Interaction of troponin I and troponin C: 19F NMR studies of the binding of the inhibitory troponin I peptide to turkey skeletal troponin C.

We have used 19F nuclear magnetic resonance spectroscopy to study the interaction of the inhibitory region of troponin (TnI) with apo- and calcium(II)-saturated turkey skeletal troponin C (TnC), using the synthetic TnI analogue N alpha-acetyl[19FPhe106]TnI(104-115)amide. Dissociation constants of Kd = (3.7 +/- 3.1) x 10(-5) M for the apo interaction and Kd = (4.8 +/- 1.8) x 10(-5) M for the calcium(II)-saturated interaction were obtained using a 1:1 binding model of peptide to protein. The 19F NMR chemical shifts for the F-phenylalanine of the bound peptide are different from the apo- and calcium-saturated protein, indicating a different environment for the bound peptide. The possibility of 2:1 binding of the peptide to Ca(II)-saturated TnC was tested by calculating the fit of the experimental titration data to a series of theoretical binding curves in which the dissociation constants for the two hypothetical binding sites were varied. We obtained the best fit for 0.056 mM less than or equal to Kd1 less than or equal to 0.071 mM and 0.5 mM less than or equal to Kd2 less than or equal to 2.0 mM. These results allow the possibility of a second peptide binding site on calcium(II)-saturated TnC with an affinity 10- to 20-fold weaker than that of the first site.     

Energetics of the binding of calcium and troponin I to troponin C from rabbit skeletal muscle.

We determined the free energy of interaction between rabbit skeletal troponin I (TNI) and troponin C (TNC) at 10 degrees and 20 degrees C with fluorescently labeled proteins. The sulfhydryl probe 5-iodoacetamidoeosin (IAE) was attached to cysteine (Cys)-98 of TNC and to Cys-133 of TNI, and each of the labeled proteins was titrated with the other unlabeled protein. The association constant for formation of the complex between labeled TNC (TNC*) and TNI was 6.67 X 10(5) M-1 in 0.3 M KCl, and pH 7.5 at 20 degrees C. In the presence of bound Mg2+, the binding constant increased to 4.58 X 10(7) M-1 and in the presence of excess of Ca2+, the association constant was 5.58 X 10(9) M-1. Very similar association constants were obtained when labeled TNI was titrated with unlabeled TNC. The energetics of Ca2+ binding to TNC* and the complex TNI X TNC* were also determined at 20 degrees C. The two sets of results were used to separately determine the coupling free energy for binding TNI and Mg2+, or Ca2+ to TNC. The results yielded a total coupling free energy of -5.4 kcal. This free energy appeared evenly partitioned into the two species: TNI X TNC(Mg)2 or TNI X TNC(Ca)2, and TNI X TNC(Ca)4. The first two species were each stabilized by -2.6 kcal, with respect to the Ca2+ free TNI X TNC complex, and TNI X TNC(Ca)4 was stabilized by -2.8 kcal, respect to TNI X TNC(Ca)2 or TNI X TNC(Mg)2. The coupling free energy was shown to produce cooperatively complexes formed between TNI and TNC in which the high affinity sites were initially saturated as a function of free Ca2+ to yield TNI X TNC(Ca)4. This saturation occurred in the free Ca2+ concentration range 10(-7) to 10(-5) M. The cooperative strengthening of the linkage between TNI and TNC induced by Ca2+ binding to the Ca2+-specific sites of TNC may have a direct relationship to activation of actomyosin ATPase. The nature of the forces involved in the Ca2+-induced strengthening of the complex is discussed.     

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.     

Interaction of troponin C and troponin C fragments with troponin I and the troponin I inhibitory peptide.

We have quantitated the interactions of two rabbit skeletal troponin C fragments with troponin I and the troponin I inhibitory peptide. The calcium binding properties of the fragments and the ability of the fragments to exert control in the regulated actomyosin ATPase assay have also been studied. The N- and C-terminal divalent metal binding domains of rabbit skeletal troponin C, residues 1-97 and residues 98-159, respectively, were prepared by specific cleavage at cysteine-98 and separation by gel exclusion chromatography. Both of the troponin C fragments bind calcium. The calcium affinity of the weak sites within the N-terminal fragment is about an order of magnitude greater than is reported for these sites in troponin C, suggesting interaction between the calcium-saturated strong sites and the weak sites. Stoichiometric binding (1:1) of the troponin I inhibitory peptide to each fragment and to troponin C increased the calcium affinities of the fragments and troponin C. Complex formation was detected by fluorescence quenching or enhancement using dansyl-labeled troponin C (and fragments) or tryptophan-labeled troponin I inhibitory peptide. The troponin C fragments bind to troponin I with 1:1 stoichiometry and approximately equal affinities (1.6 x 10(6) M-1) which are decreased 4-fold in the presence of magnesium versus calcium. These calcium effects are much smaller than is observed for troponin C. The summed free energies for the binding of the troponin C fragments to troponin I are much larger than the free energy of binding troponin C. This suggests a large positive interaction free energy for troponin C binding to troponin I relative to the fragments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium binding of troponin C. I. A potentiometric titration study.

