©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Functional Role of the Domains of Troponin-C Investigated with Thrombin Fragments of Troponin-C Reconstituted into Skinned Muscle Fibers (*)

(Received for publication, January 24, 1995; and in revised form, April 5, 1995)

Jean-Marie Francois(§)(¶) Zelin Sheng (§) Danuta Szczesna James D. Potter (**)

From the Department of Molecular & Cellular Pharmacology, University of Miami School of Medicine, Miami, Florida 33101

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Proteolysis of rabbit fast skeletal troponin-C (RSTnC) with thrombin produces four separate fragments containing the following Ca-binding site(s): TH(1) (residues 1-120) sites I-III; TH(2)(121-159) site IV; TH(3)(1-100) sites I and II; and TH(4)(101-120) site III. We studied the ability of these fragments to restore the steady state isometric force in TnC-depleted skinned skeletal muscle fibers. Interestingly, we found that all investigated fragments of RSTnC possessed some of the properties of native RSTnC, but none of them could fully regulate contraction in the fibers like intact RSTnC. TH(1) was the most effective in the force restoration (65%) whereas the smaller fragments developed about 50% (TH(3) and TH(4)) or 20% (TH(2)) of the initial force of unextracted fibers. Additionally, much higher concentrations of TH(2), TH(3), and TH(4) compared to RSTnC or TH(1) were necessary for force development suggesting a decreased affinity of these fragments to their binding site(s) in the fibers. Like intact RSTnC, TH(1) was able to interact with the fibers in a Ca-independent (Mg-dependent) manner, indicating that at a minimum, Ca-binding site III is required for this type of binding. The initial binding of the other fragments to the TnC-depleted fibers occurred only in the presence of Ca. TH(2) and TH(4) appeared to bind to two different binding sites in the fibers. The binding to one of the sites caused partial force restoration. This binding of TH(2) and TH(4) was abolished when Ca was removed. TH(2) and TH(4) binding to the second site required Ca initially but was maintained in the presence of Mg. This interaction of TH(2) and TH(4) partially blocked the rebinding of RSTnC to the fibers. The latter results suggest that site III or IV in these small fragments, when removed from the constraints of the parent protein, may assume conformations that allow them to function, to a certain extent, like both the regulatory sites (I and II) and the Ca-Mg sites (III and IV) of TnC.


INTRODUCTION

In vertebrate muscle, the binding of Ca to troponin-C (TnC), (^1)the Ca binding subunit of troponin, triggers a cascade of events that ultimately leads to muscle contraction. TnC is the critical component of the contractile apparatus directly responsible for the Ca regulation of muscle activation. The crystallographic structure of fast skeletal muscle TnC reveals that it is a dumbbell-shaped protein containing two globular domains linked by a central helix(1, 2, 3) . TnC consists of four EF hand type Ca-binding sites(4, 5, 6) . The NH(2)-terminal domain of TnC contains two low affinity Ca-binding sites, specific for Ca, designated as site I and site II and referred to as the Ca-specific sites(7) . These sites are thought to be directly involved in the regulation of muscle contraction (7, 8) . The COOH-terminal domain contains two high affinity Ca-binding sites that bind Mg competitively, designated as site III and site IV and referred to as the Ca-Mg sites(7, 9) . This domain has been demonstrated to play a structural role in anchoring TnC to the Tn complex in muscle(8, 10) . Recently, site-directed mutagenesis studies of the Ca-Mg sites of TnC showed that the inactivation of either site III or IV greatly increased the concentration of TnC required to inhibit myofibrillar ATPase activity by Tn and also to develop steady state force in skinned skeletal muscle fibers(11, 12) .

