(Received for publication, August 22, 1995; and in revised form, October 20, 1995)
From the
Exposure of an N-terminal hydrophobic region in troponin C is
thought to be important for the regulation of contraction in striated
muscle. To test this hypothesis, single Cys residues were engineered at
positions 45, 81, 84, or 85 in the N-terminal hydrophobic region of
cardiac troponin C (cTnC) to provide specific sites for attachment of
blocking groups. A synthetic peptide,
Ac-Val-Arg-Ala-Ile-Gly-Lys-Leu-Ser-Ser, or biotin was coupled to these
Cys residues, and the covalent adducts were tested for activity in
TnC-extracted myofibrils. Covalent modification of cTnC(C45) had no
effect on maximal myofibril ATPase activity. Greatly decreased
myofibril ATPase activity (70-80% inhibited) resulted when the
peptide was conjugated to Cys-81 in cTnC(C81), while a lesser degree of
inhibition (10-25% inhibited) resulted from covalent modification
of cTnC(C84) and cTnC(C85). Inhibition was not due to an altered
affinity of the cTnC(C81)/peptide conjugate for the myofibrils, and the
Ca dependence of ATPase activity was essentially
identical to the unmodified protein. Thus, a subregion of the
N-terminal hydrophobic region in cTnC is sensitive to disruption, while
other regions are less important or can adapt to rather bulky blocking
groups. The data suggest that Ca
-sensitizing drugs
may bind to the N-terminal hydrophobic region on cTnC but not interfere
with transmission of the Ca
signal.
Regulation of contraction and relaxation in vertebrate striated
muscle relies on the binding and release of Ca from
troponin C (TnC) (
)in the troponin complex. Kinetics studies (1, 2) and site-directed mutagenesis (3, 4) have demonstrated that the N-terminal
Ca
binding sites I and II in skeletal TnC (sTnC) and
site II in cardiac TnC (cTnC) are primarily responsible for this
regulation. Binding of Ca
or Mg
to
the C-terminal high affinity sites in TnC facilitates tight association
of TnC with thin filaments in the relaxed
state(5, 6, 7) .
Although the molecular
mechanism by which TnC regulates muscle contraction has not been
rigorously defined, it is thought to involve two steps. The first step
is based on the molecular modeling studies of Herzberg et
al.(8) , which predicted that helices B and C of TnC move
away from helices A and D upon Ca binding to the low
affinity sites, thereby exposing a hydrophobic surface. This step of
the model has found experimental support(9, 10, 11) and has recently been confirmed by resolution of the
solution structures for apo- and Ca
-bound
sTnC(12) . In the second step of the model, the exposed
N-terminal hydrophobic region in TnC is thought to associate with a
complimentary region in TnI, thereby releasing inhibition of actomyosin
ATPase. This is an attractive concept, since it is analogous to the
interaction of calmodulin (CaM) with a subset of target proteins.
If
an exposed hydrophobic region participates in the second step of the
model, and is important for transmission of the Ca signal, then it is logical to predict that regulation of
contraction would be disrupted by ligands which bind to and block the
hydrophobic surface. A number of small aromatic drugs which bind to and
inhibit the activity of CaM also bind to the N-terminal hydrophobic
region of isolated cTnC(13, 14, 15) .
However, these compounds sensitize, rather than inhibit, cardiac muscle
contraction (16, 17, 18) . Thus, the role of
the N-terminal hydrophobic region in the mechanism of action of sTnC or
cTnC is not clear.
Site-specific mutation of multiple hydrophobic
amino acids in the N-terminal domain of cTnC could potentially test the
importance of this region, but significant artifacts could result from
abnormal protein folding. To overcome these problems, we chose to
covalently attach bulky groups to specific sites in the N-terminal
hydrophobic region of cTnC. We then compared the functional properties
of the protein conjugates with the unmodified proteins. Attachment of a
synthetic peptide or biotin to single Cys residues placed at position
45, 84, or 85 had little or no functional consequence. In contrast, the
activity of cTnC was greatly inhibited by coupling of these blocking
groups to Cys at position 81. Thus only limited portions of the
N-terminal hydrophobic region appear to be important for transmission
of the Ca signal.
Figure 1:
Location of
Cys residues and nomenclature for monocysteine mutants. A lists the nomenclature and specific mutations in the cTnC proteins
used in this study. B shows a space filling model of the
peptide backbone of amino acids 1-95 from the
Ca-bound form of cTnC as well as the side chains of
the Cys residues. The relative positions of the sulfur atoms are
indicated in black. This structure was generated using a model
of cTnC reported previously(24) . The space filling model on
the right represents the peptide backbone of a nine amino acid
peptide in an
-helical conformation.
