Tài liệu Báo cáo khoa học: Comparative studies on the functional roles of N- and C-terminal regions of molluskan and vertebrate troponin-I pdf

Comparative studies on the functional roles of N- and
C-terminal regions of molluskan and vertebrate troponin-I
Hiroyuki Tanaka1, Yuhei Takeya1, Teppei Doi1, Fumiaki Yumoto2,3, Masaru Tanokura3,
Iwao Ohtsuki2, Kiyoyoshi Nishita1 and Takao Ojima1
1 Laboratory of Biotechnology and Microbiology, Graduate School of Fisheries Sciences, Hokkaido University, Japan
2 Laboratory of Physiology, The Jikei University School of Medicine, Tokyo, Japan
3 Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Japan

Keywords
invertebrate; mollusk; regulatory
mechanism; troponin; troponin-I
Correspondence
Takao Ojima, Laboratory of Biochemistry
and Biotechnology, Graduate School of
Fisheries Sciences, Hokkaido University,
Hakodate, Hokkaido 041–8611, Japan
Tel ⁄ Fax: +81 138 408800
E-mail: ojima@fish.hokudai.ac.jp
Note
The nucleotide sequences of cDNAs encoding Akazara scallop 52K-TnI and 19K-TnI
are available in DDBJ ⁄ EMBL ⁄ GenBank
databases under accession numbers,
AB206837 and AB206838, respectively
(Received 24 March 2005, revised 13 June
2005, accepted 15 July 2005)
doi:10.1111/j.1742-4658.2005.04866.x

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 Ca2+-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 (ATnI232)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 Ca2+. These results indicated that the mechanism involved in the alternative binding of this region was not observed in molluskan troponin. On
the other hand, ATnI130)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 Ca2+-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 TnCbinding site to the inhibitory region of TnI. Together with the observation
that corresponding rabbit TnI-fragment (RTnI1)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 Ca2+-dependent activation.

Troponin is a Ca2+-dependent regulatory protein complex, which constitute thin filaments together with
actin and tropomyosin [1]. It is composed of three distinct subunits: troponin-C (TnC), which binds Ca2+,
troponin-T (TnT), which binds tropomyosin, and troponin-I (TnI), which binds actin and inhibits actin–myosin interaction [2–4]. In relaxed muscle, TnI binds to
actin and inhibits contraction. Upon muscle stimulation, Ca2+ binds to TnC and induces the release of the
inhibition by TnI, resulting in muscle contraction. To

understand the molecular mechanisms of this Ca2+
switching, extensive studies of the structure, function,
and Ca2+-dependent conformational changes of troponin subunits have been carried out.
In vertebrate muscles, TnC has a dumbbell-like
shape with the N- and C-terminal globular domains
linked by a central helix [5,6]. Each domain contains
two EF-hand Ca2+-binding motifs [7], thus TnC has
four possible Ca2+-binding sites, sites I and II in the
N-domain and sites III and IV in the C-domain [8,9].

Abbreviations
TnC, troponin-C; TnI, troponin-I; TnT, troponin-T; IPTG, isopropyl-1-thio-b-D-galactopyranoside; PMSF, phenylmethylsulfonyl fluoride.

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Functional regions of molluskan TnI

Sites III and IV also show affinity for Mg2+ and are
thought to be always occupied by sarcoplasmic Mg2+,
whereas Ca2+ binding to site I and ⁄ or II is believed
to trigger muscle contraction [10]. TnC interacts with
both TnI and TnT. The TnC–TnI interaction and
changes in the interaction upon Ca2+ binding to TnC
have been intensively studied as the central mechanisms of Ca2+ switching. It has been revealed that TnI
has three major TnC-binding sites [11–14], namely a
structural TnC-binding site (residues 1–30 in rabbit
fast skeletal TnI), an inhibitory region (residues 104–
115), and a regulatory TnC-binding site (residues 116–
131). In the relaxed state, the inhibitory region binds
to actin and inhibits actin–myosin interaction [11,12],
while in the contractile state, Ca2+-binding to site I
and ⁄ or II of TnC causes the exposure of a hydrophobic patch on the surface of the N-domain [15], resulting in hydrophobic interaction between the N-domain
and the regulatory TnC-binding site [16]. This interaction induces the dissociation of the inhibitory region,
which is adjacent to the regulatory TnC-binding site,
from actin, resulting in the release of the inhibition
and muscle contraction [17]. The structural TnC-binding site interacts with the C-domain of TnC in both
the relaxed and contractile states, which plays a role
in maintaining the structural integrity of the troponin
complex [17,18]. These switching mechanisms were

recently confirmed by crystallographic studies of vertebrate troponins [19,20], which demonstrated that the
Ca2+-saturated N- and C-domains of TnC bind to the
regulatory and structural TnC-binding sites, respectively, of TnI, and suggested that the C-terminal region
of TnI (including the inhibitory region and the regulatory TnC-binding site) exhibits a positional change
from actin-tropomyosin filament to the N-domain of
TnC in a Ca2+-dependent manner.
However, a significant discrepancy exists between
the above schemes and the structural and functional
features of some invertebrate troponins. Molluskan
TnC binds only one mole of Ca2+ per mole of protein
at site IV in the C-domain because of amino acid substitutions at sites I–III [21,22]. Nevertheless, ternary
troponin complex combined with molluskan tropomyosin can regulate the Mg-ATPase activity of vertebrate
actomyosin in a physiologically significant Ca2+dependent manner [21]. Moreover, the troponin regulates the ATPase of molluskan myofibril together with
a well known myosin light chain-linked regulatory system, especially under low temperature conditions [23].
Therefore, the molecular mechanisms of regulation by
molluskan troponin are expected to be somewhat different from those described above. A previous study
revealed that the C-domain of molluskan TnC is
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H. Tanaka et al.

