From the Departments of Internal Medicine and Biochemistry, University of Iowa, Iowa City, Iowa 52242
Received for publication, September 9, 2002, and in revised form, October 28, 2002
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ABSTRACT |
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Troponin contains a globular
Ca2+-binding domain and an elongated tail domain
composed of the N terminus of subunit troponin T (TnT). The tail domain
anchors troponin to tropomyosin and actin, modulates myosin function,
and is a site of cardiomyopathy-inducing mutations. Critical
interactions between tropomyosin and troponin are proposed to depend on
tail domain residues 112-136, which are highly conserved across phyla.
Most cardiomyopathy mutations in TnT flank this region. Three such
mutations were examined and had contrasting effects on peptide
TnT-(1-156), promoting folding and thermal stability assessed by
circular dichroism (F110I) or weakening folding and stability (T104V
and to a small extent R92Q). Folding of both TnT-(1-156) and whole
troponin was promoted by replacing bovine TnT Thr-104 with human TnT
Ala-104, further indicating the importance of this cardiomyopathy site
residue for protein folding. Mutation F110I markedly stabilized the
troponin tail but weakened binding of holo-troponin to
actin-tropomyosin 8-fold, suggesting that loss of flexibility impairs
troponin tail function. The effect of the F110I mutation on
troponin-tropomyosin binding to actin was much less, indicating this
flexibility is particularly important for the interactions of troponin
with tropomyosin. We suggest that most cardiomyopathic mutations in the
troponin tail alter muscle function indirectly, by perturbing
interactions between troponin and tropomyosin requisite for the complex
effects of these proteins on myosin.
In striated muscles, including the heart and skeletal muscle,
contraction is tightly regulated by the reversible binding of Ca2+ to the thin filament protein troponin. Tight and
specific attachment of troponin to the thin filament is mediated by the
troponin tail domain, which is composed of the N-terminal portion of
TnT1 and interacts with the
tropomyosin C terminus. Hydrodynamic studies (1), rotary-shadowed
electron micrographs of troponin (2), and intermediate resolution
studies of both troponin-tropomyosin (3) and TnT-tropomyosin
co-crystals (4) indicate that the tail domain is highly asymmetric and
~160 Å in length. Electron microscopy of tropomyosin-TnT co-crystals
suggests that a long region of the tropomyosin C terminus may interact
with the troponin tail (4). However, most of this extended interaction
may be very weak, because a variety of other evidence suggests that
only the C terminus of tropomyosin binds strongly to troponin (reviewed in Ref. 5). Recently, an x-ray crystallographic study of the tropomyosin C terminus identified and determined the structure of an
18-residue tropomyosin region that comprises a critical TnT-binding
site (6). The TnT element that binds to this tropomyosin region is unknown.
A new approach to these important interactions has been provided by the
discovery that any of several mutations in the troponin tail region can
cause the autosomal dominant disorder, familial hypertrophic
cardiomyopathy (FHC). Regardless whether because of mutations in thick
filament or thin filament components of the cardiac sarcomere, the
characteristic finding in FHC patients is missense or mild truncation
mutations (7-13) that generally alter rather than abolish protein
function. As many as 15% of FHC kindreds have cardiac TnT mutations,
and the largest portion of these are in the troponin tail (see below).
The disease phenotype has high penetrance, including myofibrillar
disarray and risk of sudden death by arrhythmia. Therefore, troponin
tail function must be altered and protein function studies, if
sufficiently sensitive, are likely to identify abnormalities. Indeed,
beginning in 1996 (14), numerous studies have described effects on
unloaded sliding speed, force, Ca2+ affinity,
Ca2+ sensitivity, and cooperativity (15-24). These effects
support the view that the troponin tail has subtle effects on myosin, in addition to its critical function as an anchoring domain.
The present work concerns three FHC-inducing TnT mutations that are of
interest because of their location in the important but poorly
understood troponin tail region, and because their unexpected effects
on troponin solubility suggested alterations in protein folding. In an
earlier publication (18), we found that mutations R92Q and F110I (11)
greatly impaired the solubility of bovine cardiac troponin. (Bovine TnT
amino acids are designated in the present report, unlike some of our
previous work (18, 25, 26), by the sequence positions of homologous
human cardiac TnT residues (27).) Although it was possible to
reconstitute troponin complexes containing these mutant TnTs by
gradually dialyzing away denaturant under high salt conditions, R92Q
troponin precipitated when the ionic strength was decreased below 0.5 M, and F110I troponin precipitated unless 10% glycerol was
added in addition to a high ionic strength buffer. Insoluble proteins
can cause human disease (28, 29), but there was no precedent for
protein insolubility in FHC. Therefore, it seemed plausible that the
observations were due to the use of bovine rather than human troponin.