A commercially available sulfhydryl-specific maleimide derivative of biotin was selected as one blocking group. Peptides were also considered as blocking groups, since they can be synthesized with specified lengths and chemical properties for desired solubility and coupling. Several criteria were considered in selection of the peptide sequence. Sufficient size was necessary to present an effective blocking group but not so large as to interact, either specifically or nonspecifically, with the C-terminal half of cTnC and thus interfere with binding to the thin filament. It is not necessary that the peptide have an intrinsic affinity for cTnC, but we felt that it should be designed based on available data for the interaction of peptides with either TnC or CaM rather than a more arbitrary sequence. Finally, the point of coupling should be near the middle of the peptide to prevent it from being displaced from its intended position by rotating about its end point.
Given these criteria, we chose a peptide
(Ac-Val-Arg-Ala-Ile-Gly-Lys-Leu-Ser-Ser) based on amino acids
807-815 from smooth muscle myosin light chain kinase which binds
to the N-terminal half of CaM(25, 26) . The N-terminal
Val was acetylated to provide a reactive amine on the Lys. Fig. 1B shows the relative size of the backbone of cTnC
and the peptide. The peptide is depicted in an -helix which would
represent the minimal effective volume. A derivative of the minimal
inhibitory peptide of cTnI or sTnI was not considered, since it
preferentially associates with the C-terminal domain of intact sTnC and
cTnC(27, 28, 29, 30, 31, 32) .
Such an association might result in nonspecific coupling using certain
chemistries due to localized high concentrations near the C-terminal
domain. In contrast, our synthetic MLCK peptide does not appear to have
significant affinity for cTnC, since it does not inhibit myofibril
ATPase activity at a concentration of 0.1 mM and has no effect
on the fluorescence signal from cTnC(C81) labeled with N-iodoacetyl-N`-(5-sulfo-1-naphthyl)ethylenediamine
(data not shown).
Figure 2: Reversible coupling of the peptide to cTnC. A illustrates the general strategy for coupling peptides to single Cys residues in cTnC proteins using SPDP. B shows a native polyacrylamide gel which demonstrates the molecular weight shift that results from coupling the peptide to cTnC(C81). The reversibility of the disulfide linkage is demonstrated by preincubation of cTnC(C81)-Pep with the indicated concentrations of DTT.
Biotin was coupled to cTnC by irreversible alkylation of Cys residues at pH 7.5. cTnC(A-Cys), which does not contain Cys residues, was used as a negative control, since maleimide can react with amines at a pH greater than 8.0. Native gels run in the presence or absence of avidin were used to determine the extent of coupling. Fig. 3shows that about 90% of each monocysteine protein was biotinylated, while only a trace amount of cTnC(A-Cys) was biotinylated.
Figure 3: Extent of biotin-maleimide labeling. Biotinylated proteins (10 µg) were incubated in the absence (lanes 1, 3, 5, 7, 9, and 11) or presence (lanes 2, 4, 6, 8, 10, and 12) of 20 µg of avidin prior to loading on a 15% nondenaturing gel. The gel shows biotin adducts of the following proteins: cTnC(A-Cys) (lanes 1 and 2), cTnC(C81) (lanes 3 and 4), cTnC(C84) (lanes 5 and 6), cTnC(C85) (lanes 7 and 8), cTnC(C42) (lanes 9 and 10), and cTnC(C45) (lanes 11 and 12.
Figure 4:
Myofibril ATPase activities of mutant
proteins. Extracted cardiac (A) and fast skeletal (B)
myofibrils were reconstituted with the indicated monocysteine proteins
at a final concentration of 10 µg/ml. For convenience, the prefix
cTnC has been omitted. Activity is expressed as the percent maximal
ATPase activity in the presence of Ca for myofibrils
that have been reconstituted with recombinant cTnC3. These maximal
activities were approximately 20 nmol of P
/min/mg of
cardiac myofibril protein and 120 nmol of P
/min/mg of fast
skeletal myofibril protein.