This suggests that the TnC-TnI interaction can be affected by altered Ca-binding sites in TnC. Extensive studies on the interactions of the regulatory proteins in muscle (8, 13, 14) have demonstrated that there are different factors which control the type of TnC-TnI interaction. One type of interaction, dependent on the saturation of the Ca-Mg sites of TnC by Mg or Ca, is thought to be located in the COOH-terminal domain of TnC and to play a structural role in anchoring TnC to TnI(10, 14, 15) . The second type of interaction between TnC and TnI is controlled by Ca binding to the Ca-specific sites of TnC (site I and site II) and is located in its NH(2) terminus(14) . This interaction is crucial for the regulatory function of TnC. The third interaction is thought to be independent of metal binding to TnC and also helps to maintain the stability of the whole Tn complex(14) . A model to explain the interactions between TnC and TnI has been proposed by Farah et al.(16) which is consistent with Sheng et al.(14) in which the NH(2)-terminal domain of TnI is anchored to the COOH-terminal domain of TnC in the presence of either Ca or Mg, while the inhibitory and COOH-terminal regions of TnI interact with the regulatory NH(2)-terminal domain of TnC in a Ca-dependent manner.

Extensive structure-function studies of TnC have been carried out through the use of site-directed mutagenesis(14, 17, 18, 19, 20, 21, 22) . In another approach, the effects of TnC(s) from different species or tissues have been functionally investigated on a well characterized contractile system. For example, the effects of TnC from barnacle muscle, which contains only two functional Ca-binding sites(23) , and the effects of cardiac TnC, which has three functional Ca-binding sites, were investigated in the TnC-depleted skinned rabbit fiber system(18, 19, 24, 25) . A structural comparison of these TnC(s) from different species or tissues with the well studied and characterized fast skeletal muscle TnC from rabbit has made it possible to draw basic inferences concerning the regulation of muscle contraction by TnC. Finally, a third approach has utilized either synthetic peptides or proteolytic fragments of TnC to study the regulation of contraction. This approach has been particularly useful in investigating the function of proteins, like TnC, which contain several domains and interaction sites.

Among these approaches, we have chosen the last one that has been studied by others using other systems(26, 27, 28, 29, 30) . We felt that the use of proteolytic fragments of TnC combined with their reincorporation into TnC-depleted skinned skeletal muscle fibers (10, 24) would be a useful extension of previous studies and might give further insight into the mechanisms of muscle regulation. Indeed, TnC proteolytic fragments have not been previously used in a contractile system such as TnC-depleted skinned fibers. This system has been proven to be a useful, functional test to study the contractile properties of muscle proteins. In contrast to reconstituted thin filaments or even to myofibrils, the structure of TnC-depleted skinned fibers is well preserved and is an ``in vitro system'' which closely mimics in vivo conditions, where steady state force development (rather than ATPase hydrolysis) can be directly measured.

In this work, we have used four thrombin fragments of rabbit skeletal TnC (RSTnC) to assess the role of the different regions of TnC in the activation of muscle contraction. Thrombin digestion of RSTnC provides fragments which are particularly suitable for studying the function of the major TnC domains and Ca-binding sites. Our results show that the whole intact structure of TnC is required in order to maintain the full regulatory function of TnC and support the hypothesis that the NH(2)-terminal half of TnC plays a regulatory role in muscle contraction, while the COOH-terminal half is primarily responsible for anchoring TnC to the other troponin subunits.


MATERIALS AND METHODS

Preparations

Rabbit skeletal muscle TnC (RSTnC) was purified as described earlier(31) . Bovine calmodulin and carp parvalbumins (with no isotype separation) were prepared according to the procedure of Amphlett et al.(32) .

Thrombin fragments of RSTnC were prepared according to Leavis et al.(33) with the modifications described below. RSTnC was digested with 5 NIH units of thrombin/mg of protein in 20 mM NH(4)HCO(3), 5 mM EDTA, and 5 mM beta-mercaptoethanol, pH 8.0, for 72 h at room temperature. In order to increase the efficiency of the TH(1) preparation (residues 1-120), the digestion time was shortened to 3 h. For all other fragments, the incubation time of RSTnC with thrombin was 72 h. The fragments were purified on a Sephadex G-50 column in 50 mM NH(4)HCO(3), pH 8.2, at 4 °C. The final separation of TH(1) from RSTnC was achieved on a high performance liquid chromatography gel filtration column (Phenomenex G2000 WS).

