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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 Tanaka
1
, Yuhei Takeya
1
, Teppei Doi
1
, Fumiaki Yumoto
2,3
, Masaru Tanokura
3
,
Iwao Ohtsuki
2
, Kiyoyoshi Nishita
1
and Takao Ojima
1
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
Troponin is a Ca
2+
-dependent regulatory protein com-
plex, which constitute thin filaments together with
actin and tropomyosin [1]. It is composed of three dis-
tinct subunits: troponin-C (TnC), which binds Ca
2+
,
troponin-T (TnT), which binds tropomyosin, and trop-
onin-I (TnI), which binds actin and inhibits actin–myo-
sin interaction [2–4]. In relaxed muscle, TnI binds to
actin and inhibits contraction. Upon muscle stimula-
tion, Ca
2+
binds to TnC and induces the release of the
inhibition by TnI, resulting in muscle contraction. To
understand the molecular mechanisms of this Ca
2+
switching, extensive studies of the structure, function,
and Ca
2+
-dependent conformational changes of tropo-
nin 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 Ca
2+
-binding motifs [7], thus TnC has
four possible Ca
2+
-binding sites, sites I and II in the
N-domain and sites III and IV in the C-domain [8,9].
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 enco-
ding 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 bind-
ing of the C-terminal region of the inhibitory subunit, troponin-I (TnI), to
actin or troponin-C (TnC) in a Ca
2+
-dependent manner. To elucidate the
molecular mechanisms of this regulation by molluskan troponin, we com-
pared the functional properties of the recombinant fragments of Akazara
scallop TnI and rabbit fast skeletal TnI. The C-terminal fragment of Akaz-
ara scallop TnI (ATnI
232)292
), which contains the inhibitory region (resi-
dues 104–115 of rabbit TnI) and the regulatory TnC-binding site (residues
116–131), bound actin-tropomyosin and inhibited actomyosin-tropomyosin
Mg-ATPase. However, it did not interact with TnC, even in the presence
of Ca
2+
. These results indicated that the mechanism involved in the alter-
native binding of this region was not observed in molluskan troponin. On
the other hand, ATnI
130)252
, which contains the structural TnC-binding site
(residues 1–30 of rabbit TnI) and the inhibitory region, bound strongly to
both actin and TnC. Moreover, the ternary complex consisting of this frag-
ment, troponin-T, and TnC activated the ATPase in a Ca
2+
-dependent
manner almost as effectively as intact Akazara scallop troponin. Therefore,
Akazara scallop troponin regulates the contraction through the activating
mechanisms that involve the region spanning from the structural TnC-
binding site to the inhibitory region of TnI. Together with the observation
that corresponding rabbit TnI-fragment (RTnI
1)116
) shows similar activa-
ting effects, these findings suggest the importance of the TnI N-terminal
region not only for maintaining the structural integrity of troponin com-
plex but also for Ca
2+
-dependent activation.
Abbreviations
TnC, troponin-C; TnI, troponin-I; TnT, troponin-T; IPTG, isopropyl-1-thio-b-
D-galactopyranoside; PMSF, phenylmethylsulfonyl fluoride.
FEBS Journal 272 (2005) 4475–4486 ª 2005 FEBS 4475
Sites III and IV also show affinity for Mg
2+
and are
thought to be always occupied by sarcoplasmic Mg
2+
,
whereas Ca
2+
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 Ca
2+
binding to TnC
have been intensively studied as the central mecha-
nisms of Ca
2+
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, Ca
2+
-binding to site I
and ⁄ or II of TnC causes the exposure of a hydropho-
bic patch on the surface of the N-domain [15], result-
ing in hydrophobic interaction between the N-domain
and the regulatory TnC-binding site [16]. This inter-
action 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-bind-
ing 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 ver-
tebrate troponins [19,20], which demonstrated that the
Ca
2+
-saturated N- and C-domains of TnC bind to the
regulatory and structural TnC-binding sites, respect-
ively, of TnI, and suggested that the C-terminal region
of TnI (including the inhibitory region and the regula-
tory TnC-binding site) exhibits a positional change
from actin-tropomyosin filament to the N-domain of
TnC in a Ca
2+
-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 Ca
2+
per mole of protein
at site IV in the C-domain because of amino acid sub-
stitutions at sites I–III [21,22]. Nevertheless, ternary
troponin complex combined with molluskan tropomyo-
sin can regulate the Mg-ATPase activity of vertebrate
actomyosin in a physiologically significant Ca
2+
-
dependent manner [21]. Moreover, the troponin regu-
lates the ATPase of molluskan myofibril together with
a well known myosin light chain-linked regulatory sys-
tem, especially under low temperature conditions [23].