Table 1compares the activities of the cross-linked proteins
relative to the corresponding unmodified proteins. Covalent coupling of
the peptide to Cys-81 resulted in 70-80% inhibition of cTnC
activity in cardiac and skeletal myofibrils, respectively. The residual
activity was not due to a significant amount of unmodified protein,
since greater than 95% of cTnC(C81) was coupled to the peptide. The
decreased Ca-dependent activity of cTnC(C81)-Pep
results from a 10% increase in Ca
-independent
activity and a 60-70% decrease in total activity. Although
residues 45, 84, and 85 are all predicted to form the N-terminal
hydrophobic surface, coupling the peptide to these residues had
resulted in little or no decrease in activity. Coupling the peptide to
residue 42 resulted in a slight increase in activity.
Preliminary experiments showed that binding avidin to cTnC(C42)-biotin had no effect on function, while avidin further decreased the activity of cTnC(C81)-biotin (data not shown). Additional experiments using avidin were not pursued, since labeling with biotin was not quantitative (see Fig. 3) and since the large size of avidin (60,000 Da) increases the possibility that observed functional consequences are due to disruption of interactive sites that are distal to the site of attachment.
Figure 5:
Effect of increasing concentrations of
TnC(C81)-Pep on myofibril ATPase activity. TnC-extracted cardiac or
fast skeletal myofibrils were reconstituted with the indicated
concentrations of cTnC(C81)-Pep which had been preincubated with
(+) or without(-) DTT. Ca-dependent
activity is defined as the difference in specific activity at pCa 4.0 and pCa 8.0. The activity is expressed as the
percent of the maximal Ca
-dependent activity observed
using unmodified cTnC(C81).
Conversion of nonpolar residues to polar residues in
the N-terminal hydrophobic region of sTnC was shown to increase the
Ca affinity of the low affinity sites in the isolated
sTnC (33) and to increase the pCa50 of skinned fiber
contraction(34) . Therefore, it was of interest to determine if
covalent coupling of the peptide to cTnC(C81) affected the
Ca
sensitivity of myofibril ATPase activity. Fast
skeletal myofibrils were used for these experiments, since the specific
activity of these preparations is about 10-fold greater than cardiac
myofibrils. Fig. 6shows that coupling the peptide to cTnC(C81)
does not significantly alter the Ca
sensitivity of
myofibril ATPase activity.
Figure 6:
Ca sensitivity of
myofibrils reconstituted with cTnC(C81) or cTnC(C81)-Pep. ATPase
activity was measured in the presence of increasing concentrations of
free Ca
. The data are expressed as percent of the
maximal Ca
-dependent activity for each protein. The
maximal activity of cTnC(C81)-Pep was about 5-fold lower that for
cTnC(C81). The data were fitted by least squares minimization to the
following form of the Hill equation, A/A
= 1/(1 + 10
(logCa
-
logCa)), where A is Ca
-dependent activity at
a given concentration of Ca
, A
is the maximal activity, n is the Hill coefficient and
Ca
is the Ca
concentration at which the
activity is half-maximal. Ca
-dependent activity is
defined as the difference in specific activity at the indicated pCa and pCa 8.0. The pCa
values, which is defined as -logCa
, are given
in the figure. The Hill coefficients for cTnC(C81) and cTnC(C81)-Pep
were 2.1 ± 0.1 and 1.9 ± 0.3,
respectively.
Figure 7:
SDS-PAGE of cTnC(C81)-BP cross-linked to
cTnI or cTnT. Free cTnC(C81)-BP (lanes 2-4),
cTnC(C81)-BP-cTnI binary complexes (lanes 5-7), and
cTnC(C81)-BP-cTnI-cTnT ternary complexes (lanes 8-10)
were prepared as described under ``Materials and Methods.''
One aliquot of each preparation was maintained in the dark (lanes
2, 5, and 8), a second aliquot was irradiated with UV
light in the presence of 0.1 mM Ca (lanes 3, 6, and 9), and a third aliquot was
irradiated with UV light in the presence of 2 mM EGTA (lanes 4, 7, and 10). All samples were analyzed by
SDS-PAGE. Lane 1, molecular mass
standard.
Cross-linking of cTnC(C81)-BP to cTnI is
Ca independent (Fig. 7, lanes 6, 7 and 9, 10), and the rates of cross-linking in the
presence or absence of Ca
were indistinguishable
(data not shown). It is difficult to interpret the significance of this
observation with respect to Ca
-dependent and
-independent interactions between cTnC and cTnI. The cross-linker may
not be able to differentiate between subtle but distinct apo and
Ca
-bound conformations, due to its the flexibility
and length (about 9 Å). Also, conformational mobility of the
cTnC-cTnI complex during the time frame of the cross-linking reaction
may prevent identification of distinct Ca
-bound and
apo conformations.
