(Received for publication, October 1, 1996, and in revised form, January 22, 1997)
From the Muscle Research Group, Boston Biomedical
Research Institute, Boston, Massachusetts 02114, the ¶ Department
of Neurology, Harvard Medical School, Boston, Massachusetts 02115, and
the
Department of Biochemistry, Tufts University School of
Medicine, Boston, Massachusetts 02111
Calponin is a 33-kDa smooth muscle-specific
protein that has been suggested to play a role in muscle contractility.
It has previously been shown to interact with actin, tropomyosin, and calmodulin. More recently we showed that calponin also interacts with
myosin (Szymanski, P. T., and Tao, T. (1993) FEBS Lett.
331, 256-259). In the present study we used a combination of
co-sedimentation and fluorescence assays to localize the regions in
myosin and calponin that are involved in the interaction between these
two proteins. We found that recombinant chicken gizzard -calponin co-sediments with myosin rod and, to a lesser extent, with light meromyosin. Fluorescently labeled recombinant calponin shows
interaction with heavy meromyosin and myosin subfragment 2 but not
subfragment 1. A fragment comprising residues 7-182 and a synthetic
peptide spanning residues 146-176 of calponin co-sediment with myosin, but fragments comprising residues 7-144 and 183-292 do not. Our results indicate that there are calponin binding sites in the subfragment 2 and light meromyosin regions of myosin, and that the
region comprising residues 145-182 of calponin mediates its interaction with myosin.
Calponin (CaP)1 is a 33-kDa smooth muscle-specific, thin filament-associated protein that has been suggested to play a role in muscle contractility (2, 3). It is capable of binding to actin (2, 4-6), tropomyosin (7-10), and calmodulin (4, 6, 11). The binding sites for these proteins are all located in the NH2-terminal portion of CaP (residues 7-182) (12).
CaP inhibits the actin-activated myosin ATPase in solution (3, 13), unloaded shortening velocity, and, to a lesser extent, isometric force in permeabilized smooth muscle fibers (14-17). It is commonly assumed that these effects are exerted via CaP binding to actin and competition of CaP with myosin heads for binding to actin. On the other hand, observations that CaP inhibits actin translocation over myosin heads (18, 19) and enhances the affinity between actin and myosin in in vitro motility assays (19) suggest that CaP interacts simultaneously with both actin and myosin. Previous studies showed that CaP indeed binds to isolated smooth muscle myosin (1, 20). This interaction was found to be reversible upon the addition of Ca2+-calmodulin (1) and partially abolished upon phosphorylation of CaP by protein kinase C.2
In this study we further investigated the interaction between CaP and
myosin. Using a combination of co-sedimentation and fluorescence
assays, we localized the regions in these two proteins that are
involved in their interaction. Fragments of chicken gizzard myosin and
recombinant chicken gizzard -CaP (R
CaP) were generated by
proteolytic digestion. This allowed us to isolate the major functional
portions of these two proteins, viz. S1, S2, rod, HMM, and
LMM of myosin and the NH2-terminal fragment (residues
7-182), the NH2-terminal fragment without the central
portion (residues 7-144), and the COOH-terminal fragment (residues
183-292) of CaP.
Our data show that there are CaP binding sites in the S2 and LMM regions of myosin and that the region in CaP that contains a so-called actin-binding domain (residues 145-176) (4) is primarily involved in the interaction of CaP with myosin.
-Chymotrypsin, papain, and other commonly used
reagents were from Sigma. Precasted polyacrylamide gradient (4-20%)
Tris-glycine gels were from Novex. The rapid-Ag-stain kit was from ICN,
and other materials for gel electrophoresis were from Bio-Rad.
Chicken gizzard myosin was prepared according to
Ikebe et al. (21). Fragmentation of myosin into rod and S1
by digestion with papain and into LMM and HMM by digestion with
-chymotrypsin was performed as described (22). S2 was generated from
HMM by
-chymotrypsin cleavage (23).
Expression and purification of RCaP was as described previously
(24). Digestion of R
CaP with
-chymotrypsin using a protease to
substrate weight ratios of 1:1000 and 1:100 were performed according to
Mezgueldi et al. (4). Labeling of R
CaP with
N-iodoacetyl-N
-(5-sulfo-1-naphthyl)ethylenediamine (Aldrich) was carried out by incubating R
CaP (40 µM)
with a 3-fold molar excess of
N-iodoacetyl-N
-(5-sulfo-1-naphthyl)ethylenediamine in 20 mM Hepes, 0.1 M NaCl, pH 7.5, for 3 h at room temperature. Dithiothreitol (1 mM) was added to
quench the reaction, followed by dialysis to remove excess
reagents.
The polypeptide EKQQRRFQPEKLREGRNIIGLQMGTNKFAC corresponding to residues 146-176 of CaP and a COOH-terminal Cys was synthesized on an ABI protein synthesizer and purified by high pressure liquid chromatography using conventional methods.
Protein concentrations were determined spectrophotometrically at 280 nm, using A (1%, 1 cm) values of 7.4 for RCaP, 4.5 for smooth muscle myosin, 2.2 for rod, 3.0 for LMM, 6.5 for HMM, 7.0 for
S2, and 7.7 for S1; an extinction coefficient of 5600 M
1 cm
1 was used for the
synthetic polypeptide.