Purified proteins and thrombin fragments were tested on 15% SDS-polyacrylamide gel electrophoresis, performed according to Laemmli (34) . The fragments were analyzed for their amino acid composition and their identity verified by several steps of the automatic Edman degradation method or by digestion with carboxypeptidases A and B, according to Dopheide et al.(35) . Skeletal muscle fibers were obtained from rabbit psoas muscle and chemically skinned as described by Kerrick and Krasner(36) .

Steady State Isometric Force Measurements

The skinned fibers (2, 3, 4) were mounted to a force transducer using stainless steel clips(37) . The initial steady state force was recorded after immersion of the fibers in the contracting solution (pCa 4) containing: 10M [Ca], 5 mM [Mg], 4 mM EGTA, 10 mM imidazole, 5 mM MgATP, and 20 mM creatine phosphate, pH 7.0, ionic strength = 200 mM. Fibers were then relaxed in the same solution except the free Ca concentration was 10M (pCa 8, relaxing solution). Extraction of the endogenous TnC from the skinned fibers was achieved by incubation in a solution containing 2 mM EDTA, pH 7.8, for 20 min. This treatment resulted in a residual force of less than 20% of the initial force. Reincorporation of RSTnC or its thrombin fragments to the TnC-depleted skinned fibers was performed by incubation of the fibers in the relaxing (pCa 8) or contracting (pCa 4) solutions containing various concentrations of the proteins, for 20 min at room temperature. The restored force developed by the reconstituted fibers was calculated as follows: force restored (%) = (restored force - residual force)/(initial force - residual force) 100%.

In order to determine the inhibitory effect caused by the reincorporated thrombin fragments on the rebinding of RSTnC to the fibers, they were washed in relaxing solution and then incubated with RSTnC in the pCa 8 solution for 20 min. Then, the force developed by the RSTnC reconstituted fibers was measured in the pCa 4 solution. Due to the competition between reincorporated thrombin fragments and RSTnC for binding to the fibers, they were only partially reconstituted with the RSTnC. To measure the total possible restoration of force in the fibers, the final reextraction with the EDTA solution was performed to remove any bound thrombin fragments or rebound RSTnC. The fibers were then incubated in a new RSTnC solution for 20 min followed by washing with pCa 8 relaxing solution to remove excess unbound proteins. Then the final steady state force was measured. The difference between the two force measurements, after the first and the second RSTnC incorporation into the fibers, defines the ability of the investigated proteins or fragments to prevent the rebinding of RSTnC to the fibers. The blocking effect of the RSTnC rebinding was calculated as follows: blocking of RSTnC rebinding (%) = [1 - (force after the 1 RSTnC incorporation - residual force)/(force after the 2 RSTnC incorporation - residual force)] 100%.


RESULTS

Thrombin Fragments of RSTnC

Thrombin digestion of RSTnC produced four fragments: TH(1) (residues 1-120), TH(2) (residues 121-159), TH(3) (residues 1-100), and TH(4) (residues 101-120). In Fig. 1we show these fragments in a conformation which corresponds to the domains of intact RSTnC, utilizing the crystal structure of turkey TnC by Herzberg and James(1, 2) . The pattern of the purity of the obtained peptides on 15% SDS-polyacrylamide gel electrophoresis is demonstrated in Fig. 2. The TH(2) fragment was identified by performing about 20 steps of the automatic Edman degradation procedure and analyzed for its amino acid composition. TH(4) was completely sequenced. The TH(1) and TH(3) fragments were also analyzed for their amino acid composition. Their COOH-terminal residues were identified after cleavage with carboxypeptidases A and B. The Edman degradation procedure could not be directly applied here because of the acetylated form of the NH(2) terminus of TnC(38) .


Figure 1: Molecular models of the RSTnC thrombin fragments. These molecular models are based on the crystal structure of TnC(1, 2) . The NH(2)-terminal domains of RSTnC, TH(1), and TH(3) contain Ca-specific sites I and II, whereas the COOH-terminal domains of RSTnC, TH(1), TH(2), and TH(4) contain sites III (TH(1) and TH(4)) and IV (TH(2)).