Structural changes of troponin C on calcium binding were studied by hydrogen ion titration, circular dichroism, and fluorescence measurements. The potentiometric titration curves in the carboxyl region are shifted towards lower pH with calcium binding. The intrinsic pK of the carboxyl groups at the calcium binding sites decreases by 0.8 pK unit on calcium binding; on the other hand, magnesium ions have little effect on the intrinsic pK of the carboxyl groups. The intrinsic pK of the imidazole group is not affected by calcium binding. The value of w, an electrostatic interaction factor, is identical for calcium-free and calcium-bound troponin C and is about half of the value calculated assuming a compact sphere. The results of difference titration on the calcium binding indicate that the pH of troponin C solution increases on addition of CaCl2 up to 2 mol of Ca2+ per mol of troponin C and then decreases on further addition of CaCl2. The pH increase is depressed in the presence of MgCl2, in the low pH region, or at high ionic strength. The pH increase is also observed on addition of MgCl2. The ellipticity at 222 nm was measured under the same conditions as the difference titration measurements, and the relation between the pH change and the conformational change of troponin C on calcium binding is discussed based on the results obtained. The number of calcium binding sites and the binding constants estimated by analysis of these difference titration curves were in agreement with the results of Potter and Gergely (22). No magnesium binding site was observed. The tyrosine fluorescence measurements indicated that the binding site near tyrosine-109 is one of the high affinity sites.     

Mapping subdomains in the C-terminal region of troponin I involved in its binding to troponin C and to thin filament.

Troponin I (TnI) is the inhibitory component of troponin, the ternary complex that regulates skeletal and cardiac muscle contraction. Previous work showed that the C-terminal region of TnI, when linked to the "inhibitory region" (residues 98-116), possesses the major regulatory functions of the molecule (Farah, C. S., Miyamoto, C. A., Ramos, C. H. I., Silva, A. C. R., Quaggio, R. B., Fujimori, K., Smillie, L. B., and Reinach, F. C. (1994) J. Biol. Chem. 269, 5230-5240). To investigate these functions in more detail, serial deletion mutants of the C-terminal region of TnI were constructed. These experiments showed that longer C-terminal deletions result in lower inhibition of the actomyosin ATPase activity and weaken the interaction with the N-terminal domain of troponin C (TnC), consistent with the antiparallel model for the interaction between these two proteins. The conclusion is that the whole C-terminal region of TnI is necessary for its full regulatory activity. The region between residues 137 and 144, which was shown to have homology with residues 108-115 in the inhibitory region (Farah, C. S., and Reinach, F. C. (1995) FASEB J. 9, 755-767), is involved in the binding to TnC. The region between residues 98 and 129 is involved in modulating the affinity of TnC for calcium. The C-terminal residues 166-182 are involved in the binding of TnI to thin filament. A model for the function of TnI is discussed.     

The biological importance of each amino acid residue of the troponin I inhibitory sequence 104-115 in the interaction with troponin C and tropomyosin-actin

To systematically evaluate the contribution of each amino acid residue of the troponin I (TnI) inhibitory region (104-115), 14 synthetic analogs were synthesized by the solid-phase method. The analogs consisted of either single glycine or multiglycine replacements. The importance of the substituted amino acid(s) was determined from the extent of inhibition of the acto-S1 ATPase activity and the strength of binding to a troponin C (TnC) high pressure liquid chromatography affinity column of each synthetic analog. Every residue of the TnI sequence (104-115) is necessary to achieve maximum inhibition of the ATPase activity. However, the analogs quantitatively differed in the amount of inhibition induced. The TnI analogs bound less tightly to the TnC affinity column than the native synthetic peptide indicating that all residues in the TnI sequence contribute to the binding of TnC in the presence of Mg2+ or Ca2+. In the presence of Ca2+, there is a definite increase in the strength of the interaction between most analogs and TnC. This is accompanied with a shift toward a more specific interaction with the C terminus of the TnI inhibitory sequence.     

