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

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

Modulation of cardiac troponin C function by the cardiac-specific N-terminus of troponin I: influence of PKA phosphorylation and involvement in cardiomyopathies

The cardiac-specific N-terminus of cardiac troponin I (cTnI) is known to modulate the activity of troponin upon phosphorylation with protein kinase A (PKA) by decreasing its Ca²⁺ affinity and increasing the relaxation rate of the thin filament. The molecular details of this modulation have not been elaborated to date. We have established that the N-terminus and the switch region of cTnI bind to cNTnC [the N-domain of cardiac troponin C (cTnC)] simultaneously and that the PKA signal is transferred via the cTnI N-terminus modulating the cNTnC affinity toward cTnI_147-163 but not toward Ca²⁺. The K_d of cNTnC for cTnI_147-163 was found to be 600 μM in the presence of cTnI_1-29 and 370 μM in the presence of cTn1_1-29PP, which can explain the difference in muscle relaxation rates upon the phosphorylation with PKA in experiments with cardiac fibers. In the light of newly found mutations in cNTnC that are associated with cardiomyopathies, the important role played by the cTnI N-terminus in the development of heart disorders emerges. The mutants studied, L29Q (the N-domain of cTnC containing mutation L29Q) and E59D/D75Y (the N-domain of cTnC containing mutation E59D/D75Y), demonstrated unchanged Ca²⁺ affinity per se and in complex with the cTnI N-terminus (cTnI_1-29 and cTnI_1-29PP). The affinity of L29Q and E59D/D75Y toward cTnI_147-163 was significantly perturbed, both alone and in complex with cTnI_1-29 and cTnI_1-29PP, which is likely to be responsible for the development of malfunctions.     

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.     

Characterization of the cardiac holotroponin complex reconstituted from native cardiac troponin T and recombinant I and C.

Cardiac troponin I (cTnI), the inhibitory subunit of cardiac troponin (cTn), is phosphorylated by the cAMP-dependent protein kinase A at two adjacently located serine residues within the heart-specific N-terminal elongation. Four different phosphorylation states can be formed. To investigate each monophosphorylated form cTnI mutants, in which each of the two serine residues is replaced by an alanine, were generated. These mutants, as well as the wild-type cardiac troponin I (cTnI-WT) have been expressed in Escherichia coli, purified and characterized by isoelectric focusing, MS and CD-spectroscopy. Monophosphorylation induces conformational changes within cTnI that are different from those induced by bisphosphorylation. Functionality was assessed by measuring the calcium dependence of myosin S1 binding to thin filaments containing reconstituted native, wild-type and mutant cTn complexes. In all cases a functional holotroponin complex was obtained. Upon bisphosphorylation of cTnI-WT the pCa curve was shifted to the right to the same extent as that observed with bisphosphosphorylated native cTnI. However, the absolute values for the midpoints were higher when recombinant cTn subunits were used for reconstitution. Reconstitution itself changed the calcium affinity of cTnC: pCa50-values were higher than those obtained with the native cardiac holotroponin complex. Apparently only bisphosphorylation of cTnI influences the calcium sensitivity of the thin filament, thus monophosphorylation has a function different from that of bisphosphorylation; this function has not yet been identified.     

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

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

An interdomain distance in cardiac troponin C determined by fluorescence spectroscopy

The distance between Ca2+-binding site III in the C-terminal domain and Cys35 in the N-terminal domain in cardiac muscle troponin C (cTnC) was determined with a single-tryptophan mutant using bound Tb3+ as the energy donor and iodoacetamidotetramethylrhodamine linked to the cysteine residue as energy acceptor. The luminescence of bound Tb3+ was generated through sensitization by the tryptophan located in the 12-residue binding loop of site III upon irradiation at 295 nm, and this sensitized luminescence was the donor signal transferred to the acceptor. In the absence of bound cation at site II, the mean interdomain distance was found to be 48-49 A regardless of whether the cTnC was unbound or bound to cardiac troponin I, or reconstituted into cardiac troponin. These results suggest that cTnC retains its overall length in the presence of bound target proteins. The distribution of the distances was wide (half-width >9 A) and suggests considerable interdomain flexibility in isolated cTnC, but the distributions became narrower for cTnC in the complexes with the other subunits. In the presence of bound cation at the regulatory site II, the interdomain distance was shortened by 6 A for cTnC, but without an effect on the half-width. The decrease in the mean distance was much smaller or negligible when cTnC was complexed with cTnI or cTnI and cTnT under the same conditions. Although free cTnC has considerable interdomain flexibility, this dynamics is slightly reduced in troponin. These results indicate that the transition from the relaxed state to an activated state in cardiac muscle is not accompanied by a gross alteration of the cTnC conformation in cardiac troponin.     

