Functional importance of Ca2+-deficient N-terminal lobe of molluscan troponin C in troponin regulation

Ca(2+)-binding sites I and II in the N-terminal lobe of molluscan troponin C (TnC) have lost the ability to bind Ca(2+) due to substitutions of the amino acid residues responsible for Ca(2+) liganding. To evaluate the functional importance of the Ca(2+)-deficient N-terminal lobe in the Ca(2+)-regulatory function of molluscan troponin, we constructed chimeric TnCs comprising the N-terminal lobes from rabbit fast muscle and squid mantle muscle TnCs and the C-terminal lobe from akazara scallop TnC, TnC(RA), and TnC(SA), respectively. We characterized their biochemical properties as compared with those of akazara scallop wild-type TnC (TnC(AA)). According to equilibrium dialysis using (45)Ca(2+), TnC(RA), and TnC(SA) bound stoichiometrically 3 mol Ca(2+)/mol and 1 mol Ca(2+)/mol, respectively, as expected from their primary structures. All the chimeric TnCs exhibited difference-UV-absorption spectra at around 280-290 nm upon Ca(2+) binding and formed stable complexes with akazara scallop troponin I, even in the presence of 6M urea, if Ca(2+) was present. However, when the troponin complexes were constructed from chimeric TnCs and akazara scallop troponin T and troponin I, they showed different Ca(2+)-regulation abilities from each other depending on the TnC species. Thus, the troponin containing TnC(SA) conferred as high a Ca(2+) sensitivity to Mg-ATPase activity of rabbit actomyosin-akazara scallop tropomyosin as did the troponin containing TnC(AA), whereas the troponin containing TnC(RA) conferred virtually no Ca(2+) sensitivity. Our findings indicate that the N-terminal lobe of molluscan TnC plays important roles in molluscan troponin regulation, despite its inability to bind Ca(2+).     

Engineered troponin C constructs correct disease-related cardiac myofilament calcium sensitivity

Aberrant myofilament Ca(2+) sensitivity is commonly observed with multiple cardiac diseases, especially familial cardiomyopathies. Although the etiology of the cardiomyopathies remains unclear, improving cardiac muscle Ca(2+) sensitivity through either pharmacological or genetic approaches shows promise of alleviating the disease-related symptoms. Due to its central role as the Ca(2+) sensor for cardiac muscle contraction, troponin C (TnC) stands out as an obvious and versatile target to reset disease-associated myofilament Ca(2+) sensitivity back to normal. To test the hypothesis that aberrant myofilament Ca(2+) sensitivity and its related function can be corrected through rationally engineered TnC constructs, three thin filament protein modifications representing different proteins (troponin I or troponin T), modifications (missense mutation, deletion, or truncation), and disease subtypes (familial or acquired) were studied. A fluorescent TnC was utilized to measure Ca(2+) binding to TnC in the physiologically relevant biochemical model system of reconstituted thin filaments. Consistent with the pathophysiology, the restrictive cardiomyopathy mutation, troponin I R192H, and ischemia-induced truncation of troponin I (residues 1-192) increased the Ca(2+) sensitivity of TnC on the thin filament, whereas the dilated cardiomyopathy mutation, troponin T ΔK210, decreased the Ca(2+) sensitivity of TnC on the thin filament. Rationally engineered TnC constructs corrected the abnormal Ca(2+) sensitivities of the thin filament, reconstituted actomyosin ATPase activity, and force generation in skinned trabeculae. Thus, the present study provides a novel and versatile therapeutic strategy to restore diseased cardiac muscle Ca(2+) sensitivity.     

Functional and evolutionary relationships of troponin C.

Striated muscle contraction is initiated when, following membrane depolarization, Ca(2+) binds to the low-affinity Ca(2+) binding sites of troponin C (TnC). The Ca(2+) activation of this protein results in a rearrangement of the components (troponin I, troponin T, and tropomyosin) of the thin filament, resulting in increased interaction between actin and myosin and the formation of cross bridges. The functional properties of this protein are therefore critical in determining the active properties of striated muscle. To date there are 61 known TnCs that have been cloned from 41 vertebrate and invertebrate species. In vertebrate species there are also distinct fast skeletal muscle and cardiac TnC proteins. While there is relatively high conservation of the amino acid sequence of TnC homologs between species and tissue types, there is wide variation in the functional properties of these proteins. To date there has been extensive study of the structure and function of this protein and how differences in these translate into the functional properties of muscles. The purpose of this work is to integrate these studies of TnC with phylogenetic analysis to investigate how changes in the sequence and function of this protein, integrate with the evolution of striated muscle.     