Figure 2: RSTnC thrombin fragments on 15% SDS-polyacrylamide gel electrophoresis. The gel was stained with Coomassie Blue. Lane 1, RSTnC; lane 2, TH(1); lane 3, TH(3); lane 4, TH(2); lane 5, TH(4).



As shown in Fig. 1, TH(1) contains the complete NH(2)-terminal domain of RSTnC, the interconnecting helix (D/E) and the major part of calcium-binding site III (helix E, loop III, and most of helix F). In comparison to TH(1), the TH(3) fragment is shorter by 20 residues and contains the NH(2)-terminal domain, the interconnecting helix D/E and a part of the helix E. The TH(2) fragment contains the whole calcium binding loop IV with helices G and H and 2 residues of helix F which borders site III of RSTnC. TH(4), the shortest peptide studied, contains Ca-binding loop III, one turn of helix E at the NH(2)-terminal part of the peptide and two turns of helix F at its COOH-terminal region.

Effects of RSTnC, Calmodulin, and RSTnC Fragments on the Steady State Force Restoration in TnC-depleted Skinned Muscle Fibers

Incubation of TnC-depleted skinned skeletal muscle fibers with RSTnC (8 µM) dissolved in the pCa 8 solution followed by washing of the fibers in the pCa 8 solution to remove unbound RSTnC resulted in the full restoration of the initial steady state isometric force of unextracted fibers, when tested in the pCa 4 solution (Fig. 3A and Fig. 6). The same results were obtained when the initial incubation with RSTnC was carried out in the pCa 4 solution (data not shown). The above results illustrate the Mg-dependent association of TnC with the fibers. Similar Mg-dependent binding to the TnC-depleted fibers was also observed for TH(1). However, TH(1) could only restore 65% of the initial force developed by the intact unextracted fibers (Fig. 3B and Fig. 6). Moreover, a higher concentration of TH(1) fragment compared to RSTnC was required to develop maximal isometric force (Fig. 6, Table 2).


Figure 3: Reconstitution of TnC^2-depleted skinned fibers with RSTnC (A) and TH(1) (B).A, initial steady state isometric force developed by the fibers was measured after their replacement from the relaxing (pCa 8) to contracting (pCa 4) solution. Extraction of the endogenous TnC from the fibers was achieved by a 20-min incubation in 2 mM EDTA, pH 7.8, as indicated. About 6% of the initial force remained after TnC extraction (residual force). The fibers were then incubated with 8 µM RSTnC dissolved in the pCa 8 solution for 20 min. After washing with the pCa 8 solution, the RSTnC-reconstituted fibers were tested for their contraction in the pCa 4 solution. The isometric steady state force was fully recovered to the level of intact unextracted fibers. B, TnC-depleted fibers were incubated with 25 µM TH(1) in the pCa 8 solution using the same procedure described as above. About 65% of maximal isometric force developed by intact fibers was restored as a result of the TH(1) reconstitution.




Figure 6: Concentration dependence of the activation of TnC- depleted skinned fibers by RSTnC and its thrombin fragments. Experiments were performed as described in Fig. 3-5. Varying amounts of RSTnC or thrombin fragments were bound to the TnC-depleted fibers. The level of restorable force developed by the fibers reconstituted with TH(3) and TH(4) was measured upon incubation in the pCa 4 (contraction) solution. RSTnC and TH(1) were reincorporated in the pCa 8 solution. The values of maximum force restored and the protein concentrations for 50% maximal force restoration are shown in Table 1and Table 2, respectively. , RSTnC; ▪, TH(1); , TH(3); bullet, TH(4). Each data point is the average value ± S.D. of two to five experiments.







Unlike RSTnC or TH(1), calmodulin (CaM), parvalbumin as well as all the other peptides studied were not able to restore force to the fibers when incubated in the pCa 8 solution and tested for contraction in the pCa 4 solution (Table 1). Their ability to restore force to the fibers and therefore their binding to the fibers was strictly Ca-dependent. When TnC-depleted skinned fibers were incubated in a pCa 4 solution containing CaM, most of the initial force could be restored (Fig. 4A). Upon washing the CaM incorporated fibers with the pCa 8 solution, the effect of CaM on force restoration was abolished when retested in pCa 4 solution, and the force developed by the fibers returned to the value obtained before CaM incubation (Fig. 4A). Thus, the Mg present in the pCa 8 solution was not sufficient to maintain the interaction of CaM with the fibers as with either RSTnC or TH(1).