responsible not only for Ca2+-binding but also for the
interaction with TnI, although the presence of both
the N- and C-domains is essential for full regulatory
ability [24,25].
In the present study, we compared the functional
sites of molluskan and vertebrate TnI by using the
recombinant fragments of Akazara scallop Chlamys
nipponensis TnI and rabbit fast skeletal TnI. The
results provide evidence that molluskan troponin functions through a mechanism in which the region spanning from the structural TnC-binding site to the

inhibitory region of TnI plays an important role.

Results
Escherichia coli expression of TnI-fragments
Figure 1A shows a schematic representation of the
recombinant TnI-fragments used in this study. ATnI52K, ATnI-19K and RTnI are the recombinant
Akazara scallop 52K-TnI, 19K-TnI (isoforms; see
Experimental procedures section and [27]), and rabbit
fast skeletal TnI, respectively. ATnI1)128 is the fragment corresponding to the N-terminal extending region
of 52K-TnI. ATnI130)252 and RTnI1)116 are the fragments, corresponding to the regions spanning from the
structural TnC-binding sites to the inhibitory regions
of Akazara scallop and rabbit TnI, respectively.
ATnI232)292 and RTnI96)181 correspond to the regions
spanning from the inhibitory regions to the C-termini
of these TnI. Figure 1B shows an SDS ⁄ PAGE of
these purified recombinant proteins. ATnI-52K and
ATnI1)128 showed anomalously low mobility due to
the high fraction of hydrophilic residues in the N-terminal extending region as described previously [26].
The initiator Met at the N-terminus was removed by
the bacterial cell for all these proteins except for
RTnI96)181.
Inhibition of Mg-ATPase of actomyosin
by TnI-fragments
The inhibition of actomyosin-tropomyosin Mg-ATPase
by TnI fragments was compared. The inhibitory effects
of RTnI, RTnI1)116 and RTnI96)181 differed greatly
from one another, although all of these proteins
contained the inhibitory region (Fig. 2A). RTnI1)116
inhibited only 33% of rabbit-actomyosin–rabbit-tropomyosin Mg-ATPase at a 3 : 1 molar ratio with tropomyosin, compared with 82% for RTnI. As has been
reported previously [18,28,29], weaker inhibitory effects

of RTnI1)116 revealed the importance of residues
117–181 for maximal inhibition. In particular, residues
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H. Tanaka et al.

Functional regions of molluskan TnI

A

B

Fig. 1. (A) Schematic representation of recombinant TnI-fragments. The numbers preceding and following each box indicate the amino acid
positions of Akazara scallop 52K-TnI (Swiss-Prot #Q7M3Y3) and rabbit fast skeletal TnI (Swiss-Prot #P02643). The N-terminal extending
region of 52K-TnI and the functional regions that have been previously identified in vertebrate TnI are indicated by bars. The inhibitory
regions are shaded. (B) SDS ⁄ PAGE of recombinant TnI-fragments used in this study. Each protein (1.5 lg) was run on a 10% (w/v) acrylamide gel. Molecular mass markers are also shown (M).

140–148 had been proven to bind to actin-tropomyosin
and thus are referred to as the second actin-tropomyosin-binding site [14]. Moreover, in our results, the
inhibition by RTnI96)181 was the strongest (94% of
the ATPase was inhibited), suggesting that residues
1–95 may decrease the inhibitory effects of residues
96–181.
On the other hand, Akazara scallop TnI isoforms
and their fragments showed somewhat different properties (Fig. 2B). ATnI130)252, which corresponds to
RTnI1)116, inhibited about 70% of rabbit-actomyosinscallop-tropomyosin Mg-ATPase at a 3 : 1 molar ratio
with tropomyosin. Moreover, the inhibition by
ATnI232)292, which corresponds to RTnI96)181, was
weaker (51%) than that by ATnI-19K (88%) or

ATnI130)252. Therefore, the effects of the N- or C-terminal region of TnI on the function of the inhibitory
region appeared to differ between rabbit and Akazara
scallop TnI. Interestingly, ATnI-52K showed weaker
inhibition (65%) than ATnI-19K, suggesting that
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the N-terminal extending region of 52K-TnI could
decrease the inhibitory effects, although ATnI1)128,
which corresponds to the N-terminal extending
region, on its own, exhibited neither activation nor
inhibition.
To determine whether the inhibitory effect correlates
with the binding affinity to actin-tropomyosin, we
examined each TnI for its ability to cosediment with
actin-tropomyosin. When TnI-fragments were mixed at
2 : 1 molar ratios with tropomyosin, RTnI, RTnI1)116
and RTnI96)181 cosedimented with molar ratios of
approximately 0.23, 0.048, and 0.35, respectively, to
actin. On the other hand, ATnI-19K, ATnI130)252 and
ATnI232)292 cosedimented with molar ratios of 0.49,
0.44, and 0.065, respectively, to actin (the extent of the
cosedimentation of ATnI-52K could not be determined because it precipitated even in the absence of
actin-tropomyosin in a control experiment due to the
low solubility). Therefore, the observed difference
in the inhibitory effects of TnI-fragments might be
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H. Tanaka et al.