A preferred AMPK phosphorylation site adjacent to the inhibitory loop of cardiac and skeletal troponin I

5'-AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that is activated when cellular AMP to ATP ratios rise, potentially serving as a key regulator of cellular energetics. Among the known targets of AMPK are catabolic and anabolic enzymes, but little is known about the ability of this kinase to phosphorylate myofilament proteins and thereby regulating the contractile apparatus of striated muscles. Here, we demonstrate that troponin I isoforms of cardiac (cTnI) and fast skeletal (fsTnI) muscles are readily phosphorylated by AMPK. For cTnI, two highly conserved serine residues were identified as AMPK sites using a combination of high-resolution top-down electron capture dissociation mass spectrometry, (32) P-incorporation, synthetic peptides, phospho-specific antibodies, and site-directed mutagenesis. These AMPK sites in cTnI were Ser149 adjacent to the inhibitory loop and Ser22 in the cardiac-specific N-terminal extension, at the level of cTnI peptides, the intact cTnI subunit, whole cardiac troponin complexes and skinned cardiomyocytes. Phosphorylation time-course experiments revealed that Ser149 was the preferred site, because it was phosphorylated 12-16-fold faster than Ser22 in cTnI. Ser117 in fsTnI, analogous to Ser149 in cTnI, was phosphorylated with similar kinetics as cTnI Ser149. Hence, the master energy-sensing protein AMPK emerges as a possibly important regulator of cardiac and skeletal contractility via phosphorylation of a preferred site adjacent to the inhibitory loop of the thin filament protein TnI.     

Overexpression of human cardiac troponin in Escherichia coli: its purification and characterization

All three subunits of the human cardiac troponin complex (cTn), namely the major isoform of the tropomyosin binding subunit (hcTnT3), the inhibitory subunit (cTnI), and the calcium binding subunit (cTnC), have been coexpressed in Escherichia coli. The cDNAs of each subunit have been cloned into the pSBET vector and transformed into E. coli. The coexpressed subunits assembled within the bacterial cells to form the hcTn complex (hcTnT3.hcTnI.hcTnC). The complex was isolated and purified by three chromatographic steps. Per 6-L cell culture about 10 mg of a highly purified troponin complex showing the expected 1:1:1 molar ratio of hcTnT3:cTnI:cTnC was obtained. Upon phosphorylation by protein kinase A at Ser22 and Ser23 in cTnI, this recombinant troponin complex shows a nearly identical (31)P NMR spectrum to the native one isolated from bovine heart. By measuring the rate of myosin S1 binding to reconstituted thin filaments it was shown that the dependence of the regulation of S1 binding upon calcium concentration and bisphosphorylation was comparable to the native complex.     

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.     

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

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

Structural consequences of cardiac troponin I phosphorylation

beta-Adrenergic stimulation of the heart results in bisphosphorylation of the N-terminal extension of cardiac troponin I (TnI). Bisphosphorylation of TnI reduces the affinity of the regulatory site on troponin C (TnC) for Ca(2+) by increasing the rate of Ca(2+) dissociation. What remains unclear is how the phosphorylation signal is transmitted from one subunit of troponin to another. We have produced a series of mutations in the N-terminal extension of TnI designed to further our understanding of the mechanisms involved. The ability of phosphorylation of the mutant TnIs to affect Ca(2+) sensitivity has been assessed. We find that the Pro residues found in a conserved (Xaa-Pro)(4) motif N-terminal to the phosphorylation sites are not required for the effect of the N-terminal extension on Ca(2+) binding in the presence or absence of phosphorylation. Our experiments also reveal that the full effects of phosphorylation are seen even when residues 1-15 of TnI are deleted. If further residues are removed, not only does the effect of phosphorylation diminish but deletion of the N-terminal extension mimics phosphorylation. We propose that TnI residues 16-29 bind to TnC stabilizing the "open" Ca(2+)-bound state. Phosphorylation (or deletion) prevents this binding, accelerating Ca(2+) release.     

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

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

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.     

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

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

Expression and regulation of mutant forms of cardiac TnI in a reconstituted actomyosin system: Role of kinase dependent phosphorylation