Isolation, expression, and mutation of a rabbit skeletal muscle cDNA clone for troponin I. The role of the NH2 terminus of fast skeletal muscle troponin I in its biological activity.

A cDNA for rabbit fast skeletal muscle troponin I (TnI) was isolated and sequenced. The clone contains a coding sequence predicting a 182-amino-acid protein with a molecular mass of 21,162 daltons. The translated sequence is different from that reported by Wilkinson and Grand (Wilkinson, J. M., and Grand, R. J. A. (1978) Nature 271, 31-35) in that Arg-153, Asp-154, and Leu-155 must be inserted into their original sequence. Amino acid sequencing of adult rabbit TnI confirmed this result. In order to investigate the role of the NH2 terminus of TnI in its biological activity, we have expressed a recombinant deletion mutant (TnId57), which lacks residues 1-57, in a bacterial expression system. Both wild type TnI (WTnI) and TnId57 inhibited acto-S1-ATPase activity and this inhibition could be fully reversed by troponin C (TnC) in the presence of Ca2+. Additionally both WTnI and TnId57 bound to an actin affinity column. Thus, both inhibitory actin binding and Ca(2+)-dependent neutralization by TnC were retained in TnId57. TnC affinity chromatography was used to compare the binding of TnI and TnId57 to TnC. Using this method, two types of interaction between TnC and TnI were observed: 1) one which is metal independent (or structural) and 2) one dependent on Ca2+ or Mg2+ binding to the Ca(2+)-Mg2+ sites of TnC. The same experiments with TnId57 demonstrated that the type 1 interaction was weakened, and type 2 binding was lost. This method also revealed an interaction between TnC and TnI which is dependent upon Ca2+ binding to the Ca(2+)-specific sites of TnC and which is retained in TnId57. Taken together, these results suggest that the NH2 terminus of TnI may constitute a Ca(2+)-Mg(2+)-dependent interaction site between TnC and TnI and play, in part, a structural role in maintaining the stability of the troponin complex while the COOH terminus of TnI contains a Ca(2+)-specific site-dependent interaction site for TnC as well as the previously demonstrated Ca(2+)-sensitive inhibitory and actin binding activities.     

Binding properties of the calcium-activated F2 isoform of Lethocerus troponin C

While in most muscles contraction is triggered by calcium effluxes, insect flight muscles are also activated by mechanical stretch. We are interested in understanding the role that the troponin C protein, usually the calcium sensor, plays in stretch activation. In the flight muscles of Lethocerus, a giant water bug often used as a model system, there are two isoforms of TnC, F1 and F2, present in an approximately 10:1 ratio. F1 TnC is responsible for activating the muscle following a stretch, whereas F2 TnC produces a sustained contraction, the magnitude of which depends on the concentration of Ca(2+) in the fiber. We have previously shown that F1 TnC binds only one Ca(2+) ion in its C-terminal domain and that interaction with troponin H, the insect ortholog of troponin I, is insensitive to Ca(2+). Here, we have studied the effect of Ca(2+) and Mg(2+) on the affinities of the interaction of F2 TnC with troponin H peptides. We show that the presence of two Ca(2+) ions, one in each of the globular domains, increases the affinity for TnH by at least 1 order of magnitude. The N lobe has a lower affinity for Ca(2+), but it is also sensitive to Mg(2+). The C lobe is insensitive to Mg(2+) as previously demonstrated by mutations of the individual EF-hands. The interaction with TnH seems also to have significant structural differences from that observed for the F1 TnC isoform. We discuss how our findings could account for stretch activation.     

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.     

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.     