Interactions of TnI-fragments with TnC

Fig. 2. Inhibition of actomyosin-tropomyosin Mg-ATPase by rabbit
(A) or Akazara scallop (B) TnI-fragments. The actomyosin-tropomyosin Mg-ATPase was measured at increasing ratios of TnI
or TnI-fragments to tropomyosin as indicated on the abscissa.
The measurements were performed at 15 °C. The results were
expressed as a percentage of the ATPase activity obtained in the
absence of TnI. Each point is an average of three determinations.
(A) RTnI, d; RTnI1)116, n; RTnI96)181, h. (B) ATnI-52K, d; ATnI19K, s; ATnI1)128, e; ATnI130)252, n; ATnI232)292, h.

attributable to the difference in their binding affinities
for actin-tropomyosin. In addition, ATnI1)128 did not
cosediment and remained in the supernatant (data not
shown). This suggested that the N-terminal extending
region of 52K-TnI was not involved in binding to
actin-tropomyosin, although this region showed
sequence homology to the N-terminal tropomyosin
binding site of vertebrate TnT [26].
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We compared the ability of TnI-fragments to form a
complex with TnC by alkaline urea PAGE. The experiments were performed under either 6 or 3 m urea conditions in the presence of either 2 mm EDTA or 2 mm
CaCl2. RTnI and both rabbit TnI-fragments formed a
complex with rabbit TnC in 2 mm CaCl2 but not in
2 mm EDTA under both urea conditions (Fig. 3A).
These results agreed with those reported by Farah et al.
for chicken skeletal TnI-fragments [18], and were compatible with the fact that all of these proteins have at
least two of three known TnC-binding sites, namely the

structural TnC-binding site, the inhibitory region, and
the regulatory TnC-binding site. On the other hand,
ATnI1)128 and ATnI232)292 did not form a complex with
Akazara scallop TnC under any of the tested conditions,
whereas ATnI-52K, ATnI-19K, and ATnI130)252 did
under both urea concentrations in the presence of Ca2+
(Fig. 3B). It was interesting that ATnI232)292 did not
form a complex, as ATnI232)292 corresponds to
RTnI96)181 and should have two TnC-binding sites, the
inhibitory region and the regulatory TnC-binding site.
Therefore, this suggests that TnC-binding affinities of
these regions of the Akazara scallop TnI were much
weaker than those of rabbit TnI. Moreover, under
the 3 m urea condition, ATnI-52K, ATnI-19K, and
ATnI130)252 showed complex formation even in the
absence of Ca2+ (Fig. 3B, upper panels), suggesting that
in the absence of Ca2+, the Akazara scallop TnI binds
to TnC more strongly than rabbit due to the properties
of the interaction between residues 130–252 and TnC.
We also performed affinity chromatography to confirm the interaction of TnI-fragments with immobilized
rabbit or Akazara scallop TnC under nondenaturing
conditions (Fig. 4). ATnI232)292 binding to Akazara
scallop TnC was not observed, even in the absence of
both urea and KCl and the presence of 0.5 mm CaCl2,
whereas ATnI130)252, RTnI1)116, and RTnI96)181
strongly bound to TnCs. These results suggested that
the inhibitory region and the regulatory TnC-binding
site of Akazara scallop TnI essentially cannot interact
with TnC.
Ca2+-dependent alternative binding of C-terminal

TnI fragments to actin-tropomyosin and TnC
To understand the biological significance of the difference in TnI–TnC interactions, we compared the ability
of TnC to neutralize the inhibitory effects of the C-terminal fragments in the presence and absence of Ca2+.
As has been reported for similar vertebrate TnI fragments [14,18,29], the inhibitory effect of RTnI96)181 in
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H. Tanaka et al.

A

Functional regions of molluskan TnI

B

Fig. 3. Complex formation between TnI-fragments and TnC detected by alkaline urea PAGE. TnI-fragments were combined with TnC as described under ‘Experimental procedures’. The final concentration of the proteins was 13.8 lM. Twenty-microliter aliquots of the mixture were
electrophoresed on the gel containing either 6 or 3 M urea and either 2 mM EDTA (– Ca; upper panels) or 2 mM CaCl2 (+ Ca; lower panels).
(A) Rabbit TnI or TnI-fragments were run on the gels in the absence (lanes a–c) or presence (lanes d–f) of equimolar amounts of rabbit TnC.
Lanes a and d, RTnI; lanes b and e, RTnI1)116; lanes c and f, RTnI96)181; lane g, rabbit TnC. (B) Akazara scallop TnI or TnI-fragments
were run in the absence (lanes h–l) or presence (lanes m–q) of equimolar amounts of Akazara scallop TnC. Lanes h and m, ATnI-52K; lanes
i and n, ATnI-19K; lanes j and o, ATnI1)128; lanes k and p, ATnI130)252; lanes l and q, ATnI232)292; lane r, Akazara scallop TnC. Complex formation was detected by the bands of the TnI–TnC complex (arrowheads) and weakening of the free TnC bands. Free RTnI, RTnI1)116,
RTnI96)181, ATnI-19K, ATnI130)252, and ATnI232)292 did not migrate into the gels, while free ATnI-52K and ATnI1)128 exhibited a band near the
origin and at the middle of the gel, respectively. The bands corresponding to the free rabbit or Akazara scallop TnC were found in the middle
to bottom of the gels (indicated as RTnC or ATnC, respectively).