Figure 4: Effects of CaM (A) and TH(3) (B) binding to the TnC depleted skinned fibers on steady state force restoration. A, the skinned fibers were tested for their initial steady state force development followed by extraction of endogenous TnC, as described in Fig. 3A. The residual force was 8%. The TnC-depleted fibers were then incubated with 10 µM CaM in the pCa 4 solution for 20 min (note the time scale difference during this incubation). The initial force was almost completely restored. This effect was abolished when CaM reconstituted fibers were washed with the pCa 8 solution. Further incubation with 6 µM RSTnC in the pCa 8 solution for 20 min resulted in the complete restoration of isometric force to the fibers. B, the TnC-depleted fibers were incubated with 50 µM TH(3) in the pCa 4 solution using the same procedure as above. About 50% of the initial force could be restored.



The TH(3) and TH(4) fragments demonstrated the same Ca-dependent ability to interact with the fibers as CaM (Fig. 4B, Fig. 5B, Table 1), but the maximal force developed by these fragments was only about 40-50% of the initial steady state force of intact fibers (Fig. 6). The TH(2) fragment containing Ca-binding site IV of TnC was less effective in force restoration developing not more than 20% of the force of unextracted fibers (Fig. 5A, Table 1). It also did not bind to the fibers in the absence of Ca (Table 1). In summary, the abilities of CaM, TH(2), TH(3), and TH(4) to bind and to restore steady state isometric force to the fibers was strictly Ca-dependent. However, once bound, the Mg-dependent interactions of TH(2) and TH(4) with the fibers were observed (see below).


Figure 5: Blocking of RSTnC rebinding by TH(2) (A) and TH(4) (B). A, the initial steady state force measurements and extraction procedure were performed as in Fig. 3A. After measuring the residual force (20%), the fibers were incubated with TH(2) (100 µM) in the pCa 4 solution for 20 min. About 20% of the initial force was developed by the TH(2) reconstituted fibers. After washing with the pCa 8 solution, the level of force returned to the residual force level. The fibers were then incubated with 6 µM RSTnC in the pCa 8 solution for 20 min, and the steady state isometric force was restored by 45%. The fibers were then extracted again with the EDTA solution (for 20 min) and reincubated with a new RSTnC (for 20 min). 79% of force could be restored to the fibers. The value of ``blocking of RSTnC rebinding'' was 43% (see ``Materials and Methods'' for calculation). B, under the same conditions, TH(4) (100 µM) restored 41% of the initial force level and inhibited about 50% of RSTnC rebinding.



In a control experiment, we tested the ability of fibers to be reconstituted with carp parvalbumin, a Ca-binding protein which is known not to interact with TnI(39) . No force was developed by the fibers incubated with carp parvalbumin, even though the concentration of protein was raised to 25 µM in either the pCa 4.0 or pCa 8.0 solutions.

We also determined the effect of different concentrations of RSTnC, CaM (data not shown), and the thrombin fragments on force restoration in the TnC-depleted fibers (Fig. 6). Since TH(2) was least effective in restoring force, its concentration dependence is not presented here. As shown in Table 2, higher concentrations of the thrombin fragments than RSTnC were required to reach 50% of maximal force restoration. Even using fragment concentrations as high as 100 µM, none of the fragments could achieve the same level of force restoration as intact RSTnC or CaM. The maximum force developed by the reconstituted fibers plateaued around 50% for TH(3) and TH(4) and 65% for TH(1).