The side chain of Met-45 is predicted to reside on the inner
surface of helix B in the N-terminal hydrophobic region (see Fig. 1), and NMR studies (21, 28) indicate that
the solvent accessibility of Met-45 increases upon binding
Ca. Never the less, covalent modification of residue
45 had little or no functional consequence. We cannot exclude the
possibility that blocking groups attached to cTnC(C45) can rotate about
the covalent bond, perhaps under the influence of cTnI, and assume a
position which does not interfere with transmission of the
Ca
signal. If this occurs, it is likely that the
bound peptide or biotin extends away from Met-81 (into the plane of the
page in the model shown in Fig. 1). In any event, it is clear
that localized structural alteration at residue 45 in the N-terminal
hydrophobic region has little effect on the maximal activity of cTnC.
Covalent modification of cTnC(C81) with either the peptide or biotin
resulted in significant inhibition of activity. Modification of
cTnC(C84) and cTnC(C85) lowered activity relative to the corresponding
unmodified proteins, but inhibition was much less than that observed
for cTnC(C81). These data can be interpreted with respect to the recent
model for the Ca-bound binary complex of fast
skeletal TnC and TnI proposed by Olah and Trewhella(37) . A
primary feature of this model is that sTnI wraps around the central
helix of sTnC and passes through the N- and C-terminal hydrophobic
regions. Extensive interactions between sTnC and sTnI have been
confirmed by a recent cross-linking study(38) . If this model
also applies to cardiac TnC and TnI, then the region of Met-81 and
secondarily Cys-84 and Met-85, which are predicted to be located on the
same face of helix D as Met-81, may represent an interface between
these two troponin subunits. Further experiments would be necessary to
determine if this interaction is Ca
-independent, as
suggested by Fig. 7.
The data presented here have important
implications with respect to the mechanism of action of hydrophobic
anti-CaM drugs, such as calmidazolium, bepridil, and phenothiazines,
which sensitize muscle to Ca and potentiate rather
than inhibit muscle contraction(16, 17, 18) .
MacLachlan et al.(14) and Pollesello et al.(15) reported NMR studies on the association of bepridil
and levosimendan, respectively, with the Ca
-bound
form of cTnC. Both studies concluded that these compounds establish
hydrophobic interactions with residues in the N-terminal hydrophobic
region of cTnC, including Met-81. Our data predict that strong
interaction of a drug with Met-81 would inhibit rather than enhance the
function of cTnC. This apparent inconsistency could be explained by an
incorrect assignment for the methyl proton chemical shift for Met-81 of
2.22 ppm used in the previous studies. We have assigned this chemical
shift to 1.35 ppm based on site-directed mutagenesis and
two-dimensional heteronuclear single and multiple quantum coherence
NMR(21) . Although we feel that it is likely that bepridil and
levosimendan bind to the N-terminal domain of free cTnC, they may make
primary interactions with Met residues other than Met-81.
If one
assumes that the Ca-sensitizing effect of anti-CaM
drugs results from association with the N-terminal hydrophobic region
of thin filament-bound cTnC, then at least three mechanisms that can be
considered. First, the drugs may associate with a critical hydrophobic
cTnI binding site and sensitize cTnC to Ca
, but then
be displaced by cTnI when cTnC binds Ca
. Second, the
N-terminal hydrophobic region of cTnC may be a critical
Ca
-dependent site of interaction with cTnI, yet be
able to simultaneously bind Ca
-sensitizing drugs or
the covalently bound peptide. Third, the N-terminal hydrophobic region
may not be an essential Ca
-dependent binding site for
cTnI, but can bind drugs and sensitize cTnC to Ca
.
Our data are generally consistent with the latter two mechanisms, since
they show that discrete regions on the N-terminal hydrophobic surface
can tolerate covalently bound groups, and presumably a noncovalently
bound drug, without interfering with transmission of the Ca
signal. This is also consistent with the study of Pearlstone et al.(33) which showed that conversion of selected
nonpolar residues to polar residues in the N-terminal hydrophobic
region of sTnC increased the Ca
affinity of low
affinity sites, but did not inhibit regulation. Thus, careful
consideration must be given with respect to the precise role for the
N-terminal hydrophobic region of cTnC in the regulation of cardiac
muscle contraction.