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.     

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.     

Comparative studies on the functional roles of N- and C-terminal regions of molluskan and vertebrate troponin-I

Vertebrate troponin regulates muscle contraction through alternative binding of the C-terminal region of the inhibitory subunit, troponin-I (TnI), to actin or troponin-C (TnC) in a Ca(2+)-dependent manner. To elucidate the molecular mechanisms of this regulation by molluskan troponin, we compared the functional properties of the recombinant fragments of Akazara scallop TnI and rabbit fast skeletal TnI. The C-terminal fragment of Akazara scallop TnI (ATnI(232-292)), which contains the inhibitory region (residues 104-115 of rabbit TnI) and the regulatory TnC-binding site (residues 116-131), bound actin-tropomyosin and inhibited actomyosin-tropomyosin Mg-ATPase. However, it did not interact with TnC, even in the presence of Ca(2+). These results indicated that the mechanism involved in the alternative binding of this region was not observed in molluskan troponin. On the other hand, ATnI(130-252), which contains the structural TnC-binding site (residues 1-30 of rabbit TnI) and the inhibitory region, bound strongly to both actin and TnC. Moreover, the ternary complex consisting of this fragment, troponin-T, and TnC activated the ATPase in a Ca(2+)-dependent manner almost as effectively as intact Akazara scallop troponin. Therefore, Akazara scallop troponin regulates the contraction through the activating mechanisms that involve the region spanning from the structural TnC-binding site to the inhibitory region of TnI. Together with the observation that corresponding rabbit TnI-fragment (RTnI(1-116)) shows similar activating effects, these findings suggest the importance of the TnI N-terminal region not only for maintaining the structural integrity of troponin complex but also for Ca(2+)-dependent activation.     

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.     

Calcium- and magnesium-dependent interactions between the C-terminus of troponin I and the N-terminal, regulatory domain of troponin C

The muscle thin filament protein troponin (Tn) regulates contraction of vertebrate striated muscle by conferring Ca2+ sensitivity to the interaction of actin and myosin. Troponin C (TnC), the Ca2+ binding subunit of Tn contains two homologous domains and four divalent cation binding sites. Two structural sites in the C-terminal domain of TnC bind either Ca2+ or Mg2+, and two regulatory sites in the N-terminal domain are specific for Ca2+. Interactions between TnC and the inhibitory Tn subunit troponin I (TnI) are of central importance to the Ca2+ regulation of muscle contraction and have been intensively studied. Much remains to be learned, however, due mainly to the lack of a three-dimensional structure for TnI. In particular, the role of amino acid residues near the C-terminus of TnI is not well understood. In this report, we prepared a mutant TnC which contains a single Trp-26 residue in the N-terminal, regulatory domain. We used fluorescence lifetime and quenching measurements to monitor Ca2+- and Mg2+-dependent changes in the environment of Trp-26 in isolated TnC, as well as in binary complexes of TnC with a Trp-free mutant of TnI or a truncated form of this mutant, TnI(1-159), which lacked the C-terminal 22 amino acid residues of TnI. We found that full-length TnI and TnI(1-159) affected Trp-26 similarly when all four binding sites of TnC were occupied by Ca2+. When the regulatory Ca2+-binding sites in the N-terminal domain of TnC were vacant and the structural sites in the C-terminal domain of were occupied by Mg2+, we found significant differences between full-length TnI and TnI(1-159) in their effect on Trp-26. Our results provide the first indica- tion that the C-terminus of TnI may play an important role in the regulation of vertebrate striated muscle through Ca2+-dependent interactions with the regula- tory domain of TnC.     

The calcium-induced switch in the troponin complex probed by fluorescent mutants of troponin I.

The Ca2+-induced transition in the troponin complex (Tn) regulates vertebrate striated muscle contraction. Tn was reconstituted with recombinant forms of troponin I (TnI) containing a single intrinsic 5-hydroxytryptophan (5HW). Fluorescence analysis of these mutants of TnI demonstrate that the regions in TnI that respond to Ca2+ binding to the regulatory N-domain of TnC are the inhibitory region (residues 96-116) and a neighboring region that includes position 121. Our data confirms the role of TnI as a modulator of the Ca2+ affinity of TnC; we show that point mutations and incorporation of 5HW in TnI can affect both the affinity and the cooperativity of Ca2+ binding to TnC. We also discuss the possibility that the regulatory sites in the N-terminal domain of TnC might be the high affinity Ca2+-binding sites in the troponin complex.     

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.     

