(Received for publication, December 22, 1994; and in revised form, March 27, 1995)
From the
The C-terminal region of smooth muscle caldesmon (CaD) interacts with calmodulin (CaM) and reverses CaD's inhibitory effect on the actomyosin ATPase activity. We have previously shown that the major CaM-binding site (site A) in this region is within the segment from Met-658 to Ser-666 (Zhan, Q., Wong, S. S., and Wang, C.-L. A.(1991) J. Biol. Chem. 266, 21810-21814). Recently, another segment (site B), Asn-675 to Lys-695, was reported to bind CaM (Mezgueldi, M., Derancourt, J., Calas, B., Kassab, R., and Fattoum, A. (1994) J. Biol. Chem. 269, 12824-12832). To assess the functional relevance of these two putative CaM-binding sites, we have examined three synthetic peptides regarding their effects on CaM's ability to reverse CaD-induced inhibition of actomyosin ATPase activity: GS17C (Gly-651 to Ser-667), VG29C (Val-685 to Gly-713), each containing one CaM-binding site, and MG56C (Met-658 to Gly-713), which contains both sites. We found that although VG29C did bind CaM, its affinity was weakened by GS17C, and it failed to compete with CaD for CaM under the conditions where GS17C effectively displaced CaD from CaM. MG56C had an effect similar to that of GS17C. These experiments demonstrated that site A for CaM binding is involved in regulating the inhibitory property of CaD.
Smooth muscle caldesmon (CaD) ()is an actin- and
calmodulin (CaM)-binding protein (for reviews see Marston and
Redwood(1991), Matsumura and Yamashiro(1993), and Sobue and
Sellers(1991)). Upon binding to F-actin, CaD inhibits actomyosin ATPase
activity in vitro (Marston and Lehman, 1985; Ngai and Walsh,
1984). This inhibitory effect, which is potentiated by tropomyosin
(Horiuchi et al., 1986; Sobue et al., 1985), is
reversed by Ca
/CaM (Horiuchi et al., 1986;
Smith and Marston, 1985); the reversal of inhibition may also be
achieved by other mechanisms such as phosphorylation of CaD (Adam and
Hathaway, 1993; Ngai and Walsh, 1987; Yamashiro et al., 1990).
Therefore, it has been thought that CaD plays a regulatory role in the
smooth muscle contraction. This additional thin filament-based
regulation may further modulate the well established thick
filament-based regulation, which depends on myosin light chain
phosphorylation (Adelstein and Eisenberg, 1980).
CaD is a rather
elongated molecule (Mabuchi and Wang, 1991; Fürst et al., 1986); its more than 750 amino acid residues (Bryan et al., 1989; Hayashi et al., 1989) span 74 nm in
length (Graceffa et al., 1988). The compact N- and C-terminal
regions are separated by a single helical segment of about 150 residues
(Wang et al., 1991a). Domain mapping studies reveal that both
the major actin- and CaM-binding sites are localized close to the C
terminus of the CaD molecule (Marston and Redwood, 1991); the
N-terminal region binds myosin (Hemric and Chalovich, 1988; Ikebe and
Reardon, 1988) and also appears to interact with actin (Mabuchi et
al., 1993) and CaM (Wang, 1988). C-terminal fragments of a wide
range of sizes are almost equally effective in modulation of the in
vitro actomyosin interaction; these fragments include: ()37K, from Asp-451 to the C-terminal Pro-756 (Fujii et
al., 1987; Szpacenko and Dabrowska, 1986); 27K, from Lys-579 to
Pro-756 (Wang et al., 1991b); 25K, from Cys-580 to Pro-756
(Riseman et al., 1989); 20K, from Leu-597 to Pro-756 (Velaz et al., 1990); 10K, from Trp-659 to Pro-756 (Bartegi et
al., 1990); and 7.3K, from Leu-597 to Phe-665 (Chalovich et
al., 1992), all of which share a common sequence from Trp-659 to
Phe-665.