Fig. 4. TnC-affinity chromatography of TnIfragments. The fragments of rabbit or Akazara scallop TnI were applied onto the affinity
columns prepared by immobilizing either
rabbit (A) or Akazara scallop (B) TnC on
Formyl-Cellulofine. The fragments were
eluted with a stepwise gradient of KCl

concentrations indicated at the top of the
figures. Each fraction contains 1.0 mL.
Eluted protein was detected by the method
of Bradford [40] and identified by
SDS ⁄ PAGE (data not shown). Due to low
solubility, RTnI1)116 was applied at a KCl
concentration of 0.1 M.

a 2 : 1 molar ratio with tropomyosin was effectively
neutralized by rabbit TnC in the presence of Ca2+,
but not in its absence (Fig. 5A, upper panel). In addiFEBS Journal 272 (2005) 4475–4486 ª 2005 FEBS

tion, the cosedimentation experiment performed under
a 4 : 4 : 2 : 7 molar ratio of RTnI96)181–TnC–tropomyosin–actin showed that the amount of RTnI96)181
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Functional regions of molluskan TnI

A

H. Tanaka et al.

B

Fig. 5. Functional differences between RTnI96)181 (A) and ATnI232)292 (B). Upper panels, effects of TnC on inhibition by the C-terminal TnIfragments. TnI-fragments were present at a 2 : 1 molar ratio of TnI-fragments ⁄ tropomyosin. The Mg-ATPase activity was measured at
increasing ratios of TnCs to the fragments in the presence (d) or absence (s) of Ca2+. The measurements were performed at 15 °C. The
results were expressed as a percentage of the ATPase activity obtained in the absence of both TnI and TnC. Lower panels, change in C-terminal TnI-fragment affinity for actin-tropomyosin tested by cosedimentation experiments. The fragments were added to actin-tropomyosin at
a molar ratio of 4 : 2 : 7 (fragment ⁄ tropomyosin ⁄ actin) with or without an equimolar amount of TnC in the presence or absence of Ca2+. The
pellets (P) and supernatants (S) were redissolved in equivalent volumes of 5 M urea solution and then run on SDS ⁄ PAGE. Lanes a and d, in

the absence of both TnC and Ca2+; lanes b and e, in the presence of TnC and the absence of Ca2+; lanes c and f, in the presence of both
TnC and Ca2+. Ac, actin; Tm, tropomyosin; RTnC, rabbit TnC; ATnC, Akazara scallop TnC. The relative staining intensities of the C-terminal
TnI-fragments on lanes a–c were expressed as a percentage of that on lane a and were shown on the right.

cosedimented with actin-tropomyosin was greatly
reduced in the presence of Ca2+ but not in its absence.
The amount that remained with TnC in the supernatant was greater in the presence of Ca2+ than in its
absence (Fig. 5A, lower panel). Therefore, this suggested that RTnI96)181 bound actin and TnC in the
absence and presence, respectively, of Ca2+. These
phenomena should directly reflect the mechanism of
Ca2+ switching involving the alternative binding of the
C-terminal region of TnI to actin or TnC in a Ca2+dependent manner [17,19]. On the other hand, the
inhibitory effect of ATnI232)292 was not neutralized
by adding Akazara scallop TnC, irrespective of Ca2+
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concentrations (Fig. 5B, upper panel). Moreover, the
amount of ATnI232)292 cosedimented with actin-tropomyosin was unaffected by the presence and absence of
TnC and Ca2+ (Fig. 5B, lower panel). Therefore, the
Ca2+-switching mechanisms involving the alternative
binding of the C-terminal region of TnI were not present in Akazara scallop troponin.
Ca2+-regulatory effects of troponins containing
TnI fragments
The Ca2+-regulatory effects of troponins composed of
TnI-fragments, native TnT, and TnC on actomyosinFEBS Journal 272 (2005) 4475–4486 ª 2005 FEBS


H. Tanaka et al.

Functional regions of molluskan TnI

Fig. 6. Ca2+-regulation of actomyosin-tropomyosin Mg-ATPase by rabbit (A and C) and
Akazara scallop (B and D) reconstituted troponins. The effects of the troponin containing
TnI or TnI fragments on the actomyosintropomyosin Mg-ATPase were measured as
a function of pCa ()10g[Ca2+]). The assays
were performed at 15 °C (A and B) or 25 °C
(C and D). A and C: RTn, d; RTn1)116, n;
RTn96)181, h. B and D: ATn-52K, d; ATn19K, s; ATn130)252, n; ATn232)292, h. The
activities in the absence of troponin are indicated by dashed lines.