The highly conserved COOH terminus of troponin I forms a Ca2+-modulated allosteric domain in the troponin complex

The primary structure of the COOH-terminal region of troponin I (TnI) is highly conserved among the cardiac, slow, and fast skeletal muscle TnI isoforms and across species. Although no binding site for the other thin filament proteins is found at the COOH terminus of TnI, truncations of the last 19-23 amino acid residues reduce the activity of TnI in the inhibition of actomyosin ATPase and result in cardiac muscle malfunction. We have developed a specific monoclonal antibody (mAb), TnI-1, against the conserved COOH terminus of TnI. Using this mAb, isolation of the troponin complex by immunoaffinity chromatography from muscle homogenate and immunofluorescence microscopic staining of myofibrils indicate that the COOH terminus of TnI forms an exposed structure in the muscle thin filament. Binding of this mAb to the COOH terminus of cardiac TnI induced extensive conformational changes in the protein, suggesting an allosteric role of this region in the functional integrity of troponin. In the absence of Ca2+, the binding of troponin C and troponin T to TnI had very little effect on the conformation of the COOH terminus of TnI as indicated by the unaffected mAb affinity for the TnI-1 epitope. However, Ca2+ significantly increased the accessibility of the TnI-1 epitope on TnI in the presence of troponin C and troponin T. The results provide evidence that the COOH terminus is an essential structure in TnI and participates in the allosteric switch during Ca2+ activation of contraction.     

Proximity relationships between residue 6 of troponin I and residues in troponin C: further evidence for extended conformation of troponin C in the troponin complex

Skeletal muscle troponin C (TnC) adopts an extended conformation when crystallized alone and a compact one when crystallized with an N-terminal troponin I (TnI) peptide, TnI(1-47) [Vassylyev et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 4847-4852]. The N-terminal region of TnI (residues 1-40) was suggested to play a functional role of facilitating the movement of TnI's inhibitory region between TnC and actin [Tripet et al. (1997) J. Mol. Biol. 271, 728-750]. To test this hypothesis and to investigate the conformation of TnC in the intact troponin complex and in solution, we attached fluorescence and photo-cross-linking probes to a mutant TnI with a single cysteine at residue 6. Distances from this residue to residues of TnC were measured by the fluorescence resonance energy transfer technique, and the sites of photo-cross-linking in TnC were determined by microsequencing and mass spectrometry following enzymatic digestions. Our results show that in the troponin complex neither the distance between TnI residue 6 and TnC residue 89 nor the photo-cross-linking site in TnC, Ser133, changes with Ca(2+), in support of the notion that this region plays mainly a structural rather than a regulatory role. The distances to residues 12 and 41 in TnC's N-domain are both considerably longer than those predicted by the crystal structure of TnC.TnI(1-47), supporting an extended rather than a compact conformation of TnC. In the binary TnC.TnI complex and the presence of Ca(2+), Met43 in TnC's N-domain was identified as the photo-cross-linking site, and multiple distances between TnI residue 6 and TnC residue 41 were detected. This was taken to indicate increased flexibility in TnC's central helix and that TnC dynamically changes between a compact and an extended conformation when troponin T (TnT) is absent. Our results further emphasize the difference between the binary TnC.TnI and the ternary troponin complexes and the importance of using intact proteins in the study of structure-function relationships of troponin.     

Calcium-regulated conformational change in the C-terminal end segment of troponin I and its binding to tropomyosin

The troponin complex plays an essential role in the thin filament regulation of striated muscle contraction. Of the three subunits of troponin, troponin I (TnI) is the actomyosin ATPase inhibitory subunit and its effect is released upon Ca(2+) binding to troponin C. The exon-8-encoded C-terminal end segment represented by the last 24 amino acids of cardiac TnI is highly conserved and is critical to the inhibitory function of troponin. Here, we investigated the function and calcium regulation of the C-terminal end segment of TnI. A TnI model molecule was labeled with Alexa Fluor 532 at a Cys engineered at the C-terminal end and used to reconstitute the tertiary troponin complex. A Ca(2+) -regulated conformational change in the C-terminus of TnI was shown by a sigmoid-shape fluorescence intensity titration curve similar to that of the CD calcium titration curve of troponin C. Such corresponding Ca(2+) responses are consistent with the function of troponin as a coordinated molecular switch. Reconstituted troponin complex containing a mini-troponin T lacking its two tropomyosin-binding sites showed a saturable binding to tropomyosin at pCa 9 but not at pCa 4. This Ca(2+) -regulated binding was diminished when the C-terminal 19 amino acids of cardiac TnI were removed. These results provided novel evidence for suggesting that the C-terminal end segment of TnI participates in the Ca(2+) regulation of muscle thin filament through interaction with tropomyosin.     