In this regard, it is notable that between residues 70 and 141, human
and bovine TnTs are identical except at one position: bovine Thr-104 is
Ala in the human sequence. Because this residue is located near both positions 92 and 110, the sites of mutations causing poor solubility, we created bovine TnT double mutants R92Q/T104A and F110I/T104A. As
shown below, this maneuver corrected the insolubility and permitted reconstitution of functional troponins, suggesting the importance of
residue 104 in proper folding of the troponin tail. Interestingly, there is a family with FHC due to the TnT mutation T104V (30), reinforcing the significance of this residue for folding and/or function.
The results below detail the effects of R92Q, T104A, T104V, and F110I
mutations on the folding of the troponin tail region and on its
function as an anchoring domain. The respective mutation sites either
had little effect, inhibited, or strongly promoted folding stability of
the troponin tail. Mutation F110I weakened the affinity of troponin for
the thin filament, despite promoting folding, and overall the data
suggest that troponin tail flexibility is particularly important for
the interactions of troponin with tropomyosin. In addition, multiple
vertebrate and invertebrate TnTs were subjected to amino acid sequence
alignment, revealing very high conservation of residues 112-136. In
consideration of this analysis as well as previous deletional studies,
we suggest this region is a critical anchoring element of TnT. Numerous
cardiomyopathy-inducing mutations flank this TnT sequence.
Protein Purification and Construct Design--
Bovine cardiac
tropomyosin, native troponin, TnI, and TnC were purified as described
previously (31, 32) from a heart muscle ether powder. Actin was
obtained from an acetone powder of rabbit fast skeletal muscle (33).
Whole troponin was reconstituted from denatured subunits mixed in a
1:1:1 ratio, followed by stepwise dialysis and size exclusion column
chromatography (32).
Recombinant control and mutant bovine whole TnT and TnT-(1-156) were
expressed using pET3d in DE3 cells and purified to homogeneity as
described (25). T104V or T104A mutations were introduced into bovine
cardiac TnT cDNA (18) by the same PCR-based approach used
previously to create the R92Q and F110I mutations (18). The same method
was also used to introduce the T104A mutation into TnT R92Q and F110I.
To create the various TnT-(1-156) constructs, the corresponding DNA
fragment was amplified by PCR from the various full-length plasmids and
inserted into the NcoI/BamHI sites of pET3d. All
coding sequences in expression plasmids were confirmed by automated DNA
sequencing at the University of Iowa DNA Facility.
Protein Folding and Circular Dichroism--
The ellipticity of
TnT-(1-156) was monitored as a function of temperature using an Aviv
DS65 circular dichroism spectrometer, recording from Binding of Troponin or Tropomyosin to the Thin
Filament--
Binding of radiolabeled tropomyosin to actin was
measured by cosedimentation with a TLA100 rotor in a Beckman TL100
centrifuge (34). The tropomyosin was stoichiometrically labeled under
denaturing conditions on Cys-190 with [3H]iodoacetic acid
(34). Binding was calculated from the decrease in supernatant
radioactivity following sedimentation. As described in the figures,
conditions were chosen in which the tropomyosin bound negligibly to
actin unless troponin or a troponin fragment was added.
Troponin binds very tightly to the thin filament, making its affinity
problematic to measure by sedimentation. Therefore, the relative
affinity of troponin for actin-tropomyosin was measured by its ability
to displace a control, 3H-labeled troponin from the thin
filament. Bound and free [3H]troponin were separated by
ultracentrifugation as described previously (25). By fitting the data
to Equation 1 from Ref. 25, the affinity of troponin for
actin-tropomyosin was measured, relative to the affinity of the
3H-labeled troponin. Conditions are as follows: 25 °C, 7 µM actin, 3 µM tropomyosin, 1 µM 3H-labeled troponin, 10 mM
Tris (pH 7.5), 300 mM KCl, 3 mM
MgCl2, 0.2 mM dithiothreitol, 0.3 mg/ml bovine
serum albumin, and 0.5 mM EGTA.
