(Received for publication, January 24, 1995; and in revised form, April 5, 1995)
From the
Proteolysis of rabbit fast skeletal troponin-C (RSTnC) with
thrombin produces four separate fragments containing the following
Ca-binding site(s): TH
(residues
1-120) sites I-III; TH
(121-159) site IV;
TH
(1-100) sites I and II; and
TH
(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
was the most effective in the
force restoration (65%) whereas the smaller fragments developed about
50% (TH
and TH
) or 20% (TH
) of the
initial force of unextracted fibers. Additionally, much higher
concentrations of TH
, TH
, and TH
compared to RSTnC or TH
were necessary for force
development suggesting a decreased affinity of these fragments to their
binding site(s) in the fibers. Like intact RSTnC, TH
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
and TH
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
and TH
was abolished when Ca
was removed. TH
and TH
binding to the second site required
Ca
initially but was maintained in the presence of
Mg
. This interaction of TH
and TH
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.
In vertebrate muscle, the binding of Ca to
troponin-C (TnC), (
)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
-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
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
-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
-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
-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.
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 NHHCO
, 5 mM EDTA, and 5
mM
-mercaptoethanol, pH 8.0, for 72 h at room
temperature. In order to increase the efficiency of the TH
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
HCO
, pH 8.2, at 4
°C. The final separation of TH
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) .
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%.
Figure 1:
Molecular models of the RSTnC thrombin
fragments. These molecular models are based on the crystal structure of
TnC(1, 2) . The NH-terminal domains of
RSTnC, TH
, and TH
contain
Ca
-specific sites I and II, whereas the COOH-terminal
domains of RSTnC, TH
, TH
, and TH
contain sites III (TH
and TH
) and IV
(TH
).
Figure 2:
RSTnC thrombin fragments on 15%
SDS-polyacrylamide gel electrophoresis. The gel was stained with
Coomassie Blue. Lane 1, RSTnC; lane 2,
TH; lane 3, TH
; lane 4,
TH
; lane 5,
TH
.
As shown in Fig. 1, TH contains the complete
NH
-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
, the
TH
fragment is shorter by 20 residues and contains the
NH
-terminal domain, the interconnecting helix D/E and a
part of the helix E. The TH
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
, the shortest peptide
studied, contains Ca
-binding loop III, one turn of
helix E at the NH
-terminal part of the peptide and two
turns of helix F at its COOH-terminal region.
Figure 3:
Reconstitution of TnC-depleted
skinned fibers with RSTnC (A) and TH
(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
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
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 and TH
was
measured upon incubation in the pCa 4 (contraction) solution. RSTnC and
TH
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
;
,
TH
;
, TH
. Each data point is the average
value ± S.D. of two to five
experiments.
Unlike RSTnC or TH, 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
.
Figure 4:
Effects of CaM (A) and TH (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
in the pCa 4 solution using the same procedure as above. About
50% of the initial force could be restored.
The
TH and TH
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
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
, TH
, and
TH
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
and TH
with the fibers were observed (see below).
Figure 5:
Blocking of RSTnC rebinding by TH (A) and TH
(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
(100
µM) in the pCa 4 solution for 20 min. About 20% of the
initial force was developed by the TH
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
(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 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
and TH
and
65% for TH
.
Figure 7:
Concentration dependence of blocking of
RSTnC rebinding by TH and TH
. Experiments were
carried out as described in Fig. 5at different concentrations
of the TH
and TH
fragments. The maximum value
of blocking of RSTnC rebinding was 55% for TH
and 50% for
TH
. The peptide concentrations for 50% maximal blocking
effect were 33 µM for TH
and 27.5 µM for TH
.
, TH
;
, TH
.
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 or
TH
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
and TH
is shown in Fig. 7where 50%
of the inhibitory effect was achieved with 33 µM TH
and 27.5 µM TH
(Table 2).
The
force restoration seen with TH and TH
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
and TH
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
and TH
were still bound to the fibers at a
site that blocked RSTnC rebinding. TH
and TH
were removed when the fibers were treated with EDTA implying that
the inital binding required Ca
but was maintained by
Mg
.
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
(1-120), TH
(121-159), TH
(1-100), and TH
(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
and TH
. We found that if
the digestion time of RSTnC with thrombin was significantly shortened,
the efficiency of TH
preparation greatly increased. This
was an indication that under the conditions used for digestion,
TH
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
, was
composed of residues 1-100 of the NH
-terminal region
of TnC. In our study we also purified and characterized the new
peptide, TH
, 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
and TH
) 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, 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
reversed 60% of the TnI
inhibition of actomyosin ATPase activity. The inability of TH
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
, 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
- 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 (lacking two amino acid
residues of helix F) appears to be sufficient for TH
to
bind to TnC extracted fibers. TH
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
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
-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 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
and TH
, the products of TH
digestion. They could bind to fibers only in the presence of
Ca
. Perhaps the Mg
-dependent
interaction site on TH
was somehow disturbed when the
peptide was cleaved into TH
and TH
at
Arg
-Ile
. Our data do not completely agree
with the solution study of Grabarek et al.(26) who
found the TH
-TnI interaction to be
Ca
-dependent; however, in the fiber system other Tn
subunits can affect this interaction(48) .
Another peptide
of RSTnC, TH, 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
-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 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
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
(site III) and TH
(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 and TH
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
) and IV (TH
) 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
or TH
with the fibers. The
activation by TH
, TH
, and also TH
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 and
TH
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
and TH
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-terminal domain fragments (TH
and
TH
) maintained the regulatory properties of TnC whereas the
COOH-terminal domain fragments (TH
and TH
) 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.