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Originally published In Press as doi:10.1074/jbc.M410375200 on October 24, 2004

J. Biol. Chem., Vol. 280, Issue 6, 4135-4143, February 11, 2005
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Effect of Caldesmon on the Position and Myosin-induced Movement of Smooth Muscle Tropomyosin Bound to Actin*

Philip Graceffa{ddagger} and Andrew Mazurkie

From the Boston Biomedical Research Institute, Watertown, Massachusetts 02472

Received for publication, September 9, 2004 , and in revised form, October 19, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
It is known that the actin-binding protein caldesmon inhibits actomyosin ATPase activity and might in this way take part in the thin filament regulation of smooth muscle contraction. Although the molecular mechanism of this inhibition is unknown, it is clear that the presence of actin-bound tropomyosin is necessary for full inhibition. Recent evidence also suggests that the myosin-induced movement of tropomyosin plays a key role in regulation. In this work, fluorescence studies provide evidence to show that caldesmon interacts with and alters the position of tropomyosin in a reconstituted actin thin filament and thereby limits the ability of myosin heads to move tropomyosin. Caldesmon interacts with the Cys-190 region in the COOH-terminal half of tropomyosin, resulting in the movement of this part of tropomyosin to a new position on actin. Additionally, this constrains the myosin-induced movement of this region of tropomyosin. On the other hand, caldesmon does not appear to interact with the Cys-36 region in the NH2-terminal half of tropomyosin and neither alters the position of nor significantly constrains the myosin-induced movement of this part of tropomyosin. The ability of caldesmon to limit the myosin-induced movement of tropomyosin provides a possible molecular basis for the inhibitory function of caldesmon. The different movements of the two halves of tropomyosin indicate that actin-bound tropomyosin moves as a flexible molecule and not as a rigid rod. Interestingly, caldesmon, which inhibits tropomyosin's potentiation of actomyosin ATPase activity, moves tropomyosin in one direction, whereas myosin heads, which enhance potentiation, move tropomyosin in the opposite direction.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Muscle contraction takes place when myosin heads, projecting from the thick filament, cyclically interact with actin protomers in the thin filament, causing the filaments to slide past each other with a resulting muscle shortening. Actin-activated myosin hydrolysis of ATP provides the energy for this process. Tropomyosin, a long coiled-coil dimer, which binds end-to-end on each side of the thin filament, is involved in switching the muscle on. In skeletal muscle, contraction is switched on by Ca2+ binding to actin-bound troponin, which results in the relocation of tropomyosin and thus the exposure of myosin-binding sites on actin (2–5). The fact that each tropomyosin molecule covers seven actin protomers and binds end-to-end to itself endows the switching process with cooperativity (for a review, see Refs. 6–9).

Smooth muscle does not contain troponin, and the main regulatory switch is the phosphorylation of myosin light chains (for a review, see Refs. 10 and 11). However, myosin phosphorylation does not account for all aspects of smooth muscle regulation, i.e. there can be a decoupling between force and myosin phosphorylation levels, and thus it has been put forth that the actin thin filament and its associated proteins also play a role in regulation (for a review, see Refs. 12 and 13). The actin-binding protein caldesmon has been suggested as a potential regulatory protein since it reversibly inhibits actomyosin ATPase activity and in vitro motility (reviewed in Refs. 14), both of which are in vitro analogues of contraction. Although there are physiological measurements that suggest that caldesmon may assume such a regulatory role in vivo (15, 16), in general, it is still a highly controversial question. However, in the specific case of myometrial smooth muscle, the caldesmon content increases 3–5-fold during pregnancy (17, 18), when the uterus must remain quiescent. During labor, caldesmon becomes phosphorylated (18), a process that is known to reverse the inhibitory function of caldesmon (19). Thus it appears that caldesmon may play a direct regulatory role in this muscle.