Sequence Alignment of Multiple Troponin Ts--
The strongest
interaction between troponin and tropomyosin involves the TnT N
terminus and a tropomyosin C-terminal region found specifically in
tropomyosins that bind to troponin (reviewed in Ref. 6). Moreover, a
newly reported crystallographic study of tropomyosin shows that
conserved C-terminal residues of mammalian striated muscle tropomyosin
effect a distinctive structure, a troponin T recognition site. This
structural and sequence conservation in tropomyosin suggests a similar
conservation in the protein target, i.e. in the troponin
tail. To help identify this region, a multiple sequence alignment of 15 TnTs was performed (Fig. 1). In the
figure, chordate (mammals, birds, and tunicate) TnT conservation is
indicated in yellow, and blue indicates
conservation among three invertebrate phyla (nematode, mollusks, and
insect). Regions conserved among both vertebrate and invertebrate TnTs
are indicated in green and are readily apparent.
Fig. 1 shows that no TnT region is more conserved than residues
112-136 of the troponin tail domain. These residues are 70% homologous across the analyzed sequences. Significantly, engineered cardiac troponin constructs bind tightly to the thin filament only if
they contain this entire region (see "Discussion"), regardless whether the construct is a troponin tail fragment containing only the
TnT N terminus or instead is a truncated ternary troponin complex with
an N-terminal deletion (25, 34, 35). The present sequence alignment and
these earlier deletional experiments together suggest that residues
112-136 comprise a tropomyosin-binding element. Also, note that this
region is flanked by many hypertrophic cardiomyopathy alleles,
including (# in Fig. 1) sites Arg-92, Ala-104, and Phe-110.
Although not the focus of the present study, the sequence alignment in
Fig. 1 should also be considered in relationship to a preliminary, high
resolution structure of a troponin complex containing TnC, most of TnI,
and TnT fragment 188-288 (36). Of the 101 TnT residues crystallized,
70 (residues 202-271) are ordered and identifiable in the x-ray
structure. As can be seen in the figure, these boundaries correspond to
those residues of the C-terminal half of TnT that are evolutionarily
conserved. Within this region there is particular conservation of amino
acids 226-271, which form a coiled coil with subunit TnI and also
interact with TnC (36).
Effects of Cardiomyopathy Causing TnT Mutations on Folding of the
Troponin Tail--
In an earlier study, we reported that TnT R92Q and
F110I mutations interfered with the ability of bovine cardiac troponin subunits to reconstitute into a soluble troponin complex (18). To
investigate this folding abnormality quantitatively, we isolated troponin tail fragments TnT-(1-156), finding them to be soluble regardless of mutation. No precipitation was observed in low ionic strength buffer at protein concentrations studied (~1.5 mg/ml). Troponin tail fragment TnT-(1-156) was predominantly
Human cardiac and bovine cardiac TnT residues 70-141 are identical
except at amino acid 104, which is alanine in the former and threonine
in the latter. Alanine is among the most helix-promoting amino acids,
and as described below, the solubility of bovine troponins containing
human cardiomyopathy mutations R92Q or F110I was restored by
incorporating Ala at position 104 of bovine TnT. Ala-104 improved the
folding of the troponin tail fragments (Table I and Fig.
2B), raising the melting temperature by 4-6 °C
regardless of the presence of the R92Q mutation, the F110I mutation, or
neither. This suggests the importance of residue 104 for troponin tail folding stability, and this implication is reinforced by the results with the cardiomyopathy-causing valine at this position. T104V TnT-(1-156) had the lowest stability of any of the tail fragments tested.
Fig. 2B shows that bovine TnT-(1-156) containing the double
mutation T104A/F110I was particularly stable, with a
Tm even higher than that of the T104A peptide, by
5.6 °C. In contrast, cardiomyopathic mutation R92Q had no effect on
the melting curve in the presence of the T104A mutation (Fig.
2B, circles versus triangles). The CD
data overall demonstrate that cardiomyopathy-causing mutations at the
three positions, 92, 104, and 110, had strikingly disparate effects on
the folding of the troponin tail domain. While this work was in
preparation, qualitatively similar results were reported using human
cardiac TnT fragment 70-170 (39) (see "Discussion"). Despite
substantial differences in the N and C termini of the peptides studied
in the two reports, and differences in Tm values and
ellipticity at 0 °C, the effects of the mutations on apparent
stability were very similar.