tropomyosin Mg-ATPase were compared. The assays
were performed at different temperatures, 15 °C, which
is the normal ambient temperature for Akazara scallops and is suitable for functionalizing the molluskan
troponin [23], and 25 °C, at which many assays of
Ca2+ regulation by vertebrate troponin have been conducted [14,18,28–30]. At 15 °C, all the ternary complexes consisting of rabbit TnI or TnI fragments,
rabbit TnT and TnC, regulated the ATPase, although
they exhibited quite different Ca2+-dependence curves
(Fig. 6A). The complex containing RTnI1)116 (represented as RTn1)116) showed no inhibition, even under
low Ca2+ concentrations, although it strongly activated the ATPase at Ca2+ concentrations higher than
pCa 4.5. RTn96)181 did not activate the ATPase
beyond the level observed in the absence of troponin,
even at pCa 4.0. On the other hand, the complex consisting of ATnI232)292, Akazara scallop TnT and TnC
(ATn232)292) inhibited the ATPase irrespective of Ca2+
concentration, and could not regulate it at all
(Fig. 6B). This property could be explained by the fact
that the inhibitory region and the regulatory
TnC-binding site of Akazara scallop TnI bind to actintropomyosin, but not to TnC, irrespective of Ca2+
concentration, as described above. Moreover,
ATn130)252 regulated the ATPase almost as effectively
as intact troponins (ATn-52K or ATn-19K), suggesting

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that the region spanning from the regulatory TnCbinding site to the C-terminus of Akazara scallop TnI
is not important for this regulation, and that Akazara
scallop troponin acts through mechanisms in which the
region spanning from the structural TnC-binding site
to the inhibitory region plays an important role. It
should also be mentioned that ATn-52K more strongly
activated the ATPase than ATn-19K under high Ca2+
concentrations. Thus, the N-terminal extending region
of ATnI-52K may be involved in the activation of the
ATPase in the presence of Ca2+. When we performed
similar assays at 25 °C, the regulation by RTn1)116,
which was observed at 15 °C, became unremarkable,
whereas RTn96)181 more effectively regulated the
ATPase than at 15 °C (Fig. 6C). These results
obtained at 25 °C were essentially the same as those
reported by Farah et al. [18] for the chicken skeletal
troponins containing similar TnI fragments. On the
other hand, the regulatory ability of Akazara scallop
troponins dramatically decreased (Fig. 6D), suggesting
that Akazara scallop troponin does not function at the
temperature appropriate for vertebrate troponins.

Discussion
The vertebrate TnI is known to interact with TnC in
an antiparallel manner such that the regulatory and
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Functional regions of molluskan TnI

structural TnC-binding sites of TnI interact with the
N- and C-domains, respectively, of TnC [18,19]. The
inhibitory region is known to interact with both
the N- and C-domains, but preferentially with the
C-domain [18,20,31]. In the present study, we revealed
a striking difference in the TnI–TnC interactions of
vertebrate and mollusk. We showed that ATnI232)292,
which is the Akazara scallop TnI-fragment containing
the inhibitory region and the regulatory TnC-binding
site, does not bind to Akazara scallop TnC, whereas
ATnI130)252, which contains the structural TnC-binding site and the inhibitory region, strongly binds to
TnC. The antiparallel structural features of vertebrate
TnI–TnC complex and previous observations that the
N-domain of Akazara scallop TnC did not bind to
TnI while the C-domain bound strongly [24], suggest a
single interaction between the structural TnC-binding
site of TnI and the C-domain of TnC in Akazara scallop TnI–TnC complex. Although the further verification under nondenaturing conditions is required, the
results of the alkaline urea gel electrophoresis indicate
that this interaction is strengthened by Ca2+ and is
stronger than the corresponding interaction in rabbit
TnI–TnC in the absence of divalent cation. Therefore,
this interaction potentially participates in both the
Ca2+-dependent activation of the contraction and the
maintenance of structural integrity of the troponin
complex in the relaxed state.
Troponin-tropomyosin based regulation exhibits two
components [32]: inhibition and removal of inhibition
in the absence and presence, respectively, of Ca2+,

and Ca2+-dependent activation. The regulatory mechanism involving the alternative binding of the C-terminal region of TnI to actin or TnC should be
responsible for the former. However, it cannot account
for the latter, namely the phenomenon that, in the
presence of Ca2+, troponin activates actomyosintropomyosin Mg-ATPase beyond the level observable
in the absence of troponin. This activation is prominent, especially for molluskan troponin, which confers
Ca2+ sensitivity on the ATPase predominantly
through its activation in the presence of Ca2+, rather
than by inhibition due to its absence. In contrast, the
vertebrate troponin regulates the ATPase mainly by
inhibition in the absence of Ca2+ (Fig. 6 and [21,32]).
The difference in Ca2+ sensitization between vertebrates and mollusks should also be closely related to
the difference in the inhibitory effects of vertebrate
and molluskan tropomyosins [33], which inhibit rabbit actomyosin Mg-ATPase activity to 0.043 and
0.021 lmolỈmin)1Ỉmg myosin)1, respectively, at 15°C
(Fig. 6A,B). In the present study, we compared the
functional roles of the N- and C-terminal regions of
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H. Tanaka et al.

