©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of the Functionally Relevant Calmodulin Binding Site in Smooth Muscle Caldesmon (*)

(Received for publication, December 22, 1994; and in revised form, March 27, 1995)

Shaobin Zhuang Enzhong Wang C.-L. Albert Wang (§)

From the Muscle Research Group, Boston Biomedical Research Institute, Boston, Massachusetts 02114

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

Smooth muscle caldesmon (CaD) (^1)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: (^2)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).




MATERIALS AND METHODS

Synthetic Peptides

Three synthetic peptides were used in this study: GS17C (from Gly-651 to Ser-667 (GVRNIKSMWEKGNVFSS-C)), VG29C (from Val-685 to Gly-713 (VSSRINEWLTKTPEGNKSPAPKPSDLRPG-C)), and MG56C (from Met-658 to Gly-713 (GVRNIKSMWEKGNVFSSPGGTGTPNKETAGLKVGVSSRINEWLTKTPEGNKSPAPKPSDLRPG-C)); each contains a Cys residue at the C terminus in addition to the CaD sequence. GS17C contains site A (singly underlined, Met-658 to Ser-666, see Zhan et al.(1991)), VG29C encompasses site B for CaM (doubly underlined, Ser-687 to Lys-695) on the basis of the reports by Marston et al.(1994) and Mezgueldi et al. (1994), and MG56C contains both site A and site B. All peptides were synthesized on an automated ABI peptide synthesizer (model 431A) using F (N-(9-fluorenyl)methoxycarbonyl) chemistry. After synthesis, the product (in amide form) was cleaved from the resin according to the manufacturer's instructions, followed by reversed phase high performance liquid chromatography purification on a C-8 column (Phenomenex, Torrance, CA). The purified peptides were dialyzed against water using low molecular weight (1,000) cut-off dialysis tubings (Spectrum, Houston, TX) before lyophilization. Both amino acid composition and N-terminal sequence analyses were carried out to ascertain the correctness of the peptide synthesis. Concentrations of the peptides were determined spectroscopically using extinction coefficients of 5,600 cmM for GS17C and VG29C (each containing a single tryptophan residue) and 11,200 cmM for MG56C (containing 2 Trp residues). For labeling, the cysteine-containing peptides were first reduced with dithiothreitol (DTT), dialyzed, and reacted with the labeling reagent (iodoacetamido-4-nitrobenz-2-oxa-1,3-diazole; from Molecular Probes) for 5 h at room temperature; the reaction was terminated by addition of excess DTT, followed by exhaustive dialysis. A molar extinction coefficient of 25,000 cmM was used for the nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) moiety (Rosenfeld and Taylor, 1985).

Proteins

CaD was isolated from chicken gizzard as previously described (Bretscher, 1984; Wang, 1988). Skeletal actin was prepared from rabbit skeletal muscle (Spudich and Watt, 1971). Smooth muscle myosin (Ikebe et al., 1978), MLCK (Walsh et al., 1983), and tropomyosin (Graceffa, 1987) were all purified from chicken gizzard. Recombinant chicken brain CaM was purified from Escherichia coli cells by phenyl-Sepharose column chromatography (Dedman and Kaetzel, 1983). Purified smooth muscle myosin was phosphorylated by MLCK as follows. To 3 ml of gizzard myosin (7 mg/ml), 38 ml of cold buffer A (10 mM MgCl(2), 0.1 mM CaCl(2), 0.2 mM DTT, 20 mM MOPS, pH 6.8) was added, followed by centrifugation for 15 min. The pellet was homogenized in 2 ml of buffer A. MLCK (100 µl, 50 µg/ml), CaM (30 µl, 20 µg/ml), and ATP (20 µl, 200 mM) were added to the homogenate; after 40 min of incubation at 25 °C, the mixture was diluted with 5 ml of buffer A and centrifuged again. The pellet was washed twice with 10 ml of a solution containing 35 mM NaCl, 10 mM MgCl(2), 0.2 mM DTT, and 1 mM NaHCO(3) and dissolved in 2 ml of a solution containing 0.5 M NaCl, 10 mM EDTA, 0.2 mM DTT, and 1 mM NaHCO(3). After the addition of 20 ml of a solution containing 10 mM MgCl(2), 0.2 mM DTT, and 1 mM NaHCO(3), the sample was centrifuged, and the final pellet was dissolved in a minimal volume of a solution containing 0.5 M NaCl, 0.1 mM EDTA, 0.2 mM DTT, 1 mM NaHCO(3), and 10 mM MOPS, pH 7.0.