We have previously synthesized a peptide, GS17C (Gly-651 to
Ser-667), which, like CaD, binds both CaM and actin but does not
inhibit actomyosin ATPase activity (Zhan et al., 1991).
Interestingly, when perfused into a hyperpermeabilized smooth muscle
cell, GS17C induces contraction at low Ca concentrations, such contraction being attenuated by an
increasing amount of Ca
and by pretreatment with CaM
(Katsuyama et al., 1992). The GS17C-induced activation of
muscle contraction is most likely a result of direct competition for
actin, rather than for CaM, between endogenous CaD and the added
peptide. This in turn, strongly supports the idea that in vivo CaD plays an inhibitory role in the regulation of smooth muscle
contraction, and CaM apparently neutralizes such inhibition by
interacting with CaD at the GS17C sequence.
More recently, Marston et al.(1994) suggested that there is another CaM-binding site
(referred to as ``site B'') adjacent to the GS17C sequence
(site A) and that the two sites do not compete with each other for CaM.
The fact that a synthetic peptide, NK21 (from Asn-675 to Lys-695, see Fig. 1), also binds CaM in a Ca-dependent
manner (Mezgueldi et al., 1994) supports this view.
Furthermore, Marston et al.(1994) found that although a site
A-containing peptide (M73, from Ser-657 to Gly-670) did bind CaM, it
could not compete with CaD for CaM and did not restore the inhibition
of actomyosin ATPase activity, whereas a site B-containing fragment H2
(from Thr-626 to Leu-710) did. From these results, they concluded that
it is site B, not site A, that is functionally relevant for CaD's
action (Marston et al., 1994). However, since the result of
M73 is a negative one and H2 contains both site A and site B, the
interpretation is complicated by the possibility that the M73 peptide
may be too small to show any effect, or both sites are needed to
achieve re-inhibition. It would be of interest to find out whether a
site B peptide can do the same as H2, i.e. to compete with CaD
for CaM. It is the aim of this report to address this issue. We used
two newly synthesized peptides, VG29C and MG56C, plus the earlier one,
GS17C, containing the minimum sequence of site B alone, of both site A
and site B, and of site A alone, respectively. With these synthetic
peptides, we hoped to determine which site is more relevant to the
regulatory function of CaD. Our results indicate that site A clearly
plays a more important role.
Figure 1: The position of the two CaM-binding sites and other related peptides and fragments of the C-terminal region of CaD. Amino acid sequence and numbering of residues are according to chicken gizzard CaD (Bryan et al., 1989). The peptides/fragments indicated are: GS17C, VG29C, and MG56C (this work); M73, H2, and H9 (Marston et al., 1994); and NK21 (Mezgueldi et al., 1994).
Figure 2:
Fluorescence titration of synthetic
peptides with CaM. Aliquots of CaM (300 µM) were added to
solutions containing 3 µM GS17C (closedcircles), VG29C (closedsquares), and
MG56C (opencircles) in the presence of 1 mM CaCl
; the tryptophan fluorescence was monitored with
= 295 nm and
= 320
nm. Other conditions include 50 mM KCl, 1 mM
CaCl
, and 20 mM HEPES, pH 7.5. The data were
fitted to a binding equation (see ``Materials and Methods'').
The fluorescence intensity of each peptide in the absence of CaM
(F
) was taken as 1.0.
It is noteworthy that although MG56C contains the sequences of both site A and site B, it binds only one CaM molecule, and the affinity is about the same as that of GS17C. If the two sites behave independently, one would expect either a higher stoichiometry (i.e.n = 2) or a stronger binding owing to the additivity of binding energy. One may argue that since site A is at the very end of the N terminus of MG56C, it may not fold properly without the flanking sequence, thus lowering the affinity for CaM as compared with GS17C. This, however, seems unlikely, because M73, which begins at the same residue as MG56C, does not exhibit a lowered affinity for CaM (Marston et al., 1994) (see Fig. 1). A more plausible explanation is that, since MG56C contains site B in addition to site A, both sites tend to interact with the same CaM molecule, resulting in some constraints in the peptide structure that decrease the binding affinity. An extreme case is that binding of one of the sites (e.g. site A, see below) to CaM alters the conformation of CaM or the region around the other site (i.e. site B) so much that the second site binds CaM much more weakly or not at all.