molluskan and vertebrate TnI and revealed for the
first time that (a) the alternative binding of the TnI
C-terminal region is not observed in molluskan troponin, as the C-terminal region of molluskan TnI does
not interact with TnC; and (b) molluskan troponin
regulates the ATPase by a mechanism in which the
TnI N-terminal region (from the structural TnC-binding site to the inhibitory region) participates in the
Ca2+-dependent activation. In addition, at 15°C, similar activation is observed for the troponin containing
the corresponding vertebrate TnI-fragment, suggesting
the presence of a common activating mechanism
between vertebrates and mollusks. In molluskan

troponin, the activation is probably induced by strengthening of the interaction between the structural TnCbinding site and the C-domain of TnC accompanying
Ca2+ binding to site IV of TnC. In vertebrate troponin, the activation may be a result of the interaction
between the inhibitory region and TnC accompanying
Ca2+ binding to site I or II of TnC. However, we cannot rule out the possibility that the substitution of
Mg2+ at site III or IV of vertebrate TnC with Ca2+
causes the activation in vitro. Several observations
have indicated that the N-terminal region of vertebrate
TnI is involved in the activating process [14,28,30]. In
particular, Malnic et al. [30] suggested that the activating effects of the N-terminal region of TnT are exerted in the presence of Ca2+ by the TnI N-terminal
region (from the structural TnC-binding site to the
TnT-binding site) and TnC.
In summary, we propose a novel view of the general
architecture of TnI. In vertebrate muscles, the C-terminal region plays a role in the inhibition ⁄ removal of
inhibition by alternative binding, while the N-terminal
region is responsible for the Ca2+-dependent activation. This view replaces the general and conventional
view that the N-terminal region of TnI only plays a
role in maintaining the structural integrity of the troponin complex. In molluskan muscles, the C-terminal
region does not function and troponin regulates
contraction only through the activation exerted by the
N-terminal region of TnI.

Experimental procedures
Muscle proteins
Tropomyosin, TnT, and TnC from Akazara scallop striated
adductor muscle or rabbit fast skeletal muscle were prepared by the method of Ojima and Nishita [21,34]. Rabbit
fast skeletal myosin and F-actin were prepared by the
method of Perry [35] and Spudich and Watt [36], respectively. All measures were taken to minimize pain and

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H. Tanaka et al.

discomfort of animals. The procedures were conducted in
accordance with the institutional guidelines by Hokkaido
University.

Construction of plasmids expressing TnI fragments
Based on the partial nucleotide sequence (GenBank accession number AB009368), we cloned the cDNA including
the entire coding region for Akazara scallop TnI by
5¢-RACE [37] from the striated adductor muscle. As a
result, two cDNA clones encoding isoforms, namely
52K-TnI and 19K-TnI [27], were obtained. The deduced
amino acid sequence of 19K-TnI was identical to that of
C-terminal 163 residues of 52K-TnI. The 52K-TnI-cDNA
was subcloned into pCR2.1-TOPO (Invitrogen, Carlsbad,
CA, USA), and used as a template for PCR to amplify the
DNAs encoding various regions of 52K-TnI. For the
amplification of the DNAs encoding ATnI-52K (recombinant 52K-TnI; residues 1–292), ATnI1)128 (recombinant fragment consisting of residues 1–128 of 52K-TnI), ATnI-19K
(recombinant 19K-TnI; residues 130–292), ATnI130)252
(fragment; residues 130–252), and ATnI232)292 (fragment;
residues 232–292), combinations of the forward and reverse
primers, ATnI1F (5¢-CATATCACCATGGGTTCCCTTG-3¢)
and ATnI292R (5¢-CTTGATTTGGATCCTTTAAGGTA
TAGC-3¢), ATnI1F and ATnI128R (5¢-GTTCCGGATC
CTATCTTCTGGCTTCC-3¢), ATnI130F (5¢-GCCAGAA
CCATGGCGGAGGAAC-3¢) and ATnI292R, ATnI130F
and ATnI252R (5¢-CAAGTTTGGGATCCTATTTGTTAA
CTTTTC-3¢), and ATnI232F (5¢-CGAGATTAATGCC
ATGGCACTTAAGG-3¢) and ATnI292R, respectively,

were used. These forward and reverse primers introduced
NcoI and BamHI restriction sites (underlined), respectively,
into the PCR products. These primers also introduced the
initiation or termination codons (bold), except in
ATnI292R, which would anneal to the 3¢-noncoding region.
It should be noted that in ATnI1F and ATnI232F, the Ser1
and Thr232 codons in the template were replaced by Gly1
and Ala232, respectively, in addition to introducing the
NcoI site. The PCR products were digested with NcoI and
BamHI and then ligated into the NcoI-BamHI site of the
expression vector, pET-16b (Novagen, Madison, WI,
USA).
We also cloned the cDNA encoding rabbit fast skeletal
TnI from the back muscle of rabbit by RT-PCR using the
primer set, RTnI1F (5¢-CAAACCTCACCATGGGAGAT
GAAG-3¢) and RTnI181R (5¢-CCCCGGAGCCGGATCC
CCAGCCCC-3¢). These primers were designed based on
the sequence retrieved from the GenBank database under
accession number L04347, and NcoI or BamHI sites (underlined) and the initiation codon (bolded) were introduced
into the sequences. The cDNA subcloned into pCR2.1TOPO was first subjected to mutagenesis for deactivating
the native NcoI site in the coding region by using MutanSuper Express Km kit (Takara-bio, Ohts, Japan). The