Effect of hypertrophic cardiomyopathy-linked troponin C mutations on the response of reconstituted thin filaments to calcium upon troponin I phosphorylation

The objective of this work was to investigate the effect of hypertrophic cardiomyopathy-linked A8V and E134D mutations in cardiac troponin C (cTnC) on the response of reconstituted thin filaments to calcium upon phosphorylation of cardiac troponin I (cTnI) by protein kinase A. The phosphorylation of cTnI at protein kinase A sites was mimicked by the S22D/S23D double mutation in cTnI. Our results demonstrate that the A8V and E134D mutations had no effect on the extent of calcium desensitization of reconstituted thin filaments induced by cTnI pseudophosphorylation. However, the A8V mutation enhanced the effect of cTnI pseudophosphorylation on the rate of dissociation of calcium from reconstituted thin filaments and on the calcium dependence of actomyosin ATPase. Consequently, while the A8V mutation still led to a slower rate of dissociation of calcium from reconstituted thin filaments upon pseudophosphorylation of cTnI, the ability of the A8V mutation to decrease the rate of calcium dissociation was weakened. In addition, the ability of the A8V mutation to sensitize actomyosin ATPase to calcium was weakened after cTnI was replaced by the phosphorylation mimetic of cTnI. Consistent with the hypothesis that the E134D mutation is benign, it exerted a minor to no effect on the rate of dissociation of calcium from reconstituted thin filaments or on the calcium sensitivity of actomyosin ATPase, regardless of the cTnI phosphorylation status. In conclusion, our study enhances our understanding of how cardiomyopathy-linked cTnC mutations affect the response of reconstituted thin filaments to calcium upon cTnI phosphorylation.     

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⁺.     

The regulation of myosin binding to actin filaments by Lethocerus troponin

Lethocerus indirect flight muscle has two isoforms of troponin C, TnC-F1 and F2, which are unusual in having only a single C-terminal calcium binding site (site IV, isoform F1) or one C-terminal and one N-terminal site (sites IV and II, isoform F2). We show here that thin filaments assembled from rabbit actin and Lethocerus tropomyosin (Tm) and troponin (Tn) regulate the binding of rabbit myosin to rabbit actin in much the same way as the mammalian regulatory proteins. The removal of calcium reduces the rate constant for S1 binding to regulated actin about threefold, independent of which TmTn is used. This is consistent with calcium removal causing the TmTn to occupy the B or blocked state to about 70% of the total. The mid point pCa for the switch differed for TnC-F1 and F2 (pCa 6.9 and 6.0, respectively) consistent with the reported calcium affinities for the two TnCs. Equilibrium titration of S1 binding to regulated actin filaments confirms calcium regulated binding of S1 to actin and shows that in the absence of calcium the three actin filaments (TnC-F1, TnC-F2 and mammalian control) are almost indistinguishable in terms of occupancy of the B and C states of the filament. In the presence of calcium TnC-F2 is very similar to the control with approximately 80% of the filament in the C-state and 10-15% in the fully on M-State while TnC-F1 has almost 50% in each of the C and M states. This higher occupancy of the M-state for TnC-F1, which occurs above pCa 6.9, is consistent with this isoform being involved in the calcium activation of stretch activation. However, it leaves unanswered how a C-terminal calcium binding site of TnC can activate the thin filament.     

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.     

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.     

Solution conformation of the C-terminal domain of skeletal troponin C. Cation, trifluoperazine and troponin I binding effects

Proton magnetic resonance spectroscopy has been used to study the cation (Mg2+, Ca2+)-dependent conformational states of the C-terminal domain of rabbit skeletal troponin C under a variety of solution conditions. Nuclear Overhauser data and paramagnetic probe observations provide definition of the configuration of this region of troponin C. Comparative study of homologous proteins identify common features of the tertiary structure relevant to the cation binding reaction. Complex formation with troponin I and the drug trifluoperazine is observed to adjust the solution conformation of the C-terminal domain of troponin C. The interactive conformational response to cation coordination and the binding of the drug and troponin I are discussed.     

Calcium and cadmium binding to troponin C. Evidence for cooperativity

Proton NMR is used to compare the structural changes induced in bovine cardiac troponin C on binding of cadmium and calcium ions. The same spectral changes are observed for both ion species. The rate of the conformational changes associated with cadmium binding to the two high-affinity sites is slow, that associated with cadmium ions binding to the low-affinity site is high. 113Cd-NMR spectra of cardiac troponin C feature two signals interpreted as due to cadmium ions bound to the strong sites. Strong arguments are given in favour of cooperativity in binding of the first two cadmium or calcium ions to cardiac and skeletal muscle troponin C.     

Conformational change of troponin T induced by calcium binding to troponin C

The skeletal muscle troponin complex, the troponin T subunit of which was labeled with 2-((4'-iodoacetamido)anilino)naphthalene-6-sulfonic acid, showed a fluorescence titration curve with a midpoint of around pCa 6.75. Addition of 2 mM MgCl2 had no effect on the fluorescence titration curve. Therefore, we conclude that Ca2+ binding to the low affinity Ca2+-binding sites of troponin C induces a conformational change of troponin T, but Ca2+ binding to the high affinity Ca2+-binding sites does not.     

