+ of the low-affinity sites on the troponin C molecule.     

A laser Raman spectroscopic study of Ca2+ binding to troponin C.

Laser Raman spectroscopy has been used detect structural changes in troponin C induced by Ca2+ binding. Addition of Ca2+ - Mg2+ sites produces perturbations in the amide III region of the spectrum indicative of increased alpha-helical content, and in regions of the spectrum corresponding to carboxylate, thiol, and phenol side chains. However, Ca2+ binding to the low affinity Ca2+ - specific sites is not detected by laser Raman spectral changes.     

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⁺.     

Structure of the Mg2+-loaded C-lobe of cardiac troponin C bound to the N-domain of cardiac troponin I: comparison with the Ca2+-loaded structure

Cardiac troponin C (cTnC) is the Ca(2+)-binding component of the troponin complex and, as such, is the Ca(2+)-dependent switch in muscle contraction. This protein consists of two globular lobes, each containing a pair of EF-hand metal-binding sites, connected by a linker. In the N lobe, Ca(2+)-binding site I is inactive and Ca(2+)-binding site II is primarily responsible for initiation of muscle contraction. The C lobe contains Ca(2+)/Mg(2+)-binding sites III and IV, which bind Mg(2+) with lower affinity and play a structural as well as a secondary role in modulating the Ca(2+) signal. To understand the structural consequences of Ca(2+)/Mg(2+) exchange in the C lobe, we have determined the NMR solution structure of the Mg(2+)-loaded C lobe, cTnC(81-161), in a complex with the N domain of cardiac troponin I, cTnI(33-80), and compared it with a refined Ca(2+)-loaded structure. The overall tertiary structure of the Mg(2+)-loaded C lobe is very similar to that of the refined Ca(2+)-loaded structure as evidenced by the root-mean-square deviation of 0.94 A for all backbone atoms. While metal-dependent conformational changes are minimal, substitution of Mg(2+) for Ca(2+) is characterized by condensation of the C-terminal portion of the metal-binding loops with monodentate Mg(2+) ligation by the conserved Glu at position 12 and partial closure of the cTnI hydrophobic binding cleft around site IV. Thus, conformational plasticity in the Ca(2+)/Mg(2+)-dependent binding loops may represent a mechanism to modulate C-lobe cTnC interactions with the N domain of cTnI.     

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.     

Common structural framework of the two Ca2+/Mg2+ binding loops of troponin C and other Ca2+ binding proteins

The refinement of the crystal structure of turkey skeletal muscle troponin C at 2.2-A resolution reveals that the two calcium binding loops that are occupied by Ca2+ ions adopt conformations very similar to those of the two homologous loops of parvalbumin and to that of loop III-IV of the intestinal calcium binding protein. This specific fold assures suitable spatial positioning of the Ca2+ ligands. It consists of two reverse turns, one located at each end of the loop, and four Asx turns (a cyclic hydrogen-bonded structure involving an oxygen of the side chain of residue n and the main-chain amide nitrogen of residue n + 2) whenever such a side chain coordinates to the metal ion. The fifth Ca2+ coordination position in both loops of troponin C is occupied by a water molecule that is within hydrogen-bonding distance of an aspartic acid, thus mediating indirect interaction between the cation and the negatively charged carboxylate. The same loop framework is conserved in the two Ca2+ binding loops of parvalbumin and loop III-IV of the intestinal Ca2+ binding protein in spite of the variability in the nature of the side chains at equivalent positions. The disposition of the Ca2+ and of its coordinating water molecule relative to the protein main chain is conserved in all these cases.     

The effect of troponin C removal on the Ca2+-sensitive binding of Mg2+ AMPPNP to myofibrils

It was previously shown that when rabbit skeletal myofibrils are titrated with Mg2+ AMPPNP under conditions that result in the dissociation of cross-bridges from the thin filaments (i.e. 50% ethylene glycol, 0 degrees C), Ca2+-sensitive, biphasic binding is observed. These titrations have been repeated using myofibrils from which the troponin C has been selectively removed. The disappearance of both Ca2+ sensitivity and biphasic binding is taken as evidence that the Ca2+ sensitivity is due to Ca2+ binding to troponin C and the biphasic binding of Mg2+ AMPPNP observed in intact myofibrils is not due to packing constraints or steric hindrance.     

