Characterization of the biologically important interaction between troponin C and the N-terminal region of troponin I

The N-terminal regulatory region of Troponin I, residues 1-40 (TnI 1-40, regulatory peptide) has been shown to have a biologically important function in the interactions of troponin I and troponin C. Truncated analogs corresponding to shorter versions of the N-terminal region (1-30, 1-28, 1-26) were synthesized by solid-phase methodology. Our results indicate that residues 1-30 of TnI comprises the minimum sequence to retain full biological activity as measured in the acto-S1-TM ATPase assay. Binding of the TnI N-terminal regulatory peptides (TnI 1-30 and the N-terminal regulatory peptide (residues 1-40) labeled with the photoprobe benzoylbenzoyl group, BBRp) were studied by gel electrophoresis and photochemical cross-linking experiments under various conditions. Fluorescence titrations of TnI 1-30 were carried out with TnC mutants that carry a single tryptophan fluorescence probe in either the N- or C-domain (F105W, F105W/C domain (88-162), F29W and F29W/N domain (1-90)) (Fig. 1). Low Kd values (Kd < 10(-7) M) were obtained for the interaction of F105W and F105W/C domain (88-162) with TnI 1-30. However, there was no observable change in fluorescence when the fluorescence probe was located at the N-domain of the TnC mutant (F29W and F29W/N domain (1-90)). These results show that the regulatory peptide binds strongly to the C-terminal domain of TnC.     

The role of electrostatics in the interaction of the inhibitory region of troponin I with troponin C

We have addressed the electrostatic interactions occurring between the inhibitory region of cardiac troponin I with the C-lobe of troponin C using scanning glycine mutagenesis of the inhibitory region. We report variations in the electric potentials due to mutation of charged residues within this complex based upon the solved NMR structure (1OZS). These results demonstrate the importance of electrostatics within this complex, and it is proposed that electrostatic interactions are integral to the formation and function of larger ternary troponin complexes. To address this hypothesis, we report (15)N NMR relaxation measurements, which suggest that, within a ternary complex involving the C-lobe and the N-terminal region of troponin I (residues 34-71), the inhibitory region maintains the electrostatic interactions with the E-helix of the C-lobe as observed within the binary complex. These results imply that, in solution, the cardiac troponin complex behaves in a manner consistent with that of the crystal structure of the skeletal isoform (1YTZ). A cardiac troponin complex possessing domain orientations similar to that of the skeletal isoform provides structural insights into altered troponin I activities as observed for the familial hypertrophic cardiomyopathy mutation R144G and phosphorylation of Thr142.     

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.     

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.     

Characterization of zero-length cross-links between rabbit skeletal muscle troponin C and troponin I: evidence for direct interaction between the inhibitory region of troponin I and the NH2-terminal, regulatory domain of troponin C

Interactions between troponin C (TnC) and troponin I (TnI) play an important role in the Ca2(+)-dependent regulation of vertebrate striated muscle contraction. Previous attempts to elucidate the molecular details of TnC-TnI interactions, mainly involving chemically modified proteins or fragments thereof, have led to the widely accepted idea that the "inhibitory region" (residues 96-116) of TnI binds to an alpha-helical segment of TnC comprising residues 89-100 in the nonregulatory, COOH-terminal domain. In an attempt to identify other possible physiologically important interactions between these proteins, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) was used to produce zero-length cross-links in the complex of rabbit skeletal muscle TnC and TnI. TnC was activated with EDC and N-hydroxysuccinimide (NHS) and then mixed with an equimolar amount of TnI [Grabarek, Z., & Gergely, J. (1988) Biophys. J. 53, 392a]. The resulting cross-linked TnCXI was cleaved with cyanogen bromide, trypsin, and Staphylococcus aureus V8 protease (SAP). Cross-linked peptides were purified by reverse-phase HPLC and characterized by sequence analysis. The results indicated that residues from the regulatory Ca2(+)-binding site II in the NH2-terminal domain of TnC (residues 46-78) formed cross-links with TnI segments spanning residues 92-167. The most highly cross-linked residues in TnI were Lys-105 and Lys-107, located in the inhibitory region. These results yield the first evidence for an interaction between the N-terminal domain of TnC and the inhibitory region of TnI.     

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)     

Proton-magnetic-resonance studies on the interaction of rabbit skeletal-muscle troponin I with troponin C and actin.

1. The p.m.r. spectra of the larger CNBr-cleavage peptides of troponin I from rabbit fast-twitch skeletal muscle corresponded largely to those of fairly flexible solution structures. 2. On addition of troponin C to each of the CNBr-cleavage peptides in turn, perturbations of side chains were noted only for peptides CN5 (residues 1-21) and CN4 (residues 96-116). 3. In the presence of Ca2+, troponin C induced perturbations of the side chains of threonine-11, alanine, isoleucine and arginine residues of peptide CN5. 4. In the presence of Ca2+, troponin C induced perturbations of the side chains of phenylalanine, lysine and leucine residues of peptide CN4. 5. Irrespective of the presence or absence of Ca2+, specific interaction with actin was observed only with peptide CN4. In this case the side chains of arginine residues were perturbed. 6. It is concluded that actin interacts with the C-terminal region of peptide CN4, whereas troponin C interacts with the N-terminal region of peptide CN4 and with peptide CN5.     