Biological function and site II Ca2+-induced opening of the regulatory domain of skeletal troponin C are impaired by invariant site I or II Glu mutations

To investigate the roles of site I and II invariant Glu residues 41 and 77 in the functional properties and calcium-induced structural opening of skeletal muscle troponin C (TnC) regulatory domain, we have replaced them by Ala in intact F29W TnC and in wild-type and F29W N domains (TnC residues 1-90). Reconstitution of intact E41A/F29W and E77A/F29W mutants into TnC-depleted muscle skinned fibers showed that Ca(2+)-induced tension is greatly reduced compared with the F29W control. Circular dichroism measurements of wild-type N domain as a function of pCa (= -log[Ca(2+)]) demonstrated that approximately 90% of the total change in molar ellipticity at 222 nm ([theta](222 nm)) could be assigned to site II Ca(2+) binding. With E41A, E77A, and cardiac TnC N domains this [theta](222 nm) change attributable to site II was reduced to < or =40% of that seen with wild type, consistent with their structures remaining closed in +Ca(2+). Furthermore, the Ca(2+)-induced changes in fluorescence, near UV CD, and UV difference spectra observed with intact F29W are largely abolished with E41A/F29W and E77A/F29W TnCs. Taken together, the data indicate that the major structural change in N domain, including the closed to open transition, is triggered by site II Ca(2+) binding, an interpretation relevant to the energetics of the skeletal muscle TnC and cardiac TnC systems.     

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.     

Modulation of the interaction between the two halves of troponin C by the other troponin subunits

The interactions between troponin subunits have been studied by intrinsic fluorescence and electron spin resonance (ESR) spectroscopy. The tryptophan fluorescence of troponin T (TnT) and troponin I (TnI) when complexed with troponin C (TnC) undergoes a Ca2+-dependent transition. The midpoints of such spectral changes occur at pCa approximately equal to 6, suggesting that the conformational change of TnT and TnI is induced by Ca2+ binding to the low-affinity sites of TnC. When TnC is labelled at Cys-98 with a maleimide spin probe (MSL), the spin signal is sensitive to Ca2+ binding to both the high and the low-affinity sites of TnC in the presence of either or both of the other two troponin subunits. Since Cys-98 is located in the vicinity of one of the high-affinity sites, these results are indicative of a long-range interaction between the two halves of the TnC molecule. Our earlier kinetic studies [Wang, C.-L. A., Leavis, P. C. & Gergely, J. (1983) J. Biol. Chem. 258, 9175-9177] have shown such interactions in TnC alone. Since the ESR spectral change associated with metal binding to the low-affinity sites is only observed when MSL-TnC is complexed with TnT and/or TnI, this long-range interaction within TnC appears to be mediated through the other troponin subunits.     

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.     

Calmodulin supports the force-generating function in desensitized muscle fibers

Externally added calmodulin (CaM) restored Ca2+ regulation for the tension development by skeletal muscle fibers of hamster and rabbit desensitized by the troponin C (TnC) extraction treatment. CaM produced this action by combining with the TnC-denuded sites in the fiber. However, the binding properties differed strikingly from TnC: unlike TnC, CaM binding required the continued presence of Ca2+ and the bound portion was completely released with EGTA in the physiological milieu. The maximal uptake was 1.7 g of CaM/kg of muscle in the present study. The apparent Ca2+ sensitivity for force development with 200 micrograms/ml CaM in the solution was lower than in the native fiber or in the TnC-loaded fiber. The apparent association constant for CaM binding to the TnC-denuded sites was found as 4.9 x 10(5) M-1, and the extrapolated maximum force (Fmax) with CaM was close to PO. The intrinsic CaM level in intact muscle was also measured and was 18.6 mg/kg, amounting to about 1% of the total TnC or the CaM uptake by TnC-denuded fibers. The intrinsic CaM was not dislodged by EDTA treatment, indicating tight binding and suggesting that it exists in a separate pool from the vacated TnC sites adsorbing externally added CaM. The stringent Ca+ dependence of the CaM adsorption to TnC sites in the regulatory complex in the fiber supports the view that the evolutionary replacement of residues in the amino terminus helix portion of the "EF-hand" motif of site IV may be critical for the functional specialization by TnC.     

Force-calcium relationship depends on myosin heavy chain and troponin isoforms in rat diaphragm muscle fibers.