Figure 3:
Competition between GS17C-NBD and other
synthetic peptides for CaM binding. Increasing amounts of peptides
(500 µM) or CaD (20 µM) were added to a
solution containing 1.43 µM CaM and 1.2 µM
GS17C-NBD in 1 mM CaCl
. GS17C, opencircles; VG29C, opensquares; MG56C, closedcircles; CaD, closedtriangles. NBD fluorescence was monitored with
= 490 nm and
= 540
nm. Other conditions are the same as in Fig. 2. Smooth curves
were drawn through the data points to show the trend. The original,
CaM-induced fluorescence enhancement (
F) of GS17C-NBD was taken as
100.
Both MG56C and GS17C (unlabeled) produced similar decreases in the NBD fluorescence upon displacement of the bound GS17C-NBD from CaM, consistent with their similar binding constants for CaM. Intact CaD displaced GS17C-NBD more readily than the unlabeled GS17C. VG29C, on the other hand, was a rather poor competitor of GS17C-NBD, e.g. addition of 5 mol of VG29C per mol of GS17C-NBD caused only 20% decrease of the fluorescence intensity, a change brought about by unlabeled GS17C or MG56C at a molar ratio of 0.5. The observed inefficient displacement by VG29C is qualitatively consistent with the relative affinities of the peptides, although the extent of displacement caused by VG29C is somewhat lower than what one would predict on the basis of the binding constants (Table 1). An alternative explanation is that VG29C does not bind CaM at the same site as does GS17C; the observed fluorescence decrease is in fact due to a different environment experienced by the probe in the complex formed by GS17C-NBD, VG29C, and CaM. Although the actual existence of such a ternary complex requires independent proof, this possibility could not be ruled out at the present time; if it did exist, the poor competition of VG29C would suggest its binding to CaM is weakened by GS17C, which in turn, would imply that binding of the two peptides to CaM is not completely independent. To gain further insight, the reverse experiment was carried out.
In this case CaM
was first mixed with NBD-labeled VG29C in the presence of
Ca, and a second peptide was added. We found that
both unlabeled VG29C and MG56C caused a decrease in fluorescence,
consistent with VG29C-NBD being displaced from CaM. The concentration
dependence of the fluorescence change was also roughly consistent with
the affinities of the two peptides for CaM. Interestingly, GS17C was
slightly more effective than MG56C and significantly more effective
than the unlabeled VG29C in decreasing the fluorescence (Fig. 4), as if GS17C and VG29C were competing for the same site
on CaM. These results, however, can also be explained by the assumption
that VG29C and GS17C bind at two different sites on CaM and that
binding of GS17C weakens the interaction between CaM and VG29C.
Figure 4:
Competition between VG29C-NBD and other
synthetic peptides for CaM binding. A solution containing 1.0
µM CaM and 3.5 µM VG29C-NBD in 1 mM CaCl was titrated with stock solutions (
500
µM for peptides) of GS17C (closedcircles), VG29C (opensquares), or
MG56C (opencircles), and the NBD fluorescence was
monitored.
= 490 nm;
= 540 nm. Other conditions are the same as in Fig. 2. Curves were drawn only to show the
trend.
Addition of GS17C, which contains site A and binds CaM (Zhan et al., 1991), resulted in a concentration-dependent reinhibition of the ATPase activity. At a concentration of 60-80 µM, GS17C restored most of the inhibitory effect of the originally added 2 µM CaD (Fig. 5). Since GS17C alone does not cause any inhibition (Zhan et al., 1991), the observed reversal of CaM-induced deinhibition must be due to the interaction between GS17C and CaM. Thus, GS17C is able to compete with CaD for CaM, allowing CaD to interact with F-actin and to inhibit the actomyosin ATPase activity. It should be pointed out that direct competition between GS17C and intact CaD for CaM has been previously demonstrated by using fluorescently labeled CaD (Zhan et al., 1991). This implies that site A of CaD is involved in the CaM binding that is responsible for the observed reversal of inhibition.