Effect of calcium ions on the interaction of troponin C with rabbit skeletal muscle troponin I and troponin T immobilized on polyvinyl chloride

Using a new methodological approach based on the binding of 125I-labeled troponin C to troponins I and T immobilized on polyvinylchloride, the Ca2+-dependent interaction of troponin components was investigated. In the absence of Ca2+, two types of sites of troponin C--troponin T interaction were revealed (Kd = 3.6.10(-8) M and 5.10(-7) M). It was found that Ca2+ induced the formation of a troponin I--troponin C complex which was resistant to 5 M urea (Kd = 4.10(-8) M). In the absence of Ca2+, the binary troponin T--troponin C complex also revealed two types of interaction sites (Kd = 7.1.10(-8) M and 2.10(-7) M); however, in the presence of Ca2+ only high affinity sites whose number increased almost 2-fold were revealed. The events that may take place in the whole troponin complex during Ca2+ binding by troponin C are discussed.     

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.     

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.     

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.     

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.     

Biologically important interactions between synthetic peptides of the N-terminal region of troponin I and troponin C.

The interaction between troponin I and troponin C plays a critical role in the regulation of muscle contraction. In this study the interaction between troponin C (TnC) and the N-terminal region of TnI was investigated by the synthesis of three TnI peptides (residues 1-40/Rp, 10-40, and 20-40). The regulatory peptide (Rp) on binding to TnC prevents the ability of TnC to release the inhibition of the acto-S1-tropomyosin ATPase activity caused by TnI or the TnI inhibitory peptide (Ip), residues 104-115. A stable complex between TnC and Rp in the presence of Ca2+ was demonstrated by polyacrylamide gel electrophoresis in the presence of 6 M urea. Rp was able to displace TnI from a preformed TnI.TnC complex. In the absence of Ca2+, Rp was unable to maintain a complex with TnC in benign conditions of polyacrylamide gel electrophoresis which demonstrates the Ca(2+)-dependent nature of this interaction. Size-exclusion chromatography demonstrated that the TnC.Rp complex consisted of a 1:1 complex. The results of these studies have shown that the N-terminal region of TnI (1-40) plays a critical role in modulating the Ca(2+)-sensitive release of TnI inhibition by TnC.     

A pH-sensitive interaction of troponin I with troponin C coupled with strongly binding cross-bridges in cardiac myofilament activation

Slow skeletal muscle troponin I (ssTnI) expressed predominantly in perinatal heart confers a marked resistance to acidic pH on Ca(2+) regulation of cardiac muscle contraction. To explore the molecular mechanism underlying this phenomenon, we investigated the roles of TnI isoforms (ssTnI and cardiac TnI (cTnI)) in the thin filament activation by strongly binding cross-bridges, by exchanging troponin subunits in cardiac permeabilized muscle fibers. Fetal cardiac muscle showed a marked resistance to acidic pH in activation of the thin filament by strongly binding cross-bridges compared to adult muscle. Exchanging ssTnI into adult fibers altered the pH sensitivity from adult to fetal type, indicating that ssTnI also confers a marked resistance to acidic pH on the cross-bridge-induced thin filament activation. However, the adult fibers containing ssTnI or cTnI but lacking TnC showed no pH sensitivity. These findings provide the first evidence for the coupling between strongly binding cross-bridges and a pH-sensitive interaction of TnI with TnC in cardiac muscle contraction, as a molecular basis of the mechanism conferring the differential pH sensitivity on Ca(2+) regulation.     

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.     

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.     

A 1H NMR study of a ternary peptide complex that mimics the interaction between troponin C and troponin I.

The troponin I peptide N alpha-acetyl TnI (104-115) amide (TnIp) represents the minimum sequence necessary for inhibition of actomyosin ATPase activity of skeletal muscle (Talbot, J.A. & Hodges, R.S. 1981, J. Biol. Chem. 256, 2798-3802; Van Eyk, J.E. & Hodges, R.S., 1988, J. Biol. Chem. 263, 1726-1732; Van Eyk, J.E., Kay, C.M., & Hodges, R.S., 1991, Biochemistry 30, 9974-9981). In this study, we have used 1H NMR spectroscopy to compare the binding of this inhibitory TnI peptide to a synthetic peptide heterodimer representing site III and site IV of the C-terminal domain of troponin C (TnC) and to calcium-saturated skeletal TnC. The residues whose 1H NMR chemical shifts are perturbed upon TnIp binding are the same in both the site III/site IV heterodimer and TnC. These residues include F102, I104, F112, I113, I121, I149, D150, F151, and F154, which are all found in the C-terminal domain hydrophobic pocket and antiparallel beta-sheet region of the synthetic site III/site IV heterodimer and of TnC. Further, the affinity of TnIp binding to the heterodimer (Kd = 192 +/- 37 microM) was found to be similar to TnIp binding to TnC (48 +/- 18 microM [Campbell, A.P., Cachia, P.J., & Sykes, B.D., 1991, Biochem. Cell Biol. 69, 674-681]). The results indicate that binding of the inhibitory region of TnI is primarily to the C-terminal domain of TnC. The results also indicate how well the synthetic peptide heterodimer mimics the C-terminal domain of TnC in structure and functional interactions.