Tropomyosin must be present for caldesmon to fully inhibit actomyosin ATPase activity (20–28) and in vitro motility (29). Smooth muscle tropomyosin enhances actomyosin ATPase activity (30–32) and motility (29, 33) and presumably contractility. This enhanced ATPase activity is inhibited as a function of caldesmon concentration more steeply than is the activity in the absence of tropomyosin. The molecular mechanism whereby tropomyosin and caldesmon act in concert is unknown. The reported binding between tropomyosin and caldesmon (34–36) may possibly be involved. The understanding of this mechanism is the object of the present work.

Early studies of x-ray diffraction on "living" smooth muscles indicated that activation of contraction results in the movement of tropomyosin (37). More recently, using fluorescence resonance energy transfer (FRET)1 between donors on tropomyosin and an acceptor on actin, for in vitro reconstituted thin filaments, the binding of myosin heads to actin resulted in the movement of smooth muscle tropomyosin to a new position on actin (38–40). This movement was more efficient for phosphorylated smooth muscle heads than for unphosphorylated ones (39). Furthermore, tropomyosin's enhancement of ATPase activity increases from a very low value (under some conditions, inhibition (41, 42)) up to a plateau level as the number of myosin heads binding to actin increases (41–43). This led to the postulation that smooth muscle regulation includes the myosin-induced movement of tropomyosin to a position on actin where it can enhance the actomyosin ATPase activity (39). Furthermore, the myosin must be phosphorylated to promote such a change more easily (39). In this way, myosin phosphorylation and tropomyosin movement would act together to switch on smooth muscle contraction. Similarly, the movement of skeletal muscle tropomyosin by the binding of myosin heads to actin is thought to contribute to the switching on of skeletal muscle contraction (4, 5).

It has been suggested that the cooperativity between myosin phosphorylation levels and force development in smooth muscle tissue is mediated by tropomyosin (44), possibly involving myosin-induced tropomyosin movement (45). From modeling of this cooperativity, it was concluded that the binding of each myosin head activated seven actin protomers (44). The movement of tropomyosin by a double-headed thio-phosphorylated myosin fragment also takes place with a cooperativity of seven actin protomers (39). Consequently, a strong relationship between the myosin-induced tropomyosin movement and myosin phosphorylation-dependent force is reasonable.

The general question arises: can proteins or factors, other than myosin, control the position of tropomyosin on actin and thereby affect the regulation of smooth muscle contraction? More specifically, we ask in this study whether the ability of caldesmon to inhibit actomyosin ATPase activity could be due to its blocking of the movement of tropomyosin by myosin heads. To address this question, we used FRET to measure the effect of caldesmon on both the position of smooth muscle tropomyosin bound to actin and the movement of tropomyosin by myosin heads. Previous electron microscopy studies have demonstrated that the presence of caldesmon (CaD) changes the position of tropomyosin (Tm) on actin, but these studies showed movement averaged over the entire tropomyosin molecule (46, 47). Since we are specifically placing FRET donors in either half of tropomyosin, we can monitor possible inhomogeneous movements along the molecule. Furthermore, the microscopy studies were conducted on fixed and stained samples, whereas the present work is done in solution, which more closely approximates the dynamic situation that exists in live muscle.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
All materials, methods, and procedures have been described in detail in our previous works referred to below. Actin was prepared from rabbit skeletal muscle (48), and CaD (48) and Tm (49) were prepared from chicken gizzard smooth muscle. Myosin was prepared from rabbit skeletal muscle and its single-head subfragment 1 (S1) by chymotryptic digestion (38). Actin was labeled at Cys-374 with the FRET acceptors DAB-maleimide (39) or DDP-maleimide (50), which are not fluorescent. The Tm {alpha}{beta}-heterodimer was specifically labeled at either Cys-36 of the {beta}-chain or Cys-190 of the {alpha}-chain with the fluorescence probes IAEDANS, acrylodan, or pyrene-maleimide (38). Tm {alpha}{alpha}- and {beta}{beta}-homodimers were similarly labeled at Cys-190 or Cys-36, respectively, with the fluorescence acceptor fluorescein-maleimide (FM), except that FM was added at a ratio of FM/Tm = 6, with a resulting labeling ratio of FM/Tm of about 1.5. CaD was specifically labeled at either Cys-153 or Cys-5952 with the fluorescence donors 7-(diethylamino)-3-(4'-maleimidylphenyl)-4-methlycoumarin (CPM) or IAEDANS (50, 51). All labels, except for DDP-maleimide, which was obtained from Aldrich, were purchased from Molecular Probes.