Therefore, the molecular mechanisms of regulation by
molluskan troponin are expected to be somewhat dif-
ferent from those described above. A previous study
revealed that the C-domain of molluskan TnC is
responsible not only for Ca
2+
-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 func-
tions through a mechanism in which the region span-
ning 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. ATnI-
52K, 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. ATnI
1)128
is the frag-
ment corresponding to the N-terminal extending region
of 52K-TnI. ATnI
130)252
and RTnI
1)116
are the frag-
ments, corresponding to the regions spanning from the
structural TnC-binding sites to the inhibitory regions
of Akazara scallop and rabbit TnI, respectively.
ATnI
232)292
and RTnI
96)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
ATnI
1)128
showed anomalously low mobility due to
the high fraction of hydrophilic residues in the N-ter-
minal 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
RTnI
96)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, RTnI
1)116
and RTnI
96)181
differed greatly
from one another, although all of these proteins
contained the inhibitory region (Fig. 2A). RTnI
1)116
inhibited only 33% of rabbit-actomyosin–rabbit-tropo-
myosin Mg-ATPase at a 3 : 1 molar ratio with tropo-
myosin, compared with 82% for RTnI. As has been
reported previously [18,28,29], weaker inhibitory effects
of RTnI
1)116
revealed the importance of residues
117–181 for maximal inhibition. In particular, residues
Functional regions of molluskan TnI H. Tanaka et al.
4476 FEBS Journal 272 (2005) 4475–4486 ª 2005 FEBS
140–148 had been proven to bind to actin-tropomyosin
and thus are referred to as the second actin-tropo-
myosin-binding site [14]. Moreover, in our results, the
inhibition by RTnI
96)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 pro-
perties (Fig. 2B). ATnI
130)252
, which corresponds to
RTnI
1)116
, inhibited about 70% of rabbit-actomyosin-
scallop-tropomyosin Mg-ATPase at a 3 : 1 molar ratio
with tropomyosin. Moreover, the inhibition by
ATnI
232)292
, which corresponds to RTnI
96)181
, was
weaker (51%) than that by ATnI-19K (88%) or
ATnI
130)252
. Therefore, the effects of the N- or C-ter-
minal 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
the N-terminal extending region of 52K-TnI could
decrease the inhibitory effects, although ATnI
1)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, RTnI
1)116
and RTnI
96)181
cosedimented with molar ratios of
approximately 0.23, 0.048, and 0.35, respectively, to
actin. On the other hand, ATnI-19K, ATnI
130)252
and
ATnI
232)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 deter-
mined 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
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) acryl-
amide gel. Molecular mass markers are also shown (M).
H. Tanaka et al. Functional regions of molluskan TnI
FEBS Journal 272 (2005) 4475–4486 ª 2005 FEBS 4477
attributable to the difference in their binding affinities
for actin-tropomyosin. In addition, ATnI
1)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].
Interactions of TnI-fragments with TnC
We compared the ability of TnI-fragments to form a
complex with TnC by alkaline urea PAGE. The experi-
ments were performed under either 6 or 3 m urea condi-
tions in the presence of either 2 mm EDTA or 2 mm
CaCl
2
. RTnI and both rabbit TnI-fragments formed a
complex with rabbit TnC in 2 mm CaCl
2
but not in
2mm 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 com-
patible 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,
ATnI
1)128
and ATnI
232)292
did not form a complex with
Akazara scallop TnC under any of the tested conditions,
whereas ATnI-52K, ATnI-19K, and ATnI
130)252
did
under both urea concentrations in the presence of Ca
2+
(Fig. 3B). It was interesting that ATnI
232)292
did not
form a complex, as ATnI
232)292
corresponds to
RTnI
96)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
ATnI
130)252
showed complex formation even in the
absence of Ca
2+
(Fig. 3B, upper panels), suggesting that
in the absence of Ca
2+
, 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 con-
firm the interaction of TnI-fragments with immobilized
rabbit or Akazara scallop TnC under nondenaturing
conditions (Fig. 4). ATnI
232)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 CaCl
2
,
whereas ATnI
130)252
, RTnI
1)116
, and RTnI
96)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.