Effects of Thrombin Fragments on the Blocking of RSTnC Rebinding

The inhibitory effect, expressed as the ``blocking of RSTnC rebinding,'' is shown in Fig. 7and Table 1. It defines the ability of the investigated proteins or RSTnC fragments to compete with the intact RSTnC for rebinding to the fibers. It also characterizes their Ca- and Mg-dependent interactions with TnI in fibers. Fibers incubated with either CaM or carp parvalbumin in either the pCa 4 or pCa 8 solutions demonstrated no inhibition of RSTnC rebinding. In contrast to that, TH(3) reconstituted fibers showed a small blocking effect (less than 10%) when incubated with the fibers in the pCa 4.0 solution, and then with RSTnC in pCa 8 solution. No blocking effect was observed when TH(3) was incubated with the fibers in the pCa 8.0 solution (Table 1). For TH(1), the inhibitory effect was not determined because of its high level of force restoration that makes the interpretation of RSTnC rebinding equivocal.


Figure 7: Concentration dependence of blocking of RSTnC rebinding by TH(2) and TH(4). Experiments were carried out as described in Fig. 5at different concentrations of the TH(2) and TH(4) fragments. The maximum value of blocking of RSTnC rebinding was 55% for TH(2) and 50% for TH(4). The peptide concentrations for 50% maximal blocking effect were 33 µM for TH(2) and 27.5 µM for TH(4). , TH(2); bullet, TH(4). Each data point is the average value ± S.D. of three to four experiments.



A significant blocking of the RSTnC rebinding was observed for TH(2) or TH(4) reconstituted fibers ( Fig. 7and Table 1). Incubation of these fragments with the fibers in the pCa 4 solution followed by washing in the pCa 8 solution and then incubation with RSTnC in the pCa 8 solution resulted in about 50% blockage of RSTnC rebinding. The concentration dependence of the blocking effect for TH(2) and TH(4) is shown in Fig. 7where 50% of the inhibitory effect was achieved with 33 µM TH(2) and 27.5 µM TH(4) (Table 2).

The force restoration seen with TH(4) and TH(2) when the TnC-depleted fibers were incubated with them in the pCa 4 solution (Fig. 5) was lost when the fibers were subsequently incubated with pCa 8 and then retested in the pCa 4 solution (Fig. 5). These fibers, even though the force restoration was lost, were unable to rebind RSTnC. This blockage of RSTnC rebinding could be removed by treatment of the fibers with EDTA followed by RSTnC reincorporation (Fig. 5). These results suggest that TH(2) and TH(4) bind to two sites in the fibers and that the initial binding required Ca since no evidence of binding was observed in the pCa 8 (+Mg) solutions. As long as Ca was present the force restoration was maintained. Once Ca was removed by switching to the pCa 8 (+Mg) solution this activation was lost. Since the blocking was not abolished in the pCa 8 solution it implies that TH(2) and TH(4) were still bound to the fibers at a site that blocked RSTnC rebinding. TH(2) and TH(4) were removed when the fibers were treated with EDTA implying that the inital binding required Ca but was maintained by Mg.


DISCUSSION

The question of the functional role of each of the four Ca-binding sites in TnC has been addressed for many years, yet the answer is still not entirely known. Many different approaches have been taken to determine the functional differences between the two classes of Ca-binding sites in TnC, and also between the sites of the same class. To study the regions of functional significance in TnC, we obtained four peptides differing in their amino acid sequences: TH(1) (1-120), TH(2) (121-159), TH(3) (1-100), and TH(4) (101-120). These fragments, obtained by the proteolytic digestion of RSTnC with thrombin, were examined for their structural and regulatory roles utilizing skeletal muscle fibers in which the endogenous TnC had been extracted. These well organized skinned muscle fibers, where steady state isometric force could be readily measured, provided an excellent test for the study of the functional properties of the thrombin fragments of RSTnC.

According to Leavis et al.(33) , limited proteolysis with thrombin cleaves the bond Arg-Ala of RSTnC producing two separate fragments, TH(1) and TH(2). We found that if the digestion time of RSTnC with thrombin was significantly shortened, the efficiency of TH(1) preparation greatly increased. This was an indication that under the conditions used for digestion, TH(1) might undergo a structural rearrangement which resulted in further hydrolysis of the peptide. In fact, although the Arg-Ala bond is the preferential digestion site for thrombin in RSTnC, another cleavage site between Arg-Ile was identified by Wall et al.(27) . The isolated peptide, named TH(3), was composed of residues 1-100 of the NH(2)-terminal region of TnC. In our study we also purified and characterized the new peptide, TH(4), containing amino acid residues 101-120 of RSTnC.