Effects of the Mutations on Binding of the Troponin
Tail-Tropomyosin Complex to Actin--
Like whole troponin, troponin
tail peptide TnT-(1-156) promotes the binding of tropomyosin to actin
(25, 26), an effect most conveniently studied under high ionic strength
conditions that weaken tropomyosin-actin affinity. In the presence of
300 mM KCl and sub-micromolar tropomyosin concentrations,
negligible tropomyosin binds to actin unless either troponin or the
troponin tail peptide is added (Fig. 3).
The effect of TnT-(1-156) was independent of the bovine
versus human Thr-104 versus Ala-104 substitution
but was significantly impaired by each of the cardiomyopathy mutations,
T104V, F110I, or R92Q. Thus, function was lost not only by mutations
that impaired folding (T104V and R92Q), but also by a mutation that
strengthened folding (F110I). When the R92Q mutation was combined with
the T104A substitution (construct R92Q/T104A), troponin tail function
was restored. Because folding was also improved (Table I), this is
explainable by a higher fraction of the protein being folded and
therefore binding competent at 25 °C. However, the F110I/T104A
troponin tail fragment is particularly stable according to the data in
Fig. 2, but nevertheless had no effect on tropomyosin binding to actin.
This suggests that normal interactions of the troponin tail with its
target (actin and/or tropomyosin) require sufficient flexibility,
lacking as a consequence of the mutation.
Effects of the Mutations on Binding of Troponin-Tropomyosin to
Actin--
TnT R92Q and F110I mutations adversely affect bovine
cardiac whole troponin solubility (25), complicating assessment of the
properties of these molecules. Similarly, repeated efforts in the
present study to reconstitute bovine troponin containing the T104V
mutation produced a soluble complex only when the ionic strength was
maintained very high, with more than 1 M KCl. However, no
such problem was observed for troponins with the TnT double mutants
R92Q/T104A or F110I/T104A. In the context of the human Ala-104, which
promotes folding (Fig. 2), the cardiomyopathy mutations did not affect
troponin solubility, and ternary troponin complexes were routinely
isolated at concentrations of ~1.5 mg/ml in low ionic strength buffer.
Fig. 4 shows the same experiment as in
Fig. 3, except binding of tropomyosin to actin is promoted by whole
troponin with or without cardiomyopathy mutations, instead of by
troponin tail fragments. T104A troponin behaved similarly to control
troponin, as did R92Q/T104A troponin. Both the maximal
tropomyosin-actin binding and the troponin concentration dependence of
the effect were indistinguishable from data for control troponin. In
contrast, F110I/T104A troponin was less effective, requiring higher
concentrations to promote tropomyosin-actin binding and producing less
of a maximal effect. Each of these observations was unchanged by the
addition of calcium (bottom versus top
panel).
To delineate these effects better, and to determine whether the altered
properties of F110I/T104A troponin primarily reflected changes in
interactions with actin or instead with tropomyosin, saturating amounts
of troponin were added to variable concentrations of tropomyosin. Fig.
5 presents the binding of these
troponin-tropomyosin complexes to actin, and best fit lines to the
composite result from two experiments. (A third experiment under
slightly different conditions gave similar results.) The affinity of
tropomyosin-F110I/T104A troponin for actin was 56% the affinity found
for control tropomyosin-troponin, a readily detectable but modest
decrease. Similar results were observed in the presence of calcium
(data not shown).
Effects of the Mutations on Binding of Troponin to
Actin-Tropomyosin--
The modest effect of the F110I/T104A mutation
on troponin-tropomyosin binding to actin (Fig. 5) contrasts sharply
with the large effect (see Fig. 3) of this mutation on the properties
of TnT-(1-156), the troponin tail peptide. This suggested the
possibility that the large effect of this mutation in Fig. 2,
essentially a loss of all ability to promote tropomyosin-actin binding,
reflected only the properties of the troponin tail and not those of
whole troponin. To evaluate this possibility, the effects of the TnT mutation on whole troponin binding to actin-tropomyosin were measured.