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.     

Calcium-induced flexibility changes in the troponin C-troponin I complex

The contraction of vertebrate striated muscle is modulated by Ca(2+) binding to the regulatory protein troponin C (TnC). Ca(2+) binding causes conformational changes in TnC which alter its interaction with the inhibitory protein troponin I (TnI), initiating the regulatory process. We have used the frequency domain method of fluorescence resonance energy transfer (FRET) to measure distances and distance distributions between specific sites in the TnC-TnI complex in the presence and absence of Ca(2+) or Mg(2+). Using sequences based on rabbit skeletal muscle proteins, we prepared functional, binary complexes of wild-type TnC and a TnI mutant which contains no Cys residues and a single Trp residue at position 106 within the TnI inhibitory region. We used TnI Trp-106 as the FRET donor, and we introduced energy acceptor groups into TnC by labeling at Met-25 with dansyl aziridine or at Cys-98 with N-(iodoacetyl)-N'-(1-sulfo-5-naphthyl)ethylenediamine. Our distance distribution measurements indicate that the TnC-TnI complex is relatively rigid in the absence of Ca(2+), but becomes much more flexible when Ca(2+) binds to regulatory sites in TnC. This increased flexibility may be propagated to the whole thin filament, helping to release the inhibition of actomyosin ATPase activity and allowing the muscle to contract. This is the first report of distance distributions between TnC and TnI in their binary complex.     

Structure, dynamics, and thermodynamics of the structural domain of troponin C in complex with the regulatory peptide 1-40 of troponin I

The structure of the calcium-saturated C-domain of skeletal troponin C (CTnC) in complex with a regulatory peptide comprising residues 1-40 (Rp40) of troponin I (TnI) was determined using nuclear magnetic resonance (NMR) spectroscopy. The solution structure determined by NMR is similar to the structure of the C-domain from intact TnC in complex with TnI(1)(-)(47) determined by X-ray crystallography [Vassylyev, D. G., Takeda, S., Wakatsuki, S., Maeda, K., and Maeda, Y. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 4847-4852]. Changes in the dynamic properties of CTnC.2Ca2+ induced by Rp40 binding were investigated using backbone amide (15)N NMR relaxation measurements. Analysis of NMR relaxation data allows for extraction of motional order parameters on a per residue basis, from which the contribution of changes in picosecond to nanosecond time scale motions to the conformational entropy associated with complex formation can be estimated. The results indicate that binding of Rp40 decreases backbone flexibility in CTnC, particularly at the end of the C-terminal helix. The backbone conformational entropy change (-TDeltaS) associated with binding of Rp40 to CTnC.2Ca2+ determined from (15)N relaxation data is 9.6 +/- 0.7 kcal mol(-1) at 30 degrees C. However, estimation of thermodynamic quantities using a structural approach [Lavigne, P., Bagu, J. R., Boyko, R., Willard, L., Holmes, C. F., and Sykes, B. D. (2000) Protein Sci. 9, 252-264] reveals that the change in solvation entropy upon complex formation is dominant and overcomes the thermodynamic "cost" associated with "stiffening" of the protein backbone upon Rp40 binding. Additionally, backbone amide (15)N relaxation data measured at different concentrations of CTnC.2Ca2+.Rp40 reveal that the complex dimerizes in solution. Fitting of the apparent global rotational correlation time as a function of concentration to a monomer-dimer equilibrium yields a dimerization constant of approximately 8.3 mM.     

Residues 48 and 82 at the N-terminal hydrophobic pocket of rabbit skeletal muscle troponin-C photo-cross-link to Met121 of troponin-I.

It has been proposed [Herzberg et al. (1986) J. Biol. Chem. 261, 2638-2644], and confirmed by structural studies [Gagne et al. (1995) Nat. Struct. Biol. 2, 784-789], that the binding of Ca2+ to the triggering sites in troponin-C (TnC) causes the opening of the N-terminal hydrophobic pocket bound by the B, C, and D helices. This conformational change is believed to provide an additional binding site for troponin-I (TnI) and to lead to further events in the Ca2+ regulation process. To answer the question of which part of TnI interacts with this hydrophobic patch of TnC, we constructed two TnC mutants, each with a single cysteine, one at residue 48 between helices B and C and the other at residue 82 on the D helix. Each mutant was labeled with the photoactivatable cross-linker benzophenone-4-iodoacetamide, followed by reconstitution and UV irradiation. Studies were made in the binary complex composed of TnC and TnI, the ternary complex composed of TnC, TnI, and troponin-T (TnT), and the synthetic thin filament composed of troponin, tropomyosin, and F-actin. TnC-TnI photo-cross-linking was observed for both mutants and for all three types of complexes. Although no Ca2+ dependence in the photo-cross-linking was observed on the binary and ternary complexes, the extent of cross-linking was reduced in the absence vs the presence of Ca2+ in the thin filament. TnI Met121, five residues from the C-terminus of the inhibitory region, was identified as the cross-linking site for both TnC mutants using microsequencing and mass spectrometry following proteolysis. These results, obtained with intact TnC.TnI complexes, indicate that the TnI segment containing Met121 is in close contact with the N-terminal hydrophobic patch of TnC, and that in the thin filament the segment containing this residue moves away slightly from the hydrophobic patch in the absence of Ca2+, possibly triggering the translocation of the actin-binding region(s) of TnI toward actin.     