Ca
2+
-dependent alternative binding of C-terminal
TnI fragments to actin-tropomyosin and TnC
To understand the biological significance of the differ-
ence in TnI–TnC interactions, we compared the ability
of TnC to neutralize the inhibitory effects of the C-ter-
minal fragments in the presence and absence of Ca
2+
.
As has been reported for similar vertebrate TnI frag-
ments [14,18,29], the inhibitory effect of RTnI
96)181
in
Fig. 2. Inhibition of actomyosin-tropomyosin Mg-ATPase by rabbit
(A) or Akazara scallop (B) TnI-fragments. The actomyosin-tropo-
myosin 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; RTnI
1)116
, n; RTnI
96)181
, h. (B) ATnI-52K, d; ATnI-
19K, s; ATnI
1)128
, e; ATnI
130)252
, n; ATnI
232)292
, h.
Functional regions of molluskan TnI H. Tanaka et al.
4478 FEBS Journal 272 (2005) 4475–4486 ª 2005 FEBS
a 2 : 1 molar ratio with tropomyosin was effectively
neutralized by rabbit TnC in the presence of Ca
2+
,
but not in its absence (Fig. 5A, upper panel). In addi-
tion, the cosedimentation experiment performed under
a 4 : 4 : 2 : 7 molar ratio of RTnI
96)181
–TnC–tropo-
myosin–actin showed that the amount of RTnI
96)181
BA
Fig. 3. Complex formation between TnI-fragments and TnC detected by alkaline urea PAGE. TnI-fragments were combined with TnC as des-
cribed under ‘Experimental procedures’. The final concentration of the proteins was 13.8 l
M. 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 CaCl
2
(+ 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, RTnI
1)116
; lanes c and f, RTnI
96)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, ATnI
1)128
; lanes k and p, ATnI
130)252
; lanes l and q, ATnI
232)292
; lane r, Akazara scallop TnC. Complex forma-
tion was detected by the bands of the TnI–TnC complex (arrowheads) and weakening of the free TnC bands. Free RTnI, RTnI
1)116
,
RTnI
96)181
, ATnI-19K, ATnI
130)252
, and ATnI
232)292
did not migrate into the gels, while free ATnI-52K and ATnI
1)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 TnI-
fragments. The fragments of rabbit or Akaz-
ara 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, RTnI
1)116
was applied at a KCl
concentration of 0.1
M.
H. Tanaka et al. Functional regions of molluskan TnI
FEBS Journal 272 (2005) 4475–4486 ª 2005 FEBS 4479
cosedimented with actin-tropomyosin was greatly
reduced in the presence of Ca
2+
but not in its absence.
The amount that remained with TnC in the super-
natant was greater in the presence of Ca
2+
than in its
absence (Fig. 5A, lower panel). Therefore, this sugges-
ted that RTnI
96)181
bound actin and TnC in the
absence and presence, respectively, of Ca
2+
. These
phenomena should directly reflect the mechanism of
Ca
2+
switching involving the alternative binding of the
C-terminal region of TnI to actin or TnC in a Ca
2+
-
dependent manner [17,19]. On the other hand, the
inhibitory effect of ATnI
232)292
was not neutralized
by adding Akazara scallop TnC, irrespective of Ca
2+
concentrations (Fig. 5B, upper panel). Moreover, the
amount of ATnI
232)292
cosedimented with actin-tropo-
myosin was unaffected by the presence and absence of
TnC and Ca
2+
(Fig. 5B, lower panel). Therefore, the
Ca
2+
-switching mechanisms involving the alternative
binding of the C-terminal region of TnI were not pre-
sent in Akazara scallop troponin.