The present study examined Ca(2+) sensitivity of diaphragm muscle (Dia(m)) fibers expressing different myosin heavy chain (MHC) isoforms. We hypothesized that Dia(m) fibers expressing the MHC(slow) isoform have greater Ca(2+) sensitivity than fibers expressing fast MHC isoforms and that this fiber-type difference in Ca(2+) sensitivity reflects the isoform composition of the troponin (Tn) complex (TnC, TnT, and TnI). Studies were performed in single Triton-X-permeabilized Dia(m) fibers. The Ca(2+) concentration at which 50% maximal force was generated (pCa(50)) was determined for each fiber. SDS-PAGE and Western analyses were used to determine the MHC and Tn isoform composition of single fibers. The pCa(50) for Dia(m) fibers expressing MHC(slow) was significantly greater than that of fibers expressing fast MHC isoforms, and this greater Ca(2+) sensitivity was associated with expression of slow isoforms of the Tn complex. However, some Dia(m) fibers expressing MHC(slow) contained the fast TnC isoform. These results suggest that the combination of TnT, TnI, and TnC isoforms may determine Ca(2+) sensitivity in Dia(m) fibers.     

Modulation of the rate of cardiac muscle contraction by troponin C constructs with various calcium binding affinities

We investigated whether changing thin filament Ca(2+) sensitivity alters the rate of contraction, either during normal cross-bridge cycling or when cross-bridge cycling is increased by inorganic phosphate (P(i)). We increased or decreased Ca(2+) sensitivity of force production by incorporating into rat skinned cardiac trabeculae the troponin C (TnC) mutants V44QTnC(F27W) and F20QTnC(F27W). The rate of isometric contraction was assessed as the rate of force redevelopment (k(tr)) after a rapid release and restretch to the original length of the muscle. Both in the absence of added P(i) and in the presence of 2.5 mM added P(i) 1) Ca(2+) sensitivity of k(tr) was increased by V44QTnC(F27W) and decreased by F20QTnC(F27W) compared with control TnC(F27W); 2) k(tr) at submaximal Ca(2+) activation was significantly faster for V44QTnC(F27W) and slower for F20QTnC(F27W) compared with control TnC(F27W); 3) at maximum Ca(2+) activation, k(tr) values were similar for control TnC(F27W), V44QTnC(F27W), and F20QTnC(F27W); and 4) k(tr) exhibited a linear dependence on force that was indistinguishable for all TnCs. In the presence of 2.5 mM P(i), k(tr) was faster at all pCa values compared with the values for no added P(i) for TnC(F27W), V44QTnC(F27W), and F20QTnC(F27W). This study suggests that TnC Ca(2+) binding properties modulate the rate of cardiac muscle contraction at submaximal levels of Ca(2+) activation. This result has physiological relevance considering that, on a beat-to-beat basis, the heart contracts at submaximal Ca(2+) activation.     

Fourier transform infrared spectroscopic study on the binding of Mg2+ to a mutant Akazara scallop troponin C (E142Q)

Troponin C (TnC) is the Ca(2+)-binding regulatory protein of the troponin complex in muscle tissue. Vertebrate fast skeletal muscle TnCs bind four Ca(2+), while Akazara scallop (Chlamys nipponensis akazara) striated adductor muscle TnC binds only one Ca(2+) at site IV, because all the other EF-hand motifs are short of critical residues for the coordination of Ca(2+). Fourier transform infrared (FTIR) spectroscopy was applied to study coordination structure of Mg(2+) bound in a mutant Akazara scallop TnC (E142Q) in D(2)O solution. The result showed that the side-chain COO(-) groups of Asp 131 and Asp 133 in the Ca(2+)-binding site of E142Q bind to Mg(2+) in the pseudo-bridging mode. Mg(2+) titration experiments for E142Q and the wild-type of Akazara scallop TnC were performed by monitoring the band at about 1600 cm(-1), which is due to the pseudo-bridging Asp COO(-) groups. As a result, the binding constants of them for Mg(2+) were the same value (about 6 mM). Therefore, it was concluded that the side-chain COO(-) group of Glu 142 of the wild type has no relation to the Mg(2+) ligation. The effect of Mg(2+) binding in E142Q was also investigated by CD and fluorescence spectroscopy. The on-off mechanism of the activation of Akazara scallop TnC is discussed on the basis of the coordination structures of Mg(2+) as well as Ca(2+).     

Mutation of the high affinity calcium binding sites in cardiac troponin C.