A model for human cardiac troponin C and for modulation of its Ca2+ affinity by drugs

Calcium sensitizers are drugs which increase force development in striated muscle by sensitizing myofilaments to Ca2+. This can happen by increasing Ca2+ affinity of the regulatory domain of Ca2+ binding protein troponin C. High resolution crystal structures of two calcium binding proteins, calmodulin (Babu et al.: J. Mol. Biol. 203:191-204, 1988) and skeletal troponin C (Satyshur et al.: J. Biol. Chem. 263:1628-1647, 1988; Herzber et al.: J. Mol. Biol. 203:761-779, 1988), have recently been published. This makes it possible to model in detail the calcium-sensitizing action of drugs on troponin C. In this study a model of human cardiac troponin C in three-calcium state has been constructed. When calcium is bound to calcium site II of cardiac troponin C an open conformation of the protein results, which has a hydrophobic pocket surrounded by a few polar side chains. Complexation of three drugs, trifluoperazine, bepridil, and pimobendan, to the hydrophobic pocket is studied using energy minimization techniques. Two different binding modes are found, which differ in the location of a strong electrostatic interaction. In analogy with the crystal structure of skeletal troponin C it is hypothezed that in cardiac troponin C an interaction occurs between Gln-50 and Asp-88, which has a long-range effect on calcium binding. The binding modes of drugs, where a strong interaction with Asp-88 exists, can effectively prevent the interaction between Asp-88 and Gln-50 in the protein, and are proposed to be responsible for the calcium-sensitizing properties of the studied drugs.     

Calcium binding to troponin C. II. A Ca2+ ion titration study with a Ca2+ ion sensitive electrode

The effects of pH,Mg2+, and ionic strength on Ca2+ binding to rabbit skeletal troponin C were studied by using a Ca2+ sensitive electrode. Troponin C has two high affinity and two low affinity sites and the Ca2+ affinity of both sites was increased by increasing pH in a pH range from pH 5.6 to 10.4. The affinity was decreased by increasing ionic strength. The change of the Ca2+ affinity can be explained by the electrostatic interaction between Ca2+ and the protein. At alkaline pH, the four Ca2+ binding sites bind Ca2+ with the same affinity and the distinction between the high and the low affinity sites vanished. This result shows that the difference of the Ca2+ affinity is owing to differences of the secondary or the tertiary structure of the Ca2+ binding sites, not owing to a difference of the primary structures of the Ca2+ binding sites. The two high affinity sites bound two Ca2+ ions cooperatively in neutral pH. The cooperativity was diminished at both acidic and alkaline pH. Mg2+ ion decreased the affinity of the low affinity sites.     

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.     

Effects of cardiac thin filament Ca2+: statistical mechanical analysis of a troponin C site II mutant.

Cardiac thin filaments contain many troponin C (TnC) molecules, each with one regulatory Ca2+ binding site. A statistical mechanical model for the effects of these sites is presented and investigated. The ternary troponin complex was reconstituted with either TnC or the TnC mutant CBMII, in which the regulatory site in cardiac TnC (site II) is inactivated. Regardless of whether Ca2+ was present, CBMII-troponin was inhibitory in a thin filament-myosin subfragment 1 MgATPase assay. The competitive binding of [3H]troponin and [14C]CBMII-troponin to actin.tropomyosin was measured. In the presence of Mg2+ and low free Ca2+ they had equal affinities for the thin filament. When Ca274+ was added, however, troponin's affinity for the thin filament was 2.2-fold larger for the mutant than for the wild type troponin. This quantitatively describes the effect of regulatory site Ca2+ on troponin's affinity for actin.tropomyosin; the decrease in troponin-thin filament binding energy is small. Application of the theoretical model to the competitive binding data indicated that troponin molecules bind to interdependent rather than independent sites on the thin filament. Ca2+ binding to the regulatory site of TnC has a long-range rather than a merely local effect. However, these indirect TnC-TnC interactions are weak, indicating that the cooperativity of muscle activation by Ca2+ requires other sources of cooperativity.     

Ca2+ binding sites in calmodulin and troponin C alter interhelical angle movements

Molecular dynamics analyses were performed to examine conformational changes in the C-domain of calmodulin and the N-domain of troponin C induced by binding of Ca(2+) ions. Analyses of conformational changes in calmodulin and troponin C indicated that the shortening of the distance between Ca(2+) ions and Ca(2+) binding sites of helices caused widening of the distance between Ca(2+) binding sites of helices on opposite sides, while the hydrophobic side chains in the center of helices hardly moved due to their steric hindrance. This conformational change acts as the clothespin mechanism.     

Cooperative binding to the Ca2+-specific sites of troponin C in regulated actin and actomyosin