ATPase Assay

All of the Mg-ATPase activities of the actomyosin complex were measured in a solution containing the following ingredients: 60 mM NaCl, 20 mM Tris-HCl (pH 7.5), 0.4 mM DTT, 2 mM ATP, 4 mM MgCl(2), phosphorylated chicken gizzard myosin (1.0 mg/ml), rabbit skeletal F-actin (0.5 mg/ml), chicken gizzard tropomyosin (0.13 mg/ml), and 0.2 mM CaCl(2); 2 µM gizzard CaD was used to achieve 60% inhibition, and 10 µM CaM was added to restore the activity. Total reaction mixture was 250 µl. The mixture without ATP was first vortexed, followed by addition of ATP to start the reaction. After incubation for 40 min at 25 °C, the reaction was terminated by adding an equal volume of 10% trichloroacetic acid. The mixture was then centrifuged, and the supernatant was taken for phosphate determination (Fiske and Subbarow, 1925). To a sample of 350 µl were added 125 µl of ammonium molybdate solution, 725 µl of H(2)O, and 50 µl of 1-amino-2-naphthol-4-sulfonic acid (Kodak; 0.25% in 15% NaHSO(3)); after incubation at room temperature for 15 min, the absorbance was read at 750 nm. Mixtures without sample were used as blanks, and standardized phosphate solutions were used for calibration.

Fluorescence Titrations

Interactions between CaM and synthetic peptides were studied by adding aliquots of the CaM stock solution (typically 300 µM) to 0.6 ml of a peptide-containing solution (50 mM KCl, 1 mM CaCl(2), 20 mM HEPES, pH 7.5) and monitoring the tryptophan emission ( = 295 nm; = 320 nm) of the peptide. Since each peptide contains at least one tryptophan residue, whereas CaM contains none, changes in the Trp emission upon addition of CaM reflect interactions between CaM and the peptide. The titration data were fitted to a binding equation described by Morris and Lehrer (1984), AKv^2 - (1 + nKA + KB)v + nKB = 0, where A and B are the total concentrations of the peptide and CaM, respectively; v is the binding density of CaM to the peptide (e.g. the fluorescence enhancement), K is the binding constant, and n is the apparent stoichiometry of the saturated complex, AB(n). The curve fitting was carried out using a nonweighted nonlinear least squares method to obtain the binding parameters. Competition experiments were carried out by addition of aliquots of peptide stock solutions into a mixture containing CaM and GS17C-NBD or VG29C-NBD, and the NBD fluorescence change ( = 490 nm; = 540 nm) was monitored.


RESULTS AND DISCUSSION

Binding of CaD Peptides to CaM

Interaction between the CaD peptides and CaM was monitored by changes in tryptophan fluorescence. In the presence of 1 mM Ca, addition of saturating amounts of CaM resulted in a 3.4-, 2.4-, and 2.7-fold increase in the intensity of tryptophan emission of GS17C, VG29C, and MG56C, respectively ( Fig. 2and Table 1). Since GS17C and VG29C each contains a single tryptophan (Trp-659 and Trp-692, respectively) and MG56C contains two, the fluorescence enhancement of MG56C was intermediate between the values for the other two peptides. The fluorescence increases were completely reversed upon addition of EGTA, indicating that the interaction between CaM and the peptides is Ca dependent. The binding ratios (n) of CaM to the three peptides obtained from the titration data were all close to 1. The binding constant (K) was the lowest for VG29C, only about 20% of the value for the other two peptides (see Table 1). Binding of GS17C and VG29C to CaM was also monitored by NBD fluorescence using the respective NBD-labeled peptides. The same binding parameters were obtained (data not shown).