Phosphorylation-dependent conformational transition of the cardiac specific N-extension of troponin I in cardiac troponin

We present here the solution structure for the bisphosphorylated form of the cardiac N-extension of troponin I (cTnI(1-32)), a region for which there are no previous high-resolution data. Using this structure, the X-ray crystal structure of the cardiac troponin core, and uniform density models of the troponin components derived from neutron contrast variation data, we built atomic models for troponin that show the conformational transition in cardiac troponin induced by bisphosphorylation. In the absence of phosphorylation, our NMR data and sequence analyses indicate a less structured cardiac N-extension with a propensity for a helical region surrounding the phosphorylation motif, followed by a helical C-terminal region (residues 25-30). In this conformation, TnI(1-32) interacts with the N-lobe of cardiac troponin C (cTnC) and thus is positioned to modulate myofilament Ca2+-sensitivity. Bisphosphorylation at Ser23/24 extends the C-terminal helix (residues 21-30) which results in weakening interactions with the N-lobe of cTnC and a re-positioning of the acidic amino terminus of cTnI(1-32) for favorable interactions with basic regions, likely the inhibitory region of cTnI. An extended poly(L-proline)II helix between residues 11 and 19 serves as the rigid linker that aids in re-positioning the amino terminus of cTnI(1-32) upon bisphosphorylation at Ser23/24. We propose that it is these electrostatic interactions between the acidic amino terminus of cTnI(1-32) and the basic inhibitory region of troponin I that induces a bending of cTnI at the end that interacts with cTnC. This model provides a molecular mechanism for the observed changes in cross-bridge kinetics upon TnI phosphorylation.     

Role of the structural domain of troponin C in muscle regulation: NMR studies of Ca2+ binding and subsequent interactions with regions 1-40 and 96-115 of troponin I

The interaction between the calcium binding and inhibitory components of troponin is central to the regulation of muscle contraction. In this work, two-dimensional heteronuclear single-quantum coherence nuclear magnetic resonance (2D-?1H,15N?-HSQC NMR) spectroscopy was used to determine the stoichiometry, affinity, and mechanisms for binding of Ca2+ and two synthetic TnI peptides [TnI1-40 (or Rp40) and TnI96-115] to the isolated C-domain of skeletal troponin C (CTnC). The Ca2+ titration revealed that 2 equiv of Ca2+ binds to sites III and IV of CTnC with strong positive cooperativity and high affinity [dissociation constant (KD) 相似文献   

Binding of cardiac troponin-I147-163 induces a structural opening in human cardiac troponin-C.

The interaction of troponin-C (TnC) with troponin-I (TnI) plays a central role in skeletal and cardiac muscle contraction. We have recently shown that the binding of Ca2+ to cardiac TnC (cTnC) does not induce an "opening" of the regulatory domain in order to interact with cTnI [Sia, S. K., et al. (1997) J. Biol. Chem. 272, 18216-18221; Spyracopoulos et al. (1997) Biochemistry 36, 12138-12146], which is in contrast to the regulatory N-domain of skeletal TnC (sTnC). This implies that the mode of interaction between cTnC and cTnI may be different than that between sTnC and sTnI. In sTnI, a region downstream from the inhibitory region (residues 115-131) has been shown to bind the exposed hydrophobic pocket of Ca2+-saturated sNTnC [McKay, R. T., et al. (1997) J. Biol. Chem. 272, 28494-28500]. The present study demonstrates that the corresponding region in cTnI (residues 147-163) binds to the regulatory domain of cTnC only in the Ca2+-saturated state to form a 1:1 complex, with an affinity approximately six times weaker than that between the skeletal counterparts. Thus, while Ca2+ does not cause opening, it is required for muscle regulation. The solution structure of the cNTnC.Ca2+.cTnI147-163 complex has been determined by multinuclear multidimensional NMR spectroscopy. The structure reveals an open conformation for cNTnC, similar to that of Ca2+-saturated sNTnC. The bound peptide adopts a alpha-helical conformation spanning residues 150-157. The C-terminus of the peptide is unstructured. The open conformation for Ca2+-saturated cNTnC in the presence of cTnI (residues 147-163) accommodates hydrophobic interactions between side chains of the peptide and side chains at the interface of A and B helices of cNTnC. Thus the mechanistic differences between the regulation of cardiac and skeletal muscle contraction can be understood in terms of different thermodynamics and kinetics equilibria between essentially the same structure states.     