Fast skeletal and cardiac troponin C (TnC) contain two high affinity Ca2+/Mg2+ binding sites within the C-terminal domain that are thought to be important for association of TnC with the troponin complex of the thin filament. To test directly the function of these high affinity sites in cardiac TnC they were systematically altered by mutagenesis to generate proteins with a single inactive site III or IV (CBM-III and CBM-IV, respectively), or with both sites III and IV inactive (CBM-III-IV). Equilibrium dialysis indicated that the mutated sites did not bind Ca2+ at pCa 4. Both CBM-III and CBM-IV were similar to the wild type protein in their ability to regulate Ca(2+)-dependent contraction in slow skeletal muscle fibers, and Ca(2+)-dependent ATPase activity in fast skeletal and cardiac muscle myofibrils. The mutant CBM-III-IV is capable of regulating contraction in permeabilized slow muscle fibers but only if the fibers are maintained in a contraction solution containing a high concentration of the mutant protein. CBM-III-IV also regulates myofibril ATPase activity in fast skeletal and cardiac myofibrils but only at concentrations 10-100-fold greater than the normal protein. The pCa50 and Hill coefficient values for Ca(2+)-dependent activation of fast skeletal muscle myofibril ATPase activity by the normal protein and all three mutants are essentially the same. Competition between active and inactive forms of cardiac and slow TnC in a functional assay demonstrates that mutation of both sites III and IV greatly reduces the affinity of cardiac and slow TnC for its functionally relevant binding site in the myofibrils. The data indicate that although neither high affinity site is absolutely essential for regulation of muscle contraction in vitro, at least one active C-terminal site is required for tight association of cardiac troponin C with myofibrils. This requirement can be satisfied by either site III or IV.     

Characteristics of troponin C binding to the myofibrillar thin filament: extraction of troponin C is not random along the length of the thin filament.

Troponin C (TnC) is the Ca(2+)-sensing subunit of troponin responsible for initiating the cascade of events resulting in contraction of striated muscle. This protein can be readily extracted from myofibrils with low-ionic-strength EDTA-containing buffers. The properties of TnC extraction have not been characterized at the structural level, nor have the interactions of TnC with the native myofibrillar thin filament been studied. To address these issues, fluorescein-labeled TnC, in conjunction with high-resolution digital fluorescence microscopy, was used to characterize TnC binding to myofibrils and to determine the randomness of TnC extraction. Fluorescein-5-maleimide TnC (F5M TnC) retained biological activity, as evidenced by reconstitution of Ca(2+)-dependent ATPase activity in extracted myofibrils and binding to TnI in a Ca(2+)-sensitive manner. The binding of F5M TnC to highly extracted myofibrils at low Ca2+ was restricted to the overlap region under rigor conditions, and the location of binding was not influenced by F5M TnC concentration. The addition of myosin subfragment 1 to occupy all actin sites resulted in F5M TnC being bound in both the overlap and nonoverlap regions. However, very little F5M TnC was bound to myofibrils under relaxing conditions. These results suggest that strong binding of myosin heads enhances TnC binding. At high Ca2+, the pattern of F5M TnC binding was concentration dependent: binding was restricted to the overlap region at low F5M TnC concentration, whereas the binding propagated into the nonoverlap region at higher levels. Analysis of fluorescence intensity showed the greatest binding of F5M TnC at high Ca2+ with S1, and these conditions were used to characterize partially TnC-extracted myofibrils. Comparison of partially extracted myofibrils showed that low levels of extraction were associated with greater F5M TnC being bound in the nonoverlap region than in the overlap region relative to higher levels of extraction. These results show that TnC extraction is not random along the length of the thin filament, but occurs more readily in the nonoverlap region. This observation, in conjunction with the influence of rigor heads on the pattern of F5M TnC binding, suggests that strong myosin binding to actin stabilizes TnC binding at low Ca2+.     

Site-directed spin labeling electron paramagnetic resonance study of the calcium-induced structural transition in the N-domain of human cardiac troponin C complexed with troponin I