When phosphorylated, the inhibitory subunit of troponin (TnI) causes a loss in calcium sensitivity and a decrease in actomyosin ATPase. To examine this process, we bacterially expressed wild type TnI and TnI mutants in which serine 22 and 23, a putative protein kinase A (PKA) site, and threonine 143, a putative protein kinase C (PKC) site, were replaced by alanine S22A/23A and T143A. PKA dependent phosphorylation was ~90% reduced in the S22A/23A mutant and unaffected in T143A. PKC dependent phosphorylation was markedly reduced in T143A relative both to a wild type construct and to S22A/23A, although some residual phosphorylation (likely at sites other than T143) was seen. The calcium sensitivity (i.e. inhibition of actomyosin ATPase in the presence of EGTA) and regulation of the reconstituted actomyosin system was preserved in the absence of phosphorylation using wild type TnI or either mutant. Calcium sensitivity was decreased by both PKA and PKC with the wild type TnI but was unaffected by PKA when the S22A/23A mutant was employed and by PKC when the T143A mutant was reconstituted. The calcium dependency of the ATPase curve was substantially right shifted when PKC phosphorylated wild type TnI was employed for regulation, and this was markedly attenuated when T143 A was reassociated (although a slight rightward shift and a reduction in maximal ATPase activity was still seen). These data confirm that phosphorylation of TnI by regulatory kinases plays a major role in the regulation of myofibrillar ATPase. The N-terminal serines (22 and 23) appear to be uniquely important for the PKA response whereas threonine 143 is involved in the PKC response although other residues may also have functional significance.     

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.     

Structural transition of the inhibitory region of troponin I within the regulated cardiac thin filament

Contraction and relaxation of cardiac muscle are regulated by the inhibitory and regulatory regions of troponin I (cTnI). Our previous FRET studies showed that the inhibitory region of cTnI in isolated troponin experiences a structural transition from a beta-turn/coil motif to an extended conformation upon Ca(2+) activation. During the relaxation process, the kinetics of the reversal of this conformation is coupled to the closing of the Ca(2+)-induced open conformation of the N-domain of troponin C (cTnC) and an interaction between cTnC and cTnI in their interface. We have since extended the structural kinetic study of the inhibitory region to fully regulated thin filament. Single-tryptophan and single-cysteine mutant cTnI(L129W/S151C) was labeled with 1,5-IAEDANS at Cys151, and the tryptophan-AEDANS pair served as a donor-acceptor pair. Labeled cTnI mutant was used to prepare regulated thin filaments. Ca(2+)-induced conformational changes in the segment of Trp129-Cys151 of cTnI were monitored by FRET sensitized acceptor (AEDANS) emission in Ca(2+) titration and stopped-flow measurements. Control experiments suggested energy transfer from endogenous tryptophan residues of actin and myosin S1 to AEDANS attached to Cys151 of cTnI was very small and Ca(2+) independent. The present results show that the rate of Ca(2+)-induced structural transition and Ca(2+) sensitivity of the inhibitory region of cTnI were modified by (1) thin filament formation, (2) the presence of strongly bound S1, and (3) PKA phosphorylation of the N-terminus of cTnI. Ca(2+) sensitivity was not significantly changed by the presence of cTm and actin. However, the cTn-cTm interaction decreased the cooperativity and kinetics of the structural transition within cTnI, while actin filaments elicited opposite effects. The strongly bound S1 significantly increased the Ca(2+) sensitivity and slowed down the kinetics of structural transition. In contrast, PKA phosphorylation of cTnI decreased the Ca(2+) sensitivity and accelerated the structural transition rate of the inhibitory region of cTnI on thin filaments. These results support the idea of a feedback mechanism by strong cross-bridge interaction with actin and provide insights on the molecular basis for the fine tuning of cardiac function by beta-adrenergic stimulation.     

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.     

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.     

Effects of phosphorylation and mutation R145G on human cardiac troponin I function.

We have studied functional consequences of the mutations R145G, S22A, and S23A of human cardiac troponin I (cTnI) and of phosphorylation of two adjacent N-terminal serine residues in the wild-type cTnI and the mutated proteins. The mutation R145G has been linked to the development of familial hypertrophic cardiomyopathy. Cardiac troponin was reconstituted from recombinant human subunits including either wild-type or mutant cTnI and was used for reconstitution of thin filaments with skeletal muscle actin and tropomyosin. The Ca(2+)-dependent thin filament-activated myosin subfragment 1 ATPase (actoS1-ATPase) activity and the in vitro motility of these filaments driven by myosin were measured as a function of the cTnI phosphorylation state. Bisphosphorylation of wild-type cTnI decreases the Ca(2+) sensitivity of the actoS1-ATPase activity and the in vitro thin filament motility by about 0.15-0.21 pCa unit. The nonconservative replacement R145G in cTnI enhances the Ca(2+) sensitivity of the actoS1-ATPase activity by about 0.6 pCa unit independent of the phosphorylation state of cTnI. Furthermore, it mimics a strong suppressing effect on both the maximum actoS1-ATPase activity and the maximum in vitro filament sliding velocity which has been observed upon bisphosphorylation of wild-type cTnI. Bisphosphorylation of the mutant cTnI-R145G itself had no such suppressing effects anymore. Differential analysis of the effect of phosphorylation of each of the two serines, Ser23 in cTnI-S22A and Ser22 in cTnI-S23A, indicates that phosphorylation of Ser23 may already be sufficient for causing the reduction of maximum actoS1-ATPase activity and thin filament sliding velocity seen upon phosphorylation of both of these serines.