Inhibitory region of troponin I: Ca(2+)-dependent structural and environmental changes in the troponin-tropomyosin complex and in reconstituted thin filaments

In muscle thin filaments, the inhibitory region (residues 96-117) of troponin I (TnI) is thought to interact with troponin C (TnC) in the presence of Ca(2+) and with actin in the absence of Ca(2+). To better understand these interactions, we prepared mutant TnIs which contained a single Cys-96 or Cys-117 and labeled them with the thiol-specific fluorescent probe N-(iodoacetyl)-N'-(1-sulfo-5-naphthyl)ethylenediamine (IAEDANS). We characterized the microenvironments of the AEDANS labels on TnI in the presence and absence of Ca(2+) by measuring the extent of acrylamide quenching of fluorescence and lifetime-resolved anisotropy. In the troponin-tropomyosin (Tn-Tm) complex, the AEDANS labels on both Cys-96 and Cys-117 were less accessible to solvent and less flexible in the presence of Ca(2+), reflecting closer interactions with TnC under these conditions. In reconstituted thin filaments, the environment of the AEDANS on Cys-96 was not greatly affected by Ca(2+), while the AEDANS on Cys-117 was more accessible but significantly less flexible as it moved away from actin and interacted strongly with TnC in the presence of Ca(2+). We used fluorescence resonance energy transfer (FRET) to measure distances between AEDANS on TnI Cys-96 or Cys-117 and 4-?[(dimethylamino)phenyl]azo?phenyl-4'-maleimide (DABmal) on actin Cys-374 in reconstituted thin filaments. In the absence of Ca(2+), the mean distances were 40.2 A for Cys-96 and 35.2 A for Cys-117. In the presence of Ca(2+), Cys-96 moved away from actin Cys-374 by approximately 3.6 A, while Cys-117 moved away by approximately 8 A. This suggests the existence of a flexible "hinge" region near the middle of TnI, allowing amino acid residues in the N-terminal half of TnI to interact with TnC in a Ca(2+)-independent manner, while the C-terminal half of TnI binds to actin in the absence of Ca(2+) or to TnC in the presence of Ca(2+). This is the first report to demonstrate structural movement of the inhibitory region of TnI in the thin filament.     

Solution structure of the chicken skeletal muscle troponin complex via small-angle neutron and X-ray scattering

Troponin is a Ca2+-sensitive switch that regulates the contraction of vertebrate striated muscle by participating in a series of conformational events within the actin-based thin filament. Troponin is a heterotrimeric complex consisting of a Ca2+-binding subunit (TnC), an inhibitory subunit (TnI), and a tropomyosin-binding subunit (TnT). Ternary troponin complexes have been produced by assembling recombinant chicken skeletal muscle TnC, TnI and the C-terminal portion of TnT known as TnT2. A full set of small-angle neutron scattering data has been collected from TnC-TnI-TnT2 ternary complexes, in which all possible combinations of the subunits have been deuterated, in both the +Ca2+ and -Ca2+ states. Small-angle X-ray scattering data were also collected from the same troponin TnC-TnI-TnT2 complex. Guinier analysis shows that the complex is monomeric in solution and that there is a large change in the radius of gyration of TnI when it goes from the +Ca2+ to the -Ca2+ state. Starting with a model based on the human cardiac troponin crystal structure, a rigid-body Monte Carlo optimization procedure was used to yield models of chicken skeletal muscle troponin, in solution, in the presence and in the absence of regulatory calcium. The optimization was carried out simultaneously against all of the scattering data sets. The optimized models show significant differences when compared to the cardiac troponin crystal structure in the +Ca2+ state and provide a structural model for the switch between +Ca2+ and -Ca2+ states. A key feature is that TnC adopts a dumbbell conformation in both the +Ca2+ and -Ca2+ states. More importantly, the data for the -Ca2+ state suggest a long extension of the troponin IT arm, consisting mainly of TnI. Thus, the troponin complex undergoes a large structural change triggered by Ca2+ binding.     

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.