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Functional regions of molluskan TnI

mutated DNA was cut out with NcoI and BamHI and
ligated into pET-16b for the construction of the plasmid
expressing RTnI (recombinant rabbit fast skeletal TnI; residues 1–181). The expression plasmid was also used as a
template for PCR to amplify the DNA encoding RTnI1)116

(fragment; residues 1–116 of rabbit fast skeletal TnI) and
RTnI96)181 (fragment; residues 96–181), using the primer
sets RTnI1F and RTnI116R (5¢-GAGCATGGCGGGAT
CCTACATGCGCAC-3¢) and RTnI96F (5¢-GCTGGAGG
CCATGGACCAGAAGC-3¢) and RTnI181R, respectively
(BamHI ⁄ NcoI sites and termination ⁄ initiation codons are
indicated by underlines and bold type face, respectively). In
RTnI96F the Asn96 of the template was replaced by
Asp96, and an NcoI site was introduced. The PCR products were used for the construction of expression plasmids
by the method described above.

Expression and purification of recombinant TnI
fragments
The expression plasmids were introduced into E. coli
BL21(DE3) cells (Novagen) and cultivated at 37 °C for 9 h
in LB medium, and then TnI fragments were expressed by
induction with 1 mm IPTG. The cells were harvested by
centrifugation (10 000 g, 10 min), and resuspended in STET
buffer (8% (w/v) sucrose, 50 mm Tris ⁄ HCl (pH 8.0),
50 mm EDTA, and 5% (v/v) Triton X-100), and then lysed
by three freeze-thaw cycles. After centrifugation (10 000 g,
10 min), ATnI1)128, ATnI232)292, and RTnI96)181 were
found in the supernatant, and purified by CM-Toyopearl
650 m (Tosoh, Tokyo, Japan) column chromatography in
the presence of 6 m urea [34]. ATnI-52K, ATnI-19K,
ATnI130)252, RTnI, and RTnI1)116, which were found in
the precipitate, were dissolved in 7 m guanidine hydrochloride, 10 mm Tris ⁄ HCl (pH 7.6), 1 mm EDTA, and 5 mm 2mercaptoethanol, and then subjected to CM-Toyopeal column chromatography as described above. ATnI-52K was
further purified by DEAE-Toyopearl 650 m (Tosoh) column chromatography under the conditions used for CMToyopeal chromatography. RTnI, RTnI1)116, and ATnI19K were also purified by hydroxyapatite (Wako Pure
Chemicals, Osaka, Japan) column chromatography performed using 6 m urea, 10 mm KH2PO4 (pH 7.0), 5 mm 2mercaptoethanol, and a linear gradient of 0–500 mm KCl.
The N-terminal sequences of these recombinant proteins

were analyzed on an ABI 492HT protein sequencer
(Applied Biosystems, Foster City, CA, USA).

Polyacrylamide gel electrophoresis
SDS ⁄ PAGE was carried out using the method of Porzio
and Pearson [38] on a 10% (w/v) acrylamide and 0.1% bisacrylamide slab gel. Alkaline urea PAGE was performed by
the method of Head and Perry [39] on a 6% (w/v) acryl-

4483


Functional regions of molluskan TnI

amide and 0.48% (w/v) bis-acrylamide slab gel containing
either 6 m or 3 m urea and either 2 mm CaCl2 or 2 mm
EDTA. The samples were prepared as follows: TnI-fragment and TnC were mixed to a 1 : 1 molar ratio in the
medium containing 0.125 m KCl, 10 mm Tris ⁄ HCl
(pH 7.6), and either 5 mm CaCl2 or 5 mm EDTA, and then
diluted with 1.5 volumes of either 10 or 5 m urea, 41.5 mm
Tris, 133 mm glycine (pH 8.6), 0.02% (w/v) bromophenol
blue, and 8% (v/v) 2-mercaptoetanol. The samples were
allowed to stand for 2 h on ice before application to the
gels. The electrophoresis was carried out at room temperature by using 25 mm Tris and 80 mm glycine (pH 8.6) as a
running buffer.
The gels were stained with 0.2% (w/v) Coomassie brilliant blue R250. Fluorescent staining using SYPRO Red
(Cambrex, East Rutherford, NJ, USA) was also performed
for densitometric analysis on a fluorescent imager, FLA3000G (Fuji Photo Film, Tokyo, Japan).

Affinity chromatography
Rabbit or Akazara scallop TnC was immobilized on

Formyl-Cellulofine (Chisso, Tokyo, Japan) according to
the procedure suggested by the manufacturer. The TnCCellulofine was packed into a column (0.8 · 4.0 cm) and
equilibrated with 10 mm Tris ⁄ HCl (pH 7.6) and 0.5 mm
CaCl2. About 50 nmol of TnI-fragment was dialyzed
against the same solution and then applied onto the column. The fragment was eluted with a stepwise gradient of
KCl at a flow rate of 0.16 mLỈmin)1. The fragment that
was not eluted under these conditions was removed with
6 m urea, 0.5 m KCl, 10 mm Tris ⁄ HCl (pH 7.6), and
1 mm EGTA. The proteins in the effluents were detected
by the method of Bradford [40], and identified by
SDS ⁄ PAGE. RTnI1)116, which was insoluble in 10 mm
Tris ⁄ HCl (pH 7.6) and 0.5 mm CaCl2, was applied at a
KCl concentration of 0.1 m.