The interesting result concerning this study is that all RSTnC thrombin fragments preserved some of the properties of native RSTnC, but none of them could fully restore steady state force to the TnC-depleted fibers as well as intact RSTnC. All peptides and proteins studied required the presence of either Ca or Mg for interaction with the fibers; they dissociated from the fibers upon EDTA treatment. This ability of the fragments to bind Ca or Mg may explain the intriguing ability of the smallest RSTnC thrombin peptides (TH(2) and TH(4)) to exhibit some of the properties of RSTnC when bound to the fibers.

CaM, whose overall structure is very similar to RSTnC(40, 41) , could bind and restore full force to the fibers, although a higher concentration was required when compared to RSTnC. However, unlike RSTnC, CaM was unable to bind to TnC-depleted skinned fibers in the absence of Ca (pCa 8 solution). In agreement with the Ca-dependent interaction of CaM with TnI(32, 42) , CaM could interact with the fibers only in the presence of Ca (pCa 4 solution). Babu et al.(43) also found that CaM could restore full force to the skinned skeletal muscle fibers only in the presence of Ca. Since CaM contains only one type of low affinity Ca-binding site(44) , the Ca-independent (Mg dependent) interactions between TnC and TnI might be limited to the Ca-Mg sites in TnC.

In order to rule out that RSTnC, thrombin fragments, or CaM could exert their action on the fibers through TnI-independent interactions, parvalbumin, which is structurally closely related to CaM and RSTnC, and known not to interact with TnI(39) , was studied for its ability to restore force to TnC-depleted fibers. Since no force restoration was observed with parvalbumin, we concluded that force restoration requires an interaction of the peptides with TnI.

Among the peptides studied, TH(1), containing the two Ca-specific binding sites (I and II) and one high affinity Ca-Mg site (III), was the most efficient in force restoration, reaching 65% of the maximum force developed by native unextracted fibers. Our finding is in accord with the solution study presented by Grabarek et al. (26) who showed that TH(1) reversed 60% of the TnI inhibition of actomyosin ATPase activity. The inability of TH(1) to fully restore force or ATPase activity may result from either the missing COOH terminus of TnC or from a disrupted structure. In a site-directed mutagenesis study(12) , a mutant of skeletal TnC, in which Ca-binding site IV was inactivated, was still able to bind to fibers and reactivate force. Similarly, a mutant of cardiac TnC having deactivated Ca-binding site IV was able to restore force in cardiac fibers by about 85%(11) . The fact that TH(1), even though it contains both Ca-specific sites (I and II), was unable to fully restore force to the extracted fibers may illustrate the importance of the COOH-terminal domain of TnC in Ca regulation. Perhaps, an interdomain communication between the NH(2)- and COOH-terminal regions in TnC(45, 46, 47) contributes to the full regulatory function of TnC in muscle contraction.

The almost intact helix-loop-helix structure of the high affinity site III of TH(1) (lacking two amino acid residues of helix F) appears to be sufficient for TH(1) to bind to TnC extracted fibers. TH(1) was also the only fragment examined which, similar to RSTnC, could bind initially to the fibers in a Mg-dependent (Ca-independent) manner. The strong Mg-dependent interactions of RSTnC or TH(1) with the TnC-depeleted fibers presumably come from electrostatic and/or hydrophobic interactions with TnI in the thin filament. However, our recent findings (48) have revealed an additional possible binding site on TnT that could be involved in this process. Since the TH(1)-fiber interaction was Mg dependent, we can conclude that site III in TnC is sufficient to maintain the binding of TnC to its binding site(s) in the fibers.