Figure 5: Effect of various synthetic peptides on the CaM-reactivated actomyosin ATPase activity. Aliquots of GS17C (closedsquares), VG29C (opensquares), MG56C (closedcircles), or an equimolar mixture of GS17C and VG29C (opencircles) were added to a solution containing phosphorylated gizzard myosin, rabbit skeletal actin, gizzard tropomyosin, CaD, and CaM, and the release of phosphate was measured (see ``Materials and Methods'' for experimental details). The lines were drawn through each set of data points based on least squares fits. The uninhibited actomyosin ATPase activity (100%) corresponds to 990 nmol/mg/min.
In contrast to GS17C, VG29C, which contains site B and which by itself does not inhibit the actomyosin interaction (data not shown), did not cause any significant change in the deinhibited ATPase activity. Since VG29C failed to compete with GS17C for CaM binding (Fig. 3), despite its ability to bind CaM (Table 1), the lack of an effect on the ATPase activity is most likely to be because VG29C could not interact with CaM effectively when CaM is associated with CaD. When a mixture of GS17C and VG29C (at a 1:1 ratio) was used as a control, the same effect was observed as for GS17C alone.
When MG56C was added to the reaction mixture, there was also a clear reinhibition of the ATPase activity. Since MG56C itself, too, has no effect on the actomyosin interaction, the observed inhibition was interpreted by the same mechanism as in the case of GS17C. The potency of this longer peptide, however, was somewhat lower than that of GS17C; 60 µM of MG56C only resulted in an inhibition that was attained by 40 µM of GS17C. Although MG56C contains both site A and site B, it again did not compete more effectively than GS17C with CaD for CaM. This is consistent with the results of the binding studies and the competition experiments (see above).
The apparent discrepancy between this work and the work of Marston et al.(1994) may be reconciled as follows. Although both GS17C and M73 contain only site A and both VG29C and H9 contain only site B, the peptides in each pair are not identical. In each case, different portions of the CaD sequence in addition to the CaM-binding elements are included. The fact that the experimental results obtained with the two sets of peptides (GS17C and VG29C versus M73 and H9) lead to opposite conclusions strongly suggests that binding of sites A and B to CaM is affected not only by each other but also by other parts of the CaD molecule. Thus, residues that have not been shown to be directly involved in the interaction with CaM may in fact have a profound effect on the CaM-binding characteristics of either site A or site B and the specificity of such interactions. This interpretation is consistent with our recent observation that intact CaD and its 22-kDa C-terminal fragment, but not the shorter peptides, induce an extended configuration in CaM (Mabuchi et al., 1995).
The detailed molecular structure of CaD, especially the C-terminal region that contains most of the functional properties, has remained unclear. Electron microscopic images suggest that this part of the molecule is relatively compact and floppy (Mabuchi and Wang, 1991). A compact C-terminal region was also implicated by NMR studies on gizzard CaD and its proteolytic fragments (Levine et al., 1990). In view of the fact that two peptide segments (Leu-597 to Val-629 and Arg-711 to Pro-756), separated by more than 80 amino acid residues in the sequence, can both interact with actin (Wang et al., 1991b), while a third locus (Cys-580) still farther away can also cross-link to actin (Graceffa and Jancsó, 1991; Wang, 1988), the folding of the peptide backbone must be such that all three segments can be brought into proximity to the bound actin. In this compact structure, site A and site B must also be close to each other. It is therefore plausible that binding of one site (e.g. site A) to CaM would trigger a series of conformational adjustments so that the three-dimensional structure of other portions of the molecule (including those around the actin-binding sites and those around site B) is also affected. This may lead to the weakening of actin binding affinity, and ultimately the reversal of CaD's inhibitory action on the actomyosin interactions.