Fluorescence emission or polarization spectra were recorded with either a Spex Fluorolog 2/2/2 or a Varian Eclipse fluorometer (50, 51). FRET was measured from the change in steady-state fluorescence of the donor corrected for trivial absorption, acceptor fluorescence, incomplete labeling of the acceptor protein (only necessary for FM-Tm), and the effect of unlabeled acceptor protein on donor fluorescence (38, 50). For the most part, these corrections were small. Distance change was calculated from FRET change (50) using Ro, the critical transfer distance, of AED-Tm/DAB-actin = 40 Å (38), Ro of AED-CaD/DDP-A = 27 Å (50), and Ro of CPM-CaD/FM-Tm = 50 Å (50). The determined distances are apparent, or weighted average, values since it is assumed that each donor transfers energy to a single acceptor. However, in this work, the comparative and relative change in distance, and not the absolute values, are important.

The determination of Ro involves the donor probe-acceptor probe orientation factor {kappa}2, which can be assumed to have a value of 2/3 if there is sufficient motion or randomness in the orientation of one or both probes (Ref. 50 and references therein). There have been arguments attesting to the validity of using a value of 2/3 for the AED-Tm/DAB-A system (52) and to the fact that the value of {kappa}2 does not change upon binding of myosin heads to the actin (38, 39). Furthermore, the energy transfer from each donor on tropomyosin appears to be to multiple actin acceptors (40, 53), which will add to the randomness of the system and give further support to the use of {kappa}2 = 2/3 (54).

For the CPM-CaD/FM-Tm complex bound to actin, {kappa}2 = 2/3 also appears to be a reasonable assumption based on the rather high mobility of the FM probe. The steady-state polarization of FM attached to Tm, for FM-Tm both free and bound to CaD-actin, is very low at about 0.06, as compared with the rigid limit of 0.43 for this probe (55). Thus although FM-Tm is immobilized on actin filaments (+CaD), the probe has the same low polarization, and thus mobility, as for free FM-Tm, indicating that the probe has significant motion independent of the protein to which it is attached. Similar conclusions for {kappa}2 = 2/3 for the AED-CaD/DDP-A system have also been made (50).

Fluorescence measurements in the presence of actin were conducted at 0.3–0.6 µM Tm with actin/Tm = 8/1 – 7/1 and actin/CaD = 15/1 – 5/1 in a solution containing 40 mM NaCl, 2–5 mM MgCl2, 5 mM MOPS, pH 7.5, at 20 °C. FRET between CPM-CaD and FM-Tm in the absence of actin was conducted at the same solution conditions but with an 8.3-fold molar excess of FM-Tm over CPM-CaD, which was present at about 0.15 µM. Excess FM-Tm maximized the binding of CPM-CaD. The AED and pyrene labels were excited at 340 nm, the acrylodan label was excited at 385 nm, and the CPM label was excited at 396 nm. The binding of proteins to actin was determined by densitometry of SDS-PAGE, following actin filament sedimentation (38).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Caldesmon-Tropomyosin Interaction on Actin Filament—To investigate the interaction of Tm and CaD bound to actin, we measured both the effect of CaD on the fluorescence of probes attached to Tm and the FRET between donor and acceptor probes attached to CaD and Tm, respectively. Native smooth muscle Tm is an {alpha}{beta}-heterodimer with the {alpha}-chain containing a single cysteine at position 190 in the COOH-terminal half of the molecule and the {beta}-chain with a single cysteine at position 36, near the NH2 terminus. Both chains contain 284 residues. The heterodimer or homodimers were specifically labeled at either of these cysteines with IAEDANS (AED), acrylodan, pyrene-maleimide, or FM. CaD is a long (about twice the length of Tm (56)), thin molecule, which binds to actin with an actin/CaD stoichiometry of roughly 14/1 on sections of native actin filaments (57, 58). Either of the cysteines of CaD, Cys-153 in its NH2-terminal region or Cys-595 in its COOH-terminal domain with a total of 771 amino acids in the molecule, was specifically labeled with CPM.