For sedimentation binding assays RCaP or
its fragments were incubated with myosin or its insoluble fragments
(rod or LMM) in 20 mM Hepes, 50 mM NaCl, 2 mM NaN3, 1 mM dithiothreitol, pH 7.5, for 20 min at 4 °C and then centrifuged at 80,000 rpm for 20 min at 4 °C in a Beckman Instruments TL100 ultracentrifuge using the
TL100.2 rotor. Reaction mixtures before centrifugation and pellets
solubilized with Laemmli buffer (25) were subjected to gradient
(4-20%) SDS-PAGE. The amounts of materials were quantified by
densitometry of Coomassie Blue- or silver-stained gels using a
laboratory-built image-analysis system as described earlier (1).
Standard curve for R
CaP was constructed to establish the linear
concentration range.
For binding assays in solution, DAN-RCaP (1 µM) was
incubated with increasing concentrations (0-5 µM) of the
soluble myosin fragments (HMM, S1, and S2) in 20 mM Hepes,
50 mM NaCl, 2 mM NaN3, 1 mM dithiothreitol, pH 7.5, for 20 min at 4 °C.
Fluorescence-intensity measurements were carried out on an I.S.S. K2
fluorometer (Champaign, IL) using wavelengths of 377 and 480 nm for
excitation and emission, respectively.
Student's t test was used for statistical analysis, and a confidence level of p < 0.05 was chosen as an indication of a statistically significant difference.
Addition of increasing concentrations
of HMM to a solution containing DAN-RCaP produced a
concentration-dependent and saturable increase of the
label's fluorescence intensity (Fig. 1). In contrast, S1 did not cause any change in the fluorescence of DAN-R
CaP. These
data indicate that CaP interacts with HMM but not with S1 and
indirectly implicate the S2 region of myosin as a CaP binding site. The
fluorescence titration curve of S2 is very similar to that of HMM (Fig.
1). When the data were fitted by a nonlinear regression method (26), we
obtained apparent binding constants of 4.0 ± 0.3 × 106 M
1 (n = 3)
and 4.7 ± 0.4 × 106
M
1 (n = 3) (all uncertainty
values are S.E.; n refers to the number of determinations)
for HMM and S2, respectively. Thus, isolated S2 binds DAN-R
CaP with
virtually the same affinity as HMM, providing more direct evidence that
the S2 region of myosin contains a CaP binding site.
Since it is possible for S1 to bind DAN-RCaP without affecting the
label's fluorescence, steady-state polarization and anisotropy decay
measurements were also carried out; neither showed any changes with S1
(data not shown). Also, the binding of DAN-R
CaP to myosin was found
to be the same as that of unlabeled R
CaP using the co-sedimentation
assay (see below; data not shown), indicating that the label does not
affect the capacity of R
CaP to interact with myosin.
Addition of increasing concentrations of
myosin, myosin rod, and LMM to a solution of RCaP followed by high
speed centrifugation produced a concentration-dependent
increase in sedimentation of R
CaP that approaches saturation at high
concentrations of added proteins (Fig. 2). As was found
previously for myosin (1), some amounts of R
CaP remain in the
supernatant even at the highest concentrations of the proteins used,
and about 10-20% of R
CaP sedimented in the absence of added
proteins. Nonlinear regression analysis of the data in Fig. 2 yielded
apparent binding constants of 2.6 ± 0.3 × 106
M
1 (n = 6), 1.0 ± 0.3 × 106 M
1
(n = 7), and 1.5 ± 0.5 × 106
M
1 (n = 5) for LMM, intact
myosin, and rod, respectively. These binding constants are not
statistically different (p < 0.05) from each other. We
noted, however, that the fraction of R
CaP bound at high
concentrations of added proteins is significantly lower for LMM
compared with intact myosin and rod. The analysis procedure yielded
maximal fraction bound values of 0.3, 0.7, and 0.5 for LMM, myosin, and
rod, respectively. We stress that our experimental conditions were such
that the binding curves have not reached saturation, so that the
fraction of R
CaP bound at saturation could only be determined
approximately.
The co-sedimentation results described above show that the CaP binding
function of myosin resides entirely in the rod segment, a conclusion
that is consistent with our finding that S1 does not bind CaP. One of
the CaP binding sites in the rod must be in the S2 region, as we
concluded from the fluorescence titration results. Furthermore, we
found that LMM, which lacks the S2 segment, also binds CaP. Thus, there
must be another CaP binding site in the LMM portion of the myosin rod.
Our conclusion that there are two CaP binding sites in myosin or myosin
rod and only one in LMM is consistent with our finding that the maximal
amount of bound RCaP for LMM is roughly half the amount for myosin
or rod.
As reported previously
for gizzard CaP (4), limited digestion of RCaP with
-chymotrypsin
at a low protease to substrate weight ratio of 1:1000 for 17 min at
25 °C produced two major fragments (Fig.