The Ca2+-binding component of troponin (TnC) and its proteolytic fragments containing Ca2+-binding sites I-III (TH1) or sites III and IV (TR2C) have been labeled with the fluorescent probes dansylaziridine (DANZ) at methionine 25 or 5-(iodoacetamidoethyl)amino-naphthalene-1-sulfonic acid (AEDANS) at cysteine-98. These probes report binding of Ca2+ to the low and high affinity sites, respectively. Fluorescence changes as a function of [Ca2+] were measured for the free peptides, their complexes with troponin I + troponin T, and these complexes bound to actin-tropomyosin in the presence of Mg2+ and ATP with and without myosin. An apparent Hill coefficient of 1.0-1.1 has been obtained for the Ca2+-induced fluorescence changes in TnC, its fragments, and their ternary complexes regardless of the label used. When a ternary complex containing appropriately labeled TnC or its fragment is bound to the actin-tropomyosin complex, the Hill coefficient for the titration of the low affinity sites increases to 1.5-1.6 and further increases to greater than 2 in the presence of myosin. To interpret the apparent Hill coefficients, we used a model containing two binding sites and a single reporter of the conformational change. Hill coefficients between 1.0 and 1.2 can be obtained for the fluorescence change without true cooperativity in metal binding, depending on the mechanism of the fluorescence change; i.e. the contribution of the singly or doubly occupied species to the fluorescence change. A Hill coefficient between 1.2 and 2, however, always indicates cooperativity in binding independently of the mechanism. Thus, our finding that fluorescence titrations of Ca2+ binding to TnCDANZ bound to actin-tropomyosin exhibit a Hill coefficient of 1.5 in the absence of myosin and 2.4 in its presence indicates the existence of true positive cooperativity in metal binding to sites I and II. No cooperativity was observed for AEDANS-labeled complexes that reflect Ca2+-binding to the high affinity sites. Plots of the Ca2+ dependence of myosin ATPase activity activated by actin-tropomyosin in the presence of any of the troponin complexes used had apparent Hill coefficients of approximately 4. The higher value suggests cooperative interactions in the activation of ATPase beyond those involved in Ca2+-binding to the Ca2+-specific sites.     

The effect of Mg2+ on the Ca2+ binding to troponin C in rabbit fast skeletal myofibrils.

The effect of Mg2+ on the Ca2+ binding to rabbit fast skeletal troponin C and the CA2+ dependence of myofibrillar ATPase activity was studied in the physiological state where troponin C was incorporated into myofibrils. The Ca2+ binding to troponin C in myofibrils was measured directly by 45Ca using the CDTA-treated myofibrils as previously reported (Morimoto, S. and Ohtsuki, I. (1989) J. Biochem. 105, 435-439). It was found that the Ca2+ binding to the low and high affinity sites of troponin C in myofibrils was affected by Mg2+ competitively and the Ca2(+)- and Mg2(+)-binding constants were 6.20 x 10(6) and 1.94 x 10(2) M-1, respectively, for the low affinity sites, and 1.58 x 10(8) and 1.33 x 10(3) M-1, respectively, for the high affinity sites. The Ca2+ dependence of myofibrillar ATPase was also affected by Mg2+, with the apparent Ca2(+)- and Mg2(+)-binding constants of 1.46 x 10(6) and 276 x 10(2) M-1, respectively, suggesting that the myofibrillar ATPase was modulated through a competitive action of Mg2+ on Ca2+ binding to the low affinity sites, though the Ca2+ binding to the low affinity sites was not simply related to the myofibrillar ATPase.     

Effect of troponin C on the cooperativity in Ca2+ activation of cardiac muscle

The presence of protein kinase C (PKC), a key enzyme in signal transduction, has not been investigated in fungal cells. The phorbol ester TPA, an activator of PKC, may be used as an indicator of the presence and role of PKC in Phycomyces blakesleeanus spores. Activation of spore germination by acetate was prevented by 6 nM TPA. The TPA analog 4 alpha PDD, an ineffective activator of PKC, did not affect spore germination. 3 mM dbcAMP, on the other hand, reversed the inhibition of germination caused by TPA. TPA-stimulated protein kinase activity was detected in spores. The possible relationship between PKC and the increased levels of cAMP that accompany the induction of spore germination is discussed.     

Ca(2+) binding to cardiac troponin C: effects of temperature and pH on mammalian and salmonid isoforms

A reduction in temperature lowers the Ca(2+) sensitivity of skinned cardiac myofilaments but this effect is attenuated when native cardiac troponin C (cTnC) is replaced with skeletal TnC. This suggests that conformational differences between the two isoforms mediate the influence of temperature on contractility. To investigate this phenomenon, the functional characteristics of bovine cTnC (BcTnC) and that from rainbow trout, Oncorhynchus mykiss, a cold water salmonid (ScTnC), have been compared. Rainbow trout maintain cardiac function at temperatures cardioplegic to mammals. To determine whether ScTnC is more sensitive to Ca(2+) than BcTnC, F27W mutants were used to measure changes in fluorescence with in vitro Ca(2+) titrations of site II, the activation site. When measured under identical conditions, ScTnC was more sensitive to Ca(2+) than BcTnC. At 21 degrees C, pH 7.0, as indicated by K(1/2) (-log[Ca] at half-maximal fluorescence, where [Ca] is calcium concentration), ScTnC was 2.29-fold more sensitive to Ca(2+) than BcTnC. When pH was kept constant (7.0) and temperature was lowered from 37.0 to 21.0 degrees C and then to 7.0 degrees C, the K(1/2) of BcTnC decreased by 0.13 and 0.32, respectively, whereas the K(1/2) of ScTnC decreased by 0.76 and 0.42, respectively. Increasing pH from 7.0 to 7.3 at 21.0 degrees C increased the K(1/2) of both BcTnC and ScTnC by 0.14, whereas the K(1/2) of both isoforms was increased by 1.35 when pH was raised from 7.0 to 7.6 at 7.0 degrees C.