Calcium-induced structural transition in the amino-terminal domain of troponin C (TnC) triggers skeletal and cardiac muscle contraction. The salient feature of this structural transition is the movement of the B and C helices, which is termed the "opening" of the N-domain. This movement exposes a hydrophobic region, allowing interaction with the regulatory domain of troponin I (TnI) as can be seen in the crystal structure of the troponin ternary complex [Takeda, S., Yamashita, A., Maeda, K., and Maeda, Y. (2003) Nature 424, 35-41]. In contrast to skeletal TnC, Ca(2+)-binding site I (an EF-hand motif that consists of an A helix-loop-B helix motif) is inactive in cardiac TnC. The question arising from comparisons with skeletal TnC is how both helices move according to Ca(2+) binding or interact with TnI in cardiac TnC. In this study, we examined the Ca(2+)-induced movement of the B and C helices relative to the D helix in a cardiac TnC monomer state and TnC-TnI binary complex by means of site-directed spin labeling electron paramagnetic resonance (EPR). Doubly spin-labeled TnC mutants were prepared, and the spin-spin distances were estimated by analyzing dipolar interactions with the Fourier deconvolution method. An interspin distance of 18.4 A was estimated for mutants spin labeled at G42C on the B helix and C84 on the D helix in a Mg(2+)-saturated monomer state. The interspin distance between Q58C on the C helix and C84 on the D helix was estimated to be 18.3 A under the same conditions. Distance changes were observed by the addition of Ca(2+) ions and the formation of a complex with TnI. Our data indicated that the C helix moved away from the D helix in a distinct Ca(2+)-dependent manner, while the B helix did not. A movement of the B helix by interaction with TnI was observed. Both Ca(2+) and TnI were also shown to be essential for the full opening of the N-domain in cardiac TnC.     

A cross-linking study of the N-terminal extension of human cardiac troponin I

Phosphorylation of the unique N-terminal extension of cardiac troponin I (TnI) by PKA modulates Ca(2+) release from the troponin complex. The mechanism by which phosphorylation affects Ca(2+) binding, however, remains unresolved. To investigate this question, we have studied the interaction of a fragment of TnI consisting of residues 1-64 (I1-64) with troponin C (TnC) by isothermal titration microcalorimetry and cross-linking. I1-64 binds extremely tightly to the C-terminal domain of TnC and weakly to the N-terminal domain. Binding to the N-domain is weakened further by phosphorylation. Using the heterobifunctional cross-linker benzophenone-4-maleimide and four separate cysteine mutants of I1-64 (S5C, E10C, I18C, R26C), we have probed the protein-protein interactions of the N-terminal extension. All four I1-64 mutants cross-link to the N-terminal domain of TnC. The cross-linking is enhanced by Ca(2+) and reduced by phosphorylation. By introducing the same monocysteine mutations into full-length TnI, we were able to probe the environment of the N-terminal extension in intact troponin. We find that the full length of the extension lies in close proximity to both TnC and troponin T (TnT). Ca(2+) enhances the cross-linking to TnC. Cross-linking to both TnC and TnT is reduced by prior phosphorylation of the TnI. In binary complexes the mutant TnIs cross-link to both the isolated TnC N-domain and whole TnC. Cyanogen bromide digestion of the covalent TnI-TnC complex formed from intact troponin demonstrates that cross-linking is predominantly to the N-terminal domain of TnC.     

Effects of inhibitors on luminal opening of Ca2+ binding sites in an E2P-like complex of sarcoplasmic reticulum Ca22+-ATPase with Be22+-fluoride

We document here the intrinsic fluorescence and 45Ca2+ binding properties of putative "E2P-related" complexes of Ca2+-free ATPase with fluoride, formed in the presence of magnesium, aluminum, or beryllium. Intrinsic fluorescence measurements suggest that in the absence of inhibitors, the ATPase complex with beryllium fluoride (but not those with magnesium or aluminum fluoride) does constitute an appropriate analog of the "ADP-insensitive" phosphorylated form of Ca2+-ATPase, the so-called "E2P" state. 45Ca2+ binding measurements, performed in the presence of 100 mm KCl, 5 mm Mg2+, and 20% Me2SO at pH 8, demonstrate that this ATPase complex with beryllium fluoride (but again not those with magnesium or aluminum fluoride) has its Ca2+ binding sites accessible for rapid, low affinity (submillimolar) binding of Ca2+ from the luminal side of SR. In addition, we specifically demonstrate that in this E2P-like form of ATPase, the presence of thapsigargin, 2,5-di-tert-butyl-1,4-dihydroxybenzene, or cyclopiazonic acid prevents 45Ca2+ binding (i.e. presumably prevents opening of the 45Ca2+ binding sites on the SR luminal side). Since crystals of E2P-related forms of ATPase have up to now been described in the presence of thapsigargin only, these results suggest that crystallizing an inhibitor-free E2P-like form of ATPase (like its complex with beryllium fluoride) would be highly desirable, to unambiguously confirm previous predictions about the exit pathway from the ATPase transmembrane Ca2+ binding sites to the SR luminal medium.