Fig. 1 shows the effect of CaD on the fluorescence of AED attached to either Cys-190 (AED190{alpha}{beta}Tm) or Cys-36 (AED36{alpha}{beta}Tm) of the Tm heterodimer bound to actin. CaD had very little, if any, effect on AED36{alpha}{beta}Tm fluorescence (Fig. 1B), whereas the fluorescence of AED190{alpha}{beta}Tm was significantly enhanced and blue-shifted (Fig. 1A). These results suggest that CaD binds close to, and possibly interacts with, the Cys-190 region in the COOH-terminal half of Tm in this ternary protein complex. The enhancement and blue-shifting of the probe fluorescence could be due to CaD shielding the probe from accessibility to the solvent. Such an effect is similar to that which occurs upon the binding of troponin to AED attached to Cys-190 of skeletal tropomyosin bound to actin (38, 52). Troponin is known to bind close to Cys-190 of tropomyosin (6). The fluorescence of two other probes, acrylodan and pyrene-maleimide, was also sensitive to CaD when the probes were attached to Cys-190 but not when the probes were attached to Cys-36 of the Tm heterodimer (not shown).



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  		FIG. 1.
Fluorescence of AED-labeled Tm ± CaD. Fluorescence spectrum of the AED probe attached to Tm {alpha}{beta}-heterodimer at Cys-190 (A) of the {alpha}-chain or Cys-36 (B) of the {beta}-chain, in the presence or absence of CaD, all in the presence of actin, are shown. AED excitation is at 340 nm. Actin/Tm and actin/CaD molar ratios are 8/1 and 5/1, respectively. Solution conditions are: 40 mM NaCl, 5 mM MOPS, 5 mM MgCl2, pH 7.5, 20 °C.

 
Fluorescence polarization, an indicator of probe mobility, was similarly sensitive to CaD for a probe attached to Cys-190 but not for the probe attached to Cys-36. For example, the polarization of pyrene-maleimide attached to Cys-190 of actin-bound tropomyosin changed from a value of 0.30 to a value 0.34 upon binding of CaD (actin/CaD = 5), whereas the probe at Cys-36 showed no discernable change from a value of 0.21 with the addition of CaD.

The proximity between CaD and the Cys-190 region of Tm was also observed from FRET between the two proteins bound to actin (Fig. 2). The addition of FM-labeled Cys-190 of the Tm {alpha}{alpha}-homodimer (FM{alpha}{alpha}Tm) to actin-bound CPM-labeled Cys-595 of CaD resulted in a large decrease in the CPM fluorescence due to the fluorescence resonance energy transfer from the CPM donor to the FM acceptor on Tm (Fig. 2A). The distance between the two probes was calculated to be roughly 51 Å. On the other hand, there was negligible energy transfer from CPM attached to Cys-153 of CaD to FM on Cys-190 of Tm, as evidenced by essentially no change in the CPM donor fluorescence (Fig. 2B). With the FM acceptor on Cys-36 of the {beta}{beta}-homodimer of Tm, there was negligible energy transfer to CPM on either Cys-595 (Fig. 3A) or Cys-153 (Fig. 3B) of CaD. We can conclude from these experiments that the Cys-595 region of the COOH-terminal domain of CaD is close to the Cys-190 region of the COOH-terminal half of Tm when both are bound to actin. The fact that CPM on Cys-153 of CaD is not close to FM attached to either Cys-36 or Cys-190 of Tm is consistent with previous work which has shown that, whereas the COOH-terminal domain of CaD is bound to actin/Tm, the NH2-terminal region is dissociated (50, 51). As regards the proximity of the NH2-terminal half of Tm to some part of CaD, we can only conclude that there is a lack of evidence in the experiments that we have performed.