Actin-tropomyosin centrifugation studies
The binding of the TnI-fragment to actin-tropomyosin was
analyzed by a cosedimentation assay. The assay conditions
were as follows: 0.15 mgỈmL)1 (3.6 lm) rabbit F-actin,
0.075 mgỈmL)1 (1.1 lm) rabbit or Akazara scallop tropomyosin, 2.2 lm recombinant TnI-fragment with or without
equimolar amount of TnC, 50 mm KCl, 20 mm Tris maleate
(pH 6.8), 2 mm MgCl2, and 0.2 mm EGTA (in the absence
of Ca2+) or 0.2 mm EGTA plus 0.3 mm CaCl2 (in the presence of Ca2+). The proteins were mixed in the presence of
0.3 m KCl and then diluted to the above conditions. The
samples (0.5 mL) were incubated at 15 °C for 30 min and
then centrifuged at 100 000 g for 30 min on an Optima
TL-100 ultracentrifuge (Beckman Coulter, Fullerton, CA,
USA). The pellets and supernatants were redissolved in
equivalent volumes (0.1 mL) of 5 m urea, 5 mm Tris ⁄ HCl

4484

H. Tanaka et al.

(pH 8.9), 0.5% (w ⁄ v) SDS, and 5% (v ⁄ v) 2-mercaptoethanol, and then analyzed by SDS ⁄ PAGE. The amount of the
TnI-fragment bound to actin-tropomyosin was estimated by
densitometry, using known amounts of protein run on the
same gel, as a standard. The amount of nonspecific
precipitation of the TnI-fragment was also monitored by
simultaneous centrifugation of the sample containing no
actin-tropomyosin under the same conditions.

Reconstitution of troponins
Recombinant TnI-fragment and native TnC and TnT were
mixed at a 1 : 1 : 1 molar ratio and dialyzed against 6 m
urea, 0.5 m KCl, 10 mm Tris ⁄ HCl (pH 7.6), and 5 mm
2-mercaptoethanol. The urea and KCl concentrations were
reduced stepwise by the following changes of dialysis buffer: (a) buffer B (3 m urea, 0.5 m KCl, 10 mm Tris maleate
(pH 6.8), 2 mm MgCl2, 0.2 mm EGTA, 0.3 mm CaCl2,
0.01% NaN3 (w/v), and 5 mm 2-mercaptoethanol); (b) buffer B containing 1 m urea and 0.5 m KCl; (c) buffer B containing 0.5 m KCl; and (d) buffer B containing 0.25 m KCl.
After dialysis, the complexes were centrifuged and the supernatants were used immediately.

Measurements of Mg2+-ATPase activity
The inhibition of actomyosin-tropomyosin Mg2+-ATPase
by the TnI-fragment and the release of the inhibition by
TnC were measured in the presence of 0.05 mgỈmL)1
(1.2 lm) rabbit F-actin, 0.1 mgỈmL)1 (0.19 lm) rabbit myosin, 0.025 mgỈmL)1 (0.38 lm) rabbit or Akazara scallop
tropomyosin, and various concentrations of TnI-fragment
and TnC. The assays were performed at 15 °C in a medium
containing 50 mm KCl, 2 mm MgCl2, 20 mm Tris maleate
(pH 6.8), 1 mm ATP, and 0.2 mm EGTA (in the absence of

Ca2+) or 0.2 mm EGTA plus 0.3 mm CaCl2 (in the presence of Ca2+). The Ca2+ regulatory effect of the reconstituted troponin was measured in the presence of
0.03 mgỈmL)1 (0.71 lm) rabbit F-actin, 0.06 mgỈmL)1
(0.11 lm) rabbit myosin, 0.015 mgỈmL)1 (0.23 lm) rabbit
or Akazara scallop tropomyosin, and 0.23 lm reconstituted
troponin. The assays were performed at 15 or 25 °C in a
medium containing 50 mm KCl, 2 mm MgCl2, 20 mm
Tris maleate (pH 6.8), 1 mm ATP, 0.1 mm CaCl2 and
0–3.84 mm EGTA. The concentrations of EGTA required
to attain the desired final free Ca2+ concentrations (pCa
7.5–4.0) were calculated by using the stability constant of
8.45 · 105 m)1 for the Ca2+–EGTA complex [41].
The reaction was initiated by adding 0.5 mL of 10 mm
ATP to 4.5 mL of the solution containing all the components except for ATP. After 2, 4, 6, and 8 min incubation,
1 mL aliquots were withdrawn from the reaction mixture
and added to 4 mL of acidic malachite green solution to
determine the liberated inorganic phosphate concentrations
by the method of Chan et al. [42].

FEBS Journal 272 (2005) 4475–4486 ª 2005 FEBS


H. Tanaka et al.

Functional regions of molluskan TnI

Acknowledgements
This study was supported by Special Coordination
Funds from the Ministry of Education, Culture,
Sports, Science and Technology, of the Japanese
Government.

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