Troponin binds very tightly to actin-tropomyosin, complicating
measurement of binding affinity. Therefore, the effect of the TnT
mutations on this process was measured by competition (25). T104A and
R92Q/T104A troponins displaced control, radiolabeled troponin from the
thin filament (Fig. 6), with patterns
consistent with normal (i.e. KR The results in this article substantially agree with the recent
findings of Palm et al. (39). R92Q, T104V (or A104V), and F110I mutations in TnT have similar effects on protein folding when
examined either in human cardiac TnT peptides TnT-(70-170) (39) or
instead in our bovine construct TnT-(1-156). In particular, mutant
residue Val-104 was destabilizing; Ile-110 was stabilizing, and
Gln-92 had a small effect in both studies. Nevertheless, unique features of each report result in some differences that are worth noting, and overall assessment of the mutations requires considering both studies. Our data on the T104A substitution indicate that this
residue has an unexpected importance in troponin folding, in both whole
troponin and in the troponin tail, in both control and cardiomyopathic
contexts. Further emphasizing the importance of this residue,
cardiomyopathy mutation to Val at this same position weakens stability,
shown in both reports by effects on Tm. In our study
but not in Palm et al. (39) Val-104 disrupts the adoption of
normal secondary structure at 0 °C (Fig. 2A), perhaps suggesting that residue 104 affects folding of residues 1-69 that are
included in our constructs but not in TnT-(70-170). However, a
definitive conclusion on this point would require more data on the
structure of this region of troponin, and the more notable point is
that the two reports agree qualitatively on the effects of the
mutations on folding. As discussed below, the functional implications
of Fig. 2 do not require that the troponin tail be unfolded in
vivo, which cannot be determined from the present experiments.
In both studies the F110I mutation promoted protein folding but
impaired interactions of the troponin tail with its targets, strongly
suggesting that flexibility is important for this process. Interestingly, Palm et al. (39) found similar properties for mutations R92W, R92L, and R94L, not examined here. In the present report, the effects of the mutations on whole troponin were also examined, so the mutations could be understood in the context of the
entire protein. One of several advantages is that the effects of whole
troponin on tropomyosin binding to actin are very much larger
(20-35-fold (25, 34)) than the 3-fold effect of TnT-(70-170) (39).
Furthermore, the current results allow quantitative comparison of
effects on two target interactions, binding of troponin to actin-tropomyosin versus troponin-tropomyosin binding to the
thin filament. We found that only the former process was greatly
affected by the F110I mutation. Once troponin binds to tropomyosin,
interaction with actin is little affected. This result indicates it is
troponin tail-tropomyosin interactions that are most impaired for this mutant, relatively rigid troponin. Some impairment of these
interactions has been demonstrated independently in the context of
TnT-(70-170); the F110I mutation alters salt-dependent
dissociation of the fragment from a tropomyosin affinity column and
diminishes the effect of the peptide on the ellipticity of a
tropomyosin N terminus plus tropomyosin C terminus solution (39). New
F110I observations are that this effect contrasts with a much smaller
effect on thin filament assembly once the troponin is bound to
tropomyosin, occurs with whole troponin, and overall weakens troponin
anchoring by an order of magnitude.
One possible interpretation of these findings is that the examined TnT
mutations occur in a region (residues 92-110) that directly interacts
with tropomyosin, as has been suggested (39). It is difficult to
confirm or exclude this hypothesis without a high resolution structure
of the troponin tail-tropomyosin complex. However, in our view it is
more likely that highly conserved TnT residues 112-136 are the element
that binds to the similarly conserved C-terminal region of tropomyosin.
By comparison, residues 92-110 are much less conserved, 33% in
contrast to 70% (Fig. 1). In further support of the current proposal,
troponin-thin filament binding is only modestly impaired by deleting 98 N-terminal residues of TnT from whole troponin but is profoundly
diminished by deleting 122 such residues (25). Interestingly, peptide
TnT-(1-135) by itself has no detectable interaction with tropomyosin
and actin, but TnT-(1-156) interacts very strongly (25, 26). These
results suggest that the entire 112-136 region is required for the
troponin anchoring function, although this could reflect poor folding
of the TnT-(1-135) fragment.
The strikingly disparate effects of the three mutations examined in
this report do not immediately suggest a common molecular theme for the
pathophysiology of hypertrophic cardiomyopathy due to mutations in the
troponin tail domain. Nevertheless, there are at least two reasons to
believe that many of these mutations act by similar mechanisms. First,
a high percentage of all identified TnT mutations are clustered
N-terminal to the putative tropomyosin-binding element, 112-136.