3A, lane 4). Their molecular
masses are 21.5 and 13.7 kDa based on electrophoretic mobilities. These
two fragments remain stable up to about 20 min of digestion, and then
further cleavage takes place (Fig. 3A, lane 5).
The 21.5-kDa fragment represents the NH2-terminal portion
of R
CaP spanning the region between Asn7 and
Tyr182, and the 13.7-kDa fragment spans the region between
Gly183 and the COOH terminus of intact R
CaP (4).
When the limited -chymotryptic R
CaP digest was centrifuged
together with an equimolar concentration of myosin, 76.6 ± 3.2% (n = 6) and 12.2 ± 1.0% (n = 6)
of the 21.5- and 13.7-kDa fragments, respectively, were sedimented
(Fig. 3B, lanes 10-12). When the same R
CaP
digest was centrifuged without myosin, 17.2 ± 0.9% (n = 6) and 11.3 ± 1.8% (n = 6)
of 21.5- and 13.7-kDa fragments, respectively, were found in the
pellets (Fig. 3B, lanes 7-9). Thus, although a
significant amount (~55%) of the 21.5-kDa fragment sedimented via
binding to myosin, virtually none of the 13.7-kDa fragment did. These
data indicate that the myosin binding function of CaP resides within
residues 7-182 and not residues 183-292. It is interesting to note
that actin (4, 5), tropomyosin (4, 5, 8, 9), calmodulin (5, 6, 11), and
caltropin (27) binding sites are located in the same
NH2-terminal portion of CaP.
As for native CaP (4), digestion of RCaP with
-chymotrypsin at a
high protease to substrate weight ratio (1:100) produced further
cleavage (Fig. 4A). After digestion for 30 min at 25 °C, the 13.7-kDa COOH-terminal fragment was completely
digested. The 21.5-kDa NH2-terminal fragment (residues
7-182) was cleaved between Tyr144 and Ala145
into a 15.5-kDa fragment that spans residues 7-144 and smaller segments.
When this extensive chymotryptic digest of RCaP was centrifuged with
an equimolar concentration of myosin, 15.4 ± 1.4%
(n = 6) of the 15.5-kDa fragment was sedimented
compared with the total unsedimented material (Fig. 4B,
lanes 12-14). When the same digest was centrifuged without
myosin, 13.5 ± 1.0% (n = 6) of the 15.5-kDa
R
CaP fragment was sedimented (Fig. 4B, lanes
9-11). These data clearly indicate that the R
CaP fragment that
spans residues 7-144 does not bind myosin. Since the 21.5-kDa
NH2-terminal fragment (residues 7-182) does bind
myosin, our data suggest that the myosin binding site in CaP is located
within residues 145-182.
When a synthetic peptide that contains residues 146-176 of CaP was
centrifuged with and without myosin, 51.0 ± 2.2%
(n = 6) and 14.7 ± 0.9% (n = 5),
respectively, of the peptide was sedimented (Fig.
5A). These data show more directly that the
central region of CaP spanning residues 145-182 is responsible for
binding to myosin.
To ascertain whether the binding of our synthetic CaP peptide to myosin
is specific, we centrifuged the preformed complex between myosin and
the peptide (each at 3 µM) in the presence of increasing
concentrations of RCaP. As shown in Fig. 5B (lanes 8-12), addition of R
CaP resulted in a concentration-dependent displacement of the synthetic peptide from myosin. Specifically, the
amount of peptide that sedimented with myosin decreased from 48.3 ± 2.1% (n = 3) in the absence of R
CaP to 29.7 ± 2.0% (n = 3) and 21.8 ± 1.5%
(n = 3) in the presence of 1.5 and 3.0 µM R
CaP, respectively. Our data show that the peptide competes with R
CaP for binding to myosin, indicating that the interaction between the peptide and myosin is specific.
Our results show that there are CaP binding regions in the S2 and LMM portions of myosin and that the CaP segment comprising residues 146-182 constitutes a myosin binding region. We noted that caldesmon (28-31) and telokin (32), two proteins that have been shown to regulate smooth muscle contractility (33) and myosin assembly (34), also bind to myosin in the S2 region. More interestingly, the same stretch of residues (145-182) in CaP has previously been shown to be a region of interaction with actin (4, 5). Thus, myosin and actin share (or partially share) a common binding region in CaP, so it is not likely that CaP functions as a linker between myosin and actin.
The physiological significance of the interaction between CaP and myosin is not clear at this point. It was recently reported that CaP is primarily localized in the cytoskeletal regions of chicken gizzard cells (35). However, it was recognized that some of the CaP are found near myosin filaments, suggesting that CaP may serve as a link between cytoskeletal extensions and contractile regions (35). We may speculate that a physiological function of CaP might be to link a component of the cytoskeleton (e.g. desmin intermediate filaments) with myosin in the contractile region. Under such a circumstance the local concentrations of both CaP and myosin are likely to be quite high, so that the interaction might be able to occur in situ even though it is relatively weak at physiological NaCl concentrations in vitro (1). Clearly, much additional work will be required before the true physiological role of CaP can be understood.
We thank Dr. John Gergely for critically
reviewing the manuscript and Bing Li for the preparation of RCaP and
synthetic peptide.