The fact that both TH(1) and RSTnC bound to the fibers in a Mg-dependent manner implies that both of them share the same interaction site in the fibers (possibly on TnI). This site appeared not to be present or functional in the two smaller fragments, TH(3) and TH(4), the products of TH(1) digestion. They could bind to fibers only in the presence of Ca. Perhaps the Mg-dependent interaction site on TH(1) was somehow disturbed when the peptide was cleaved into TH(3) and TH(4) at Arg-Ile. Our data do not completely agree with the solution study of Grabarek et al.(26) who found the TH(1)-TnI interaction to be Ca-dependent; however, in the fiber system other Tn subunits can affect this interaction(48) .

Another peptide of RSTnC, TH(3), containing the two low affinity Ca-specific sites I and II could restore 50% of steady state force developed by intact unextracted fibers. Both NH(2)-terminal domain peptides of TnC were quite efficient in force restoration, in agreement with the regulatory role for this domain where Ca-binding sites I and II have to be occupied by Ca to induce muscle activation.

Surprisingly, the TH(4) peptide, containing only Ca-binding site III, could also restore 50% of the initial force to TnC-depleted fibers. The same kind of effect was observed for TH(2) containing Ca-binding site IV, although it could not restore more than 20% of the initial force. The difference between the level of force restoration by TH(4) (site III) and TH(2) (site IV) suggests that these two Ca-Mg sites in RSTnC are not equal and that site III may contribute to the regulatory function of sites I and II in RSTnC.

The mechanism by which the fragments TH(2) and TH(4) activate contraction is not entirely understood. Their functional interaction with the TnC-depleted fibers can be discussed in terms of their interaction with TnI in the fibers. It is possible that their Ca-Mg-binding sites III (TH(4)) and IV (TH(2)) in the absence of their normal interactions within the intact protein assume a new conformation which is more like a Ca-specific site conformation that allows them to bind to the Ca-specific site-dependent interaction site on TnI and/or TnT and activate contraction(14, 48) . It has been shown by Shaw et al.(49) that small peptides of TnC containing sites III or IV can undergo a calcium-induced dimerization which may affect the interaction of TH(2) or TH(4) with the fibers. The activation by TH(2), TH(4), and also TH(3) was lost after incubation of the reconstituted fibers in the absence of Ca (pCa 8 solution), when subsequently checked in the pCa 4 solution. This suggests that they bound to the Ca-specific site-dependent interaction site on TnI and/or TnT(14, 48) , activated contraction, and then dissociated from this site(s) once Ca was removed.

It is interesting that the activating effect of TH(2) and TH(4) was washed out in the pCa 8 solution (containing 5 mM Mg) but that the RSTnC rebinding inhibitory effect was not abolished. This suggests that TH(2) and TH(4) remained bound to a second site, possibly on TnI in the fibers even in the absence of Ca (presence of Mg). This is consistent with the fact that they competed with RSTnC for binding to the extracted fibers, most likely through the Ca-Mg site-dependent interaction site on TnI(14) . Although Ca was required for their interaction with TnI in the fibers it is likely that the Mg present in the pCa 8 solution was all that was necessary for their continued binding. Interestingly, the inhibitory effect was removed by treatment with EDTA confirming that only Mg was required for this binding.

In conclusion, depending on the structure of the thrombin fragments generated, they retained some of the properties of intact RSTnC. The NH(2)-terminal domain fragments (TH(1) and TH(3)) maintained the regulatory properties of TnC whereas the COOH-terminal domain fragments (TH(3) and TH(4)) were mostly involved in the Ca-Mg-dependent interaction of TnC with TnI in the fibers. Moreover, our results show the possibility of the mutual role of the COOH-terminal region of TnC depending on the kind of cation bound to Ca-Mg-binding sites III and IV. In addition to the main role of this domain in maintaining the structural stability of the whole troponin complex in the thin filament, this region may contribute to the regulatory role of sites I and II of TnC in muscle contraction.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants AR37701 and AR40727. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
The first two authors contributed equally to this work.

Supported by AHA/FL Grant 89F/4.

**
To whom correspondence should be addressed.

(^1)
The abbreviations used are: TnC, troponin-C; RSTnC, rabbit fast skeletal troponin-C.


ACKNOWLEDGEMENTS

We thank Alan Mandveno for his help in creating the computer drawings of RSTnC and its thrombin fragments and Lois Rosenzweig for help in preparing the manuscript.


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