Second, although the mechanism is not understood, troponin tail
mutations seem to have one similar effect; they raise the calcium
sensitivity of thin filament activation (16, 18, 20, 21, 23)). The
validity of this generalization is supported by the finding that
increased calcium sensitivity is a typical consequence of hypertrophic
cardiomyopathy-inducing mutations in two other thin filament proteins,
tropomyosin and TnI (40-44). Interestingly, rat cardiac TnT was
employed in a study with a rare contrary result, decreased calcium
sensitivity (19) caused by the R92Q mutation. Rat cardiac TnT contains
threonine rather that alanine at position 104. As the current report
shows, this species difference can be critical. One other contrary
report employed skeletal muscle cells (15).
The present data concerning a few mutations permit one to infer more
general pathophysiological mechanisms, albeit tentatively. The T104V
and F110I mutations greatly alter protein folding stability, and so
must also affect the overall properties of the troponin tail.
Therefore, the cardiomyopathy mutations should be thought of as broadly
altering the troponin tail domain, rather than as locally changing
residues directly involved in binding to tropomyosin or actin. The
troponin tail primarily interacts with tropomyosin, and as far as is
known, its interactions with actin are indirect (5). We suggest that
the common theme of cardiomyopathy mutations in the troponin tail is a
change in the effects of troponin on tropomyosin. In some cases but not
all (e.g. not R92Q), this involves weaker binding of
troponin to tropomyosin and either sub-normal or super-normal folding
stability. Furthermore, we suggest that a result of the mutations is
that the troponin-tropomyosin strand moves more flexibly than normal,
allowing it to shift its azimuthal position on the actin filament at
lower Ca2+ concentrations. In support of this idea,
cardiomyopathy-inducing mutations in tropomyosin appear to act by this
mechanism. D175N and E160G mutations increase thin filament
Ca2+ sensitivity and decrease tropomyosin folding stability
(40, 43, 45). Tropomyosin mutations K70T, V95A, and A63V have the same
combination of effects (46).2
Thus an emerging pattern for tropomyosin mutations is an increased Ca2+ sensitivity and increased flexibility. Similarly, we
suggest that the troponin tail mutations interact abnormally with
tropomyosin, resulting in altered flexibility of the
troponin-tropomyosin strand. An increasing body of experimental (26,
47-51) and theoretical (52-54) work indicates that the cooperative
activation of the thin filament by myosin critically depends on the
flexibility with which tropomyosin shifts its azimuthal position on
actin (55-57). Although less well understood, this flexibility must
also affect activation by combined effects of both myosin and
Ca2+.
Protein-protein interactions within allosteric complexes stabilize the
assembly of those complexes, but equally significantly, they are
essential to mechanism; activating and inactivating shifts in
quaternary structure represent changes in inter-subunit interactions. In this context, it is not surprising that the troponin tail region, first identified as an anchoring or assembly domain (reviewed in Ref.
5), is now known to be an important modulator of thin filament
function. Thin filament properties and actin-myosin interactions can be
affected by troponin tail changes that are either isoform switches (32,
58, 59), missense mutations (14-18, 21-23), or truncations (60-62).
In a recent report (26), we showed that bovine cardiac troponin tail
peptide TnT-(1-156) suppresses thin filament-myosin S1 ATPase
activity, weakens myosin S1-thin filament binding, and affects
tropomyosin-binding position on actin. The skeletal troponin tail
exerts similar effects (51). It is intriguing therefore that the
mutations, in an important sense, weaken the ability of
troponin-tropomyosin to suppress contraction; dis-inhibition of
contraction by calcium occurs at lower calcium concentrations. We
suggest this is due to an increased flexibility of the
troponin-tropomyosin strand. However, the details of both normal and
abnormal troponin-tropomyosin structure remain to be elucidated at a
level of resolution allowing precise functional understanding.
In summary, critical interactions between tropomyosin and troponin are
proposed to depend on highly conserved TnT residues 112-136. Three
cardiomyopathy mutations near this region had contrasting effects,
promoting or weakening folding and thermal stability. Cardiomyopathy
site Ala-104 of normal human TnT particularly strengthened protein
folding. Mutation F110I strongly stabilized the troponin tail but
weakened troponin binding to actin-tropomyosin 9-fold, indicating that
loss of flexibility was especially disadvantageous to the interactions
of troponin with tropomyosin. Finally, the data suggest that
cardiomyopathic mutations in the troponin tail alter muscle function
indirectly, by perturbing interactions between troponin and tropomyosin.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
5 to 80 °C.