Ca
2+
-regulatory effects of troponins containing
TnI fragments
The Ca
2+
-regulatory effects of troponins composed of
TnI-fragments, native TnT, and TnC on actomyosin-
AB
Fig. 5. Functional differences between RTnI
96)181
(A) and ATnI
232)292
(B). Upper panels, effects of TnC on inhibition by the C-terminal TnI-
fragments. 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 )ofCa
2+
. 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-ter-
minal 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 Ca
2+
.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 Ca
2+
; lanes b and e, in the presence of TnC and the absence of Ca
2+
; lanes c and f, in the presence of both
TnC and Ca
2+
. 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.
Functional regions of molluskan TnI H. Tanaka et al.
4480 FEBS Journal 272 (2005) 4475–4486 ª 2005 FEBS
tropomyosin Mg-ATPase were compared. The assays
were performed at different temperatures, 15 °C, which
is the normal ambient temperature for Akazara scal-
lops and is suitable for functionalizing the molluskan
troponin [23], and 25 °C, at which many assays of
Ca
2+
regulation by vertebrate troponin have been con-
ducted [14,18,28–30]. At 15 °C, all the ternary com-
plexes consisting of rabbit TnI or TnI fragments,
rabbit TnT and TnC, regulated the ATPase, although
they exhibited quite different Ca
2+
-dependence curves
(Fig. 6A). The complex containing RTnI
1)116
(repre-
sented as RTn
1)116
) showed no inhibition, even under
low Ca
2+
concentrations, although it strongly activa-
ted the ATPase at Ca
2+
concentrations higher than
pCa 4.5. RTn
96)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 con-
sisting of ATnI
232)292
, Akazara scallop TnT and TnC
(ATn
232)292
) inhibited the ATPase irrespective of Ca
2+
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 actin-
tropomyosin, but not to TnC, irrespective of Ca
2+
concentration, as described above. Moreover,
ATn
130)252
regulated the ATPase almost as effectively
as intact troponins (ATn-52K or ATn-19K), suggesting
that the region spanning from the regulatory TnC-
binding 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 Ca
2+
concentrations. Thus, the N-terminal extending region
of ATnI-52K may be involved in the activation of the
ATPase in the presence of Ca
2+
. When we performed
similar assays at 25 °C, the regulation by RTn
1)116
,
which was observed at 15 °C, became unremarkable,
whereas RTn
96)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
Fig. 6. Ca
2+
-regulation of actomyosin-tropo-
myosin Mg-ATPase by rabbit (A and C) and
Akazara scallop (B and D) reconstituted tropo-
nins. The effects of the troponin containing
TnI or TnI fragments on the actomyosin-
tropomyosin Mg-ATPase were measured as
a function of pCa ()10g[Ca
2+
]). The assays
were performed at 15 °C (A and B) or 25 °C
(C and D). A and C: RTn, d; RTn
1)116
, n;
RTn
96)181
, h. B and D: ATn-52K, d; ATn-
19K, s; ATn
130)252
, n;ATn
232)292
, h. The
activities in the absence of troponin are indi-
cated by dashed lines.
H. Tanaka et al. Functional regions of molluskan TnI
FEBS Journal 272 (2005) 4475–4486 ª 2005 FEBS 4481
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 ATnI
232)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
ATnI
130)252
, which contains the structural TnC-bind-
ing 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 scal-
lop TnI–TnC complex. Although the further verifica-
tion under nondenaturing conditions is required, the
results of the alkaline urea gel electrophoresis indicate
that this interaction is strengthened by Ca
2+
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
Ca
2+
-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 Ca
2+
,
and Ca
2+
-dependent activation. The regulatory mech-
anism involving the alternative binding of the C-ter-
minal 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 Ca
2+
, troponin activates actomyosin-
tropomyosin Mg-ATPase beyond the level observable
in the absence of troponin. This activation is promin-
ent, especially for molluskan troponin, which confers
Ca
2+
sensitivity on the ATPase predominantly
through its activation in the presence of Ca
2+
, rather
than by inhibition due to its absence. In contrast, the
vertebrate troponin regulates the ATPase mainly by
inhibition in the absence of Ca
2+
(Fig. 6 and [21,32]).