Kinetic studies of calcium and magnesium binding to troponin C

The kinetic mechanism of calcium binding was investigated for the high-affinity calcium-magnesium sites of troponin C (TN-C), for the C-terminal fragment containing only the high-affinity sites (TR2) and for the TN-C:TN-I (where TN-I represents the inhibitory subunit of troponin) complex. Rate constants were measured by the change in fluorescence of the proteins labeled with 4-(N-iodoacetoxyethyl-N-methyl-7-nitrobenz-2-oxa-1,3-diazole at Cys 98. Rate constants for calcium dissociation were also measured using the fluorescent calcium chelating agent quin 2. Calcium binding to TR2 at 4 degrees C is a two-step process at each binding site. (formula; see text) A first order transition (k1 = 700 s-1) follows the formation of a weakly bound collision complex (K0 = 2.5 X 10(3) M-1). The two sits of the labeled protein are distinguishable because of a 2-4-fold difference in rate constants of calcium dissociation. The kinetic evidence is consistent with additive changes in structure induced by calcium binding to two identical or nearly identical high-affinity sites. The mechanism for TN-C:TN-I is similar to TR2. TN-C gave complex kinetic behavior for calcium binding but calcium dissociation occurred with the same rate constants found for TR2. Calcium binding to the high-affinity sites of TnC can be interpreted by the same mechanism as for TR2 but an additional reaction possibly arriving from calcium binding to the low-affinity sites leads to a high-fluorescence intermediate state which is detected by the fluorophore. The interactions between the two classes of sites are interpreted by a model in which calcium binding at the high-affinity sites reverses the fluorescence change induced by calcium binding at the low-affinity sites. Magnesium binding to the calcium-magnesium sites of TR2 and TN-C occurs by the same two-step binding mechanism with a smaller value for K0 and a 5-fold larger rate constant of dissociation.     

Interaction of troponin I and troponin C. Use of the two-dimensional nuclear magnetic resonance transferred nuclear Overhauser effect to determine the structure of the inhibitory troponin I peptide when bound to skeletal troponin C

We have used two-dimensional 1H nuclear magnetic resonance spectroscopy to determine the structure of the synthetic inhibitory peptide N alpha-acetyl TnI(104-115) amide bound to calcium-saturated skeletal troponin C (TnC). Conformational changes in the peptide induced by the formation of the troponin I (TnI) peptide-TnC complex were followed by the study of the transferred nuclear Overhauser effect, a technique that allows one to determine the structure of a ligand bound to a macromolecule. The structure of the bound TnI peptide reveals an amphiphilic alpha-helix, distorted around the two central proline residues. The central bend in the peptide functions to bring the residues on the hydrophobic face into closer proximity with each other, thereby forming a small hydrophobic pocket. The hydrophilic, basic residues extend off the opposite face of the peptide. Hydrophobic surfaces on TnC that become exposed upon binding of calcium are involved in the binding of the TnI peptide, but electrostatic interactions also contribute to the strength of the interaction. The role of amphiphilic helices in the targeting of calcium-binding proteins such as troponin C will be discussed.     

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.     

The mobility of troponin C and troponin I in muscle

In vertebrate skeletal muscle, contraction is initiated by the elevation of the intracellular Ca²⁺ concentration. The binding of Ca²⁺ to TnC induces a series of conformational changes which ultimately release the inhibition of the actomyosin ATPase activity by Tnl. In this study we have characterized the dynamic behavior of TnC and Tnl in solution, as well as in reconstituted fibers, using EPR and ST-EPR spectroscopy. Cys98 of TnC and Cys133 of Tnl were specifically labeled with malemide spin label (MSL) and indane dione nitroxide spin label (InVSL). In solution, the labeled TnC and Tnl exhibited fast nanosecond motion. MSL-TnC is sensitive to cation binding to the high affinity sites (τ_r increases from 1.5 to 3.7 ns), InVSL-TnC s sensitive to the replacement of Mg²⁺ by Ca²⁺ at these sites (τ_r increase from 1.7 to 6 ns). Upon reconstitution into fibers, the nanosecond mobility is reduced by interactions with other proteins. TnC and Tnl both exhibited microsecond anisotropic motion in fibers similar to that of the actin monomers within the filament. The microsecond motion of TnC was found to be modulated by the binding of Ca²⁺ and by cross-bridge attachment, but this was not the case for the global mobility of Tnl. © 1997 John Wiley & Sons, Ltd.