Two sets of protein preparations gave similar results. Conditions are
as follows: 0.1 mg/ml TnT-(1-156), 300 mM KCl, 15 mM NaH2PO4 (pH 6.5). Similar
results were also obtained for one of the sets of protein preparations,
examined in the presence of 150 mM KCl. Data were fit to a
two-state, temperature (T)-dependent transition,
with
RT lnK =
G =
Hm(1
T/Tm).
The ellipticity of the unfolded state was taken as constant, but
satisfactory fits required that the ellipticity of the folded state be
assumed to vary linearly with temperature. This procedure should be
considered as semi-empirical, because the unfolding process is not
two-state as assumed in the modeling. Differential scanning calorimetry
of wt TnT-(1-156), F110I TnT-(1-156), and of F110I/T104A TnT-(1-156)
demonstrated that the heat of unfolding was in fact gradual and could
not be described accurately with models including as many as four
states (three transitions) (data not shown).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Troponin T sequence alignment.
ClustalW was used to align the amino acid sequences of human cardiac
TnT (top line), five other vertebrate TnTs (mammalian,
avian, and fish sequences from several muscle types), one tunicate TnT
(Halocynthia roretzi, a chordate grouped here with
vertebrate sequences), and eight TnTs from species in other
invertebrate phyla: three insect, two molluscan, and three nematode
TnTs. Yellow or blue identify residues homologous
in a majority of either chordate or non-chordate sequences,
respectively. Green indicates residues that are conserved in
a majority of all examined TnTs, and in addition are found in two or
more sequences from both chordate and non-chordate groups. Red
rectangles indicate sites of hypertrophic cardiomyopathy
mutations, with rectangle height indicating one, two, or three mutant
alleles. Purple squares are sites of dilated cardiomyopathy
mutations. According to a preliminarily reported troponin atomic
structure, amino acids 227-271 (black line) form one strand
of a coiled-coil with TnI and also bind to other portions of TnI and to
TnC (36). Note that from Asn-112 through Glu-136 of cardiac TnT, there is 70% homology across phyla. From top
to bottom the TnTs and GenBankTM accession codes are
as follows: Homo sapiens cardiac (AAK92231),
Meleagris gallopavo cardiac (AY005139), Homo
sapiens slow skeletal muscle (NP003274), Oryctolagus
cuniculus fast skeletal muscle (TPRBTS), Mitu tomentosa
(AAG44259), Salmo salar (AAC23580), Halocynthia
roretzi (BAA09463), Periplaneta americana (AAD33603),
Libellula pulchella (AAD33604), Drosophila
melanogaster upheld (XP082730), Mizuhopecten yessoensis
(BAA20456), Chlamys nippoensis (JC4951),
Caenorhabditis elegans mup2 (Q27371), Caenorhabditis
elegans adult (NP509337), and Brugia pahangi partial
sequence (CAA12260). Assumed identities: K = R; D = E; S = T; I = L; and Q = N.
-helical as
assessed by circular dichroism (data not shown), as expected (37, 38).
When ellipticity was examined as a function of temperature, the
mutations caused large but disparate effects (Fig.
2A and Table
I). TnT T104V altered the ellipticity in
the manner expected for a destabilizing mutation, decreasing the
temperature of the unfolding curve midpoint (Tm) by
4.2 °C and also decreasing the absolute magnitude of the maximal
ellipticity observed at the lowest temperature examined (
5 °C).
Cardiomyopathy causing mutation R92Q similarly decreased both the
ellipticity magnitude at low temperature and the Tm,
but to lesser extents. Interestingly, TnT mutation F110I had the
opposite consequences, markedly shifting the circular dichroism results
in the direction expected for a stabilizing rather than a
de-stabilizing mutation.
View larger version (25K):
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Fig. 2.
Temperature-dependent folding of
troponin tail fragment TnT-(1-156) is altered by
cardiomyopathy-inducing mutations. The ellipticity at 222 nm of
control and mutant bovine cardiac troponin tail fragments are shown as
a function of temperature. A, effect of cardiomyopathic
mutations on ellipticity of the TnT peptide. B, effect
of the mutations on TnT peptides containing human TnT Ala-104 instead
of bovine TnT Thr-104. WT, wild type.
Thermal denaturation midpoint of bovine cardiac troponin tail peptide
TnT-(1-156) containing cardiomyopathy mutations and/or bovine versus
human difference T104A
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Fig. 3.