The difference in Ca
2+
sensitization between verte-
brates and mollusks should also be closely related to
the difference in the inhibitory effects of vertebrate
and molluskan tropomyosins [33], which inhibit rab-
bit actomyosin Mg-ATPase activity to 0.043 and
0.021 lmolÆmin
)1
Æmg 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
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 tropo-
nin, 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-bind-
ing site to the inhibitory region) participates in the
Ca
2+
-dependent activation. In addition, at 15°C, sim-
ilar 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 streng-
thening of the interaction between the structural TnC-
binding site and the C-domain of TnC accompanying
Ca
2+
binding to site IV of TnC. In vertebrate tropo-
nin, the activation may be a result of the interaction
between the inhibitory region and TnC accompanying
Ca
2+
binding to site I or II of TnC. However, we can-
not rule out the possibility that the substitution of
Mg
2+
at site III or IV of vertebrate TnC with Ca
2+
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 activa-
ting effects of the N-terminal region of TnT are exer-
ted in the presence of Ca
2+
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-ter-
minal region plays a role in the inhibition ⁄ removal of
inhibition by alternative binding, while the N-terminal
region is responsible for the Ca
2+
-dependent activa-
tion. 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 tro-
ponin 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 pre-
pared 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], respect-
ively. All measures were taken to minimize pain and
Functional regions of molluskan TnI H. Tanaka et al.
4482 FEBS Journal 272 (2005) 4475–4486 ª 2005 FEBS
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 acces-
sion 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 (recombin-
ant 52K-TnI; residues 1–292), ATnI
1)128
(recombinant frag-
ment consisting of residues 1–128 of 52K-TnI), ATnI-19K
(recombinant 19K-TnI; residues 130–292), ATnI
130)252
(fragment; residues 130–252), and ATnI
232)292
(fragment;
residues 232–292), combinations of the forward and reverse
primers, ATnI1F (5¢-CATATCA
CCATGGGTTCCCTTG-3¢)
and ATnI292R (5¢-CTTGATTT
GGATCCTTTAAGGTA
TAGC-3¢), ATnI1F and ATnI128R (5¢-GTTCC
GGATC
CTATCTTCTGGCTTCC-3¢), ATnI130F (5¢-GCCAGAA
CCATGGCGGAGGAAC-3¢) and ATnI292R, ATnI130F
and ATnI252R (5¢-CAAGTTTG
GGATCCTATTTGTTAA
CTTTTC-3¢), and ATnI232F (5¢-CGAGATTAATG
CC
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¢-CAAACCTCA
CCATGGGAGAT
GAAG-3¢) and RTnI181R (5¢-CCCCGGAGCC
GGATCC
CCAGCCCC-3¢). These primers were designed based on
the sequence retrieved from the GenBank database under
accession number L04347, and NcoIorBamHI sites (under-
lined) and the initiation codon (bolded) were introduced
into the sequences. The cDNA subcloned into pCR2.1-
TOPO was first subjected to mutagenesis for deactivating
the native NcoI site in the coding region by using Mutan-
Super Express Km kit (Takara-bio, Ohts, Japan). The
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; resi-
dues 1–181). The expression plasmid was also used as a
template for PCR to amplify the DNA encoding RTnI
1)116
(fragment; residues 1–116 of rabbit fast skeletal TnI) and
RTnI
96)181
(fragment; residues 96–181), using the primer
sets RTnI1F and RTnI116R (5¢-GAGCATGGCG
GGAT
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 prod-
ucts 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), ATnI
1)128
, ATnI
232)292
, and RTnI
96)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,
ATnI
130)252
, RTnI, and RTnI
1)116
, which were found in
the precipitate, were dissolved in 7 m guanidine hydrochlo-
ride, 10 m m Tris ⁄ HCl (pH 7.6), 1 mm EDTA, and 5 mm 2-
mercaptoethanol, and then subjected to CM-Toyopeal col-
umn chromatography as described above. ATnI-52K was
further purified by DEAE-Toyopearl 650 m (Tosoh) col-
umn chromatography under the conditions used for CM-