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(2); the tryptophan fluorescence was monitored with = 295 nm and = 320 nm. Other conditions include 50 mM KCl, 1 mM CaCl(2), 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(0)) 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.

Competition between CaD Peptides for CaM Binding

To test whether or not site A interacts with the same region of CaM as does site B, we have carried out competition experiments using NBD-labeled GS17C as a reference target, which exhibits a more than 5-fold fluorescence enhancement upon binding CaM (Zhan et al., 1991). Unlabeled CaD peptides were added to a solution containing an equimolar amount of CaM and GS17C-NBD in the presence of Ca (under these conditions the initial enhancement of NBD fluorescence was 4-fold), and the decrease in NBD fluorescence was monitored (Fig. 3).


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(2). 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 (DeltaF) 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(2) 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.



Effect of CaD Peptides on the Ability of CaM to Reverse CaD-induced Inhibition of Actomyosin ATPase Activity

To investigate the relevance of the interactions between CaM and site A or site B to the function of CaD, we set out to examine the ability of the three peptides to compete with intact CaD for CaM in the actomyosin ATPase activity assay. When purified chicken gizzard CaD was added (final concentration, 2 µM) to the mixture containing actin-tropomyosin and phosphorylated gizzard myosin, the rate of ATP hydrolysis decreased by 60%. This inhibition was totally reversed by the addition of 10 µM CaM. It was at this point that the synthetic peptides of CaD were added to see whether the ability of CaM to reverse the CaD-induced inhibition was affected.

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).

Conclusion

A recent report by Marston et al. (1994) suggested that there exists a second CaM-binding site (site B, see Fig. 1) in the C-terminal region of CaD, in addition to the previously identified CaM-binding site (site A) (Zhan et al., 1991), and that site B alone is involved in the CaM sensitivity of CaD's inhibitory action. Their viewpoint was mainly supported by two findings: (a) a recombinant fragment (H9) of CaD that lacks site A, but contains site B, shows Ca/CaM-dependent regulation of acto-S1 ATPase similar to that of intact CaD; (b) a synthetic peptide (M73), which contains site A and binds CaM but does not inhibit the acto-S1 ATPase activity, does not compete with intact CaD in the presence of CaM, and thus there is no re-inhibition. In this study, we have confirmed that the peptide stretch encompassing site B indeed binds CaM; but we found that site A is involved in the CaD-CaM interaction that is responsible for the reversal of CaD's inhibitory action of the actomyosin ATPase activity. This latter view is consistent with the findings that CaD competes with NBD-labeled GS17C (this work) and that GS17C competes with NBD-labeled CaD for CaM binding (Zhan et al., 1991).

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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant P01-AR41637. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom all correspondence should be addressed: Muscle Research Group, Boston Biomedical Research Institute, 20 Staniford St., Boston, MA 02114. Tel.: 617-742-2010 (ext. 376); Fax: 617-523-6649.

(^1)
The abbreviations used are: CaD, chicken gizzard caldesmon; CaM, calmodulin; MLCK, smooth muscle myosin light chain kinase; DTT, dithiothreitol; NBD, nitrobenz-2-oxa-1,3-diazol-4-yl; MOPS, 4-morpholinepropanesulfonic acid.

(^2)
The amino acid numbering systems are different for chicken gizzard CaD (Bryan et al., 1989) and human isoform (Humphrey et al., 1992), the latter being 57 greater than the former in the region of interest. In this work, the chicken system is used exclusively.


ACKNOWLEDGEMENTS

We express gratitude to Drs. J. Gergely, Z. Grabarek, and P. Graceffa for critical reading of this manuscript and to Dr. R. Lu and Anna Wong of the Protein Chemistry Facility Core of the Smooth Muscle Program Project Grant for help in the preparation and analysis of the synthetic peptides.


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