TnT mutations decrease the ability of the
troponin tail to induce tropomyosin-actin binding. Tropomyosin
(total 0.5 µM) binding to actin was negligible under the
examined conditions (which notably included 300 mM KCl),
unless either troponin or the troponin tail domain was added. The
cardiomyopathy mutations greatly diminished this effect of the troponin
tail. Lines are arbitrary curves. Conditions are as follows: 25 °C,
10 µM F-actin, 0.5 µM tropomyosin, 3 mM MgCl2, 300 mM KCl, 10 mM Tris-HCl (pH 7.5), 0.5 mM EGTA, 0.2 mM dithiothreitol, 0.3 mg/ml bovine serum albumin, and
between 0 and 1.6 µM troponin or TnT-(1-156).
wt, wild type.
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[in a new window]
Fig. 4.
Effect of TnT mutations on the ability of the
troponin tail to induce tropomyosin-actin binding. Increasing
concentrations of native troponin ( ), T104A troponin (
), or
R92Q/T104A troponin (
) similarly promoted binding of tropomyosin
(0.5 µM) to actin. F110I/T104A (
) troponin also
promoted tropomyosin binding to actin but to a lesser extent. Similar
effects were observed in the presence of MgCl2/EGTA
(A) or CaCl2 (B), except for the
expected, slight weakening of binding in the latter condition (63, 64).
Lines are arbitrary curves. Conditions are the same as in
Fig. 3.
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Fig. 5.
Effect of F110I/T104A mutation in TnT on
troponin-tropomyosin binding to actin. Increasing concentrations
of tropomyosin were added to actin, simultaneously with a molar excess
of either control troponin (circles) or F110I/T104A troponin
(triangles). Binding of tropomyosin to actin was determined.
Lines are best-fit curves. The apparent binding constants
and cooperativity parameters (see Ref. 32) for control
troponin-tropomyosin were (solid line) 6.1 ± 0.4 × 106 M 1 and 38 ± 11, and
for mutant troponin-tropomyosin were (dashed line) 3.4 ± 0.2 × 106 M
1 and 31 ± 9. Conditions as in Fig. 3, except the troponin and tropomyosin
concentrations were progressively increased in parallel, with troponin
maintained in 0.3 µM excess relative to tropomyosin.
Also, the actin concentration was 5 µM.
1)
binding of these complexes to the thin filament. (KR
equals the fold change in affinity, so KR = 1 indicates no effect of the mutation.) In contrast, F110I/T104A troponin
was much less effective, implying an affinity decrease of an order of
magnitude. This indicates that the mutation had a large effect on the
binding of whole troponin to the thin filament and not merely an effect
on the isolated troponin tail. Furthermore, because the F110I/T104A
mutation caused only a modest weakening of troponin-tropomyosin binding
to actin (Fig. 5), the most significant effect of the mutation, and the
basis for the KR
1 in Fig. 6, was impaired
interactions between troponin and tropomyosin rather than changes in
the actin affinity of the regulatory complex.
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Fig. 6.
Binding of troponin to actin-tropomyosin,
measured by competition. Saturating amounts of radiolabeled
control troponin were added to actin-tropomyosin, and the relative
affinity of competing, unlabeled troponins was measured by displacement
of the radiolabel from the thin filament. T104A (squares)
and R92Q/T104A (diamonds) TnT mutations did not
significantly alter the affinity of troponin for the thin filament
(KR = 0.92 ± 0.09 and 1.06 ± 0.12, respectively, where KR = 1 indicates no effect). In
contrast, F110I/Thr-104 troponin (triangles) had a
considerably decreased affinity, KR = 0.113 ± 0.015. Lines are best-fit curves.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Drs. Taylor Allen and Yu Li each for independently bringing to our attention the potential significance of conserved regions in the troponin tail domain.
![]() |
FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Internal
Medicine, University of Iowa, 200 Hawkins Dr., SE-610-GH, Iowa City, IA
52242. Tel.: 319-356-3703; Fax: 319-356-3086; E-mail: larry-tobacman@uiowa.edu.
Published, JBC Papers in Press, October 29, 2002, DOI 10.1074/jbc.M209194200
2 L. S. Tobacman, E. Homsher, and M. Heller, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: TnT, troponin T; FHC, familial hypertrophic cardiomyopathy.
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REFERENCES |
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