Toyopeal chromatography. RTnI, RTnI
1)116
, and ATnI-
19K were also purified by hydroxyapatite (Wako Pure
Chemicals, Osaka, Japan) column chromatography per-
formed using 6 m urea, 10 mm KH
2
PO
4
(pH 7.0), 5 mm 2-
mercaptoethanol, 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% bis-
acrylamide slab gel. Alkaline urea PAGE was performed by
the method of Head and Perry [39] on a 6% (w/v) acryl-
H. Tanaka et al. Functional regions of molluskan TnI
FEBS Journal 272 (2005) 4475–4486 ª 2005 FEBS 4483
amide and 0.48% (w/v) bis-acrylamide slab gel containing
either 6 m or 3 m urea and either 2 mm CaCl
2
or 2 mm
EDTA. The samples were prepared as follows: TnI-frag-
ment 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 CaCl
2
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 tempera-
ture 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 brilli-
ant blue R250. Fluorescent staining using SYPRO Red
(Cambrex, East Rutherford, NJ, USA) was also performed
for densitometric analysis on a fluorescent imager, FLA-
3000G (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 TnC-
Cellulofine was packed into a column (0.8 · 4.0 cm) and
equilibrated with 10 mm Tris ⁄ HCl (pH 7.6) and 0.5 mm
CaCl
2
. About 50 nmol of TnI-fragment was dialyzed
against the same solution and then applied onto the col-
umn. The fragment was eluted with a stepwise gradient of
KCl at a flow rate of 0.16 mLÆmin
)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
1mm EGTA. The proteins in the effluents were detected
by the method of Bradford [40], and identified by
SDS ⁄ PAGE. RTnI
1)116
, which was insoluble in 10 mm
Tris ⁄ HCl (pH 7.6) and 0.5 mm CaCl
2
, 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 mgÆmL
)1
(3.6 lm) rabbit F-actin,
0.075 mgÆmL
)1
(1.1 lm) rabbit or Akazara scallop tropo-
myosin, 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 MgCl
2
, and 0.2 mm EGTA (in the absence
of Ca
2+
) or 0.2 mm EGTA plus 0.3 mm CaCl
2
(in the pres-
ence of Ca
2+
). 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
(pH 8.9), 0.5% (w ⁄ v) SDS, and 5% (v ⁄ v) 2-mercaptoetha-
nol, 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 buf-
fer: (a) buffer B (3 m urea, 0.5 m KCl, 10 mm Tris maleate
(pH 6.8), 2 mm MgCl
2
, 0.2 mm EGTA, 0.3 mm CaCl
2
,
0.01% NaN
3
(w/v), and 5 mm 2-mercaptoethanol); (b) buf-
fer B containing 1 m urea and 0.5 m KCl; (c) buffer B con-
taining 0.5 m KCl; and (d) buffer B containing 0.25 m KCl.
After dialysis, the complexes were centrifuged and the sup-
ernatants were used immediately.
Measurements of Mg
2+
-ATPase activity
The inhibition of actomyosin-tropomyosin Mg
2+
-ATPase
by the TnI-fragment and the release of the inhibition by
TnC were measured in the presence of 0.05 mgÆmL
)1
(1.2 lm) rabbit F-actin, 0.1 mgÆmL
)1
(0.19 lm) rabbit myo-
sin, 0.025 mgÆmL
)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 MgCl
2
,20mm Tris maleate
(pH 6.8), 1 mm ATP, and 0.2 mm EGTA (in the absence of
Ca
2+
) or 0.2 mm EGTA plus 0.3 mm CaCl
2
(in the pres-
ence of Ca
2+
). The Ca
2+
regulatory effect of the recon-
stituted troponin was measured in the presence of
0.03 mgÆmL
)1
(0.71 lm) rabbit F-actin, 0.06 mgÆmL
)1
(0.11 lm) rabbit myosin, 0.015 mgÆmL
)1
(0.23 lm) rabbit
or Akazara scallop tropomyosin, and 0.23 lm reconstituted
troponin. The assays were performed at 15 or 25 °Cina
medium containing 50 mm KCl, 2 mm MgCl
2
,20mm
Tris maleate (pH 6.8), 1 mm ATP, 0.1 mm CaCl
2
and
0–3.84 mm EGTA. The concentrations of EGTA required
to attain the desired final free Ca
2+
concentrations (pCa
7.5–4.0) were calculated by using the stability constant of
8.45 · 10
5
m
)1
for the Ca
2+
–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 compo-
nents 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].
Functional regions of molluskan TnI H. Tanaka et al.
4484 FEBS Journal 272 (2005) 4475–4486 ª 2005 FEBS
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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