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  		FIG. 2.
FRET between CPM-labeled CaD and FM{alpha}{alpha}Tm bound to actin. FRET between CPM donor on CaD at Cys-595 (A) or Cys-153 (B) and FM acceptor attached to Cys-190 of Tm {alpha}{alpha}-homodimer, in the presence of actin, is shown. Excitation is at 396 nm peak absorption of CPM. Actin/Tm and actin/CaD molar ratios are 7/1 and 15/1, respectively. Solution conditions are as in Fig. 1, except that MgCl2 = 2 mM. In A, but not in B, there is a large drop in CPM fluorescence due to energy transfer to FM. FM {alpha}{alpha}Tm, Tm {alpha}{alpha}-homodimer labeled with FM at Cys-190. CPM(595)CaD, CaD labeled at Cys-595 with CPM. CPM(153)CaD, CaD labeled at Cys-153 with CPM.

 



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  		FIG. 3.
FRET between CPM-labeled CaD and FM{beta}{beta}Tm bound to actin. FRET between CPM donor on CaD at Cys-595 (A) or Cys-153 (B) and FM acceptor attached to Cys-36 of Tm {beta}{beta}-homodimer, in the presence of actin, is shown. Excitation is at 396 nm peak absorption of CPM. Actin/Tm and actin/CaD molar ratios are 7/1 and 15/1, respectively. Solution conditions are as in Fig. 1, except that MgCl2 = 2 mM. There is little or no energy transfer from CPM to FM in either A or B. FM{beta}{beta}Tm, Tm {beta}{beta}-homodimer labeled with FM at Cys-36. CPM(595)CaD, CaD labeled with CPM at Cys-595. CPM(153), CaD labeled at Cys-153 with CPM.

 
Similar FRET experiments were performed for a complex between Cys-595 donor-labeled CaD and Cys-190 acceptor-labeled Tm in the absence of actin (Fig. 4). The large fluorescence of labeled Tm is due its high excess over CaD, used to ensure maximum binding of the latter. An interprobe distance of 55 Å was obtained, close to the 51 Å when both are bound to actin. This strongly implies that CaD and Tm are interacting when bound to the actin surface and not just in close proximity.



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  		FIG. 4.
FRET between CaD labeled with CPM at Cys-595 (CPM(595)CaD) and FM{alpha}{alpha}Tm homodimer in the absence of actin. Solution conditions are as in Fig. 2 except that the Tm/CaD molar ratio is 8.3/1. There is a drop in CPM fluorescence due to energy transfer to FM. FM{alpha}{alpha}Tm, Tm {alpha}{alpha}-homodimer labeled with FM at Cys-190.

 
Caldesmon-induced Movement of Tropomyosin Bound to Actin—The effect of CaD on the position of Tm bound to actin was monitored by the FRET between an AED fluorescence donor attached to either Cys-36 or Cys-190 of the Tm {alpha}{beta}-heterodimer and a DAB acceptor attached to Cys-374 of actin. The experiments were conducted at an actin/CaD ratio of 10/1 and 5/1. It has been reported that the CaD interaction with actin-bound Tm saturates at an actin/CaD ratio of 10/1 (36). In the experiments reported here, there were no additional effects of increasing the amount of CaD from an actin/CaD ratio of 10/1 to 5/1. The actual actin-bound CaD ratios for CaD added at actin/CaD ratios of 10/1 and 5/1, determined by sedimenting actin filaments, were equivalent to an actin/CaD ratio of 11.5/1 and 6.8/1, respectively.

FRET between AED190{alpha}{beta}Tm (Fig. 5A) or AED36{alpha}{beta}Tm (Fig. 5B) and DAB-actin is evidenced by the drop in AED fluorescence upon the addition of the DAB-actin. FRET between AED190{alpha}{beta}Tm and DAB-actin is increased upon the addition of CaD (Fig. 5A), as seen by the further drop in AED donor fluorescence, resulting in a shortening of the distance between donor and acceptor by about 3 Å. Note that this change in FRET is opposite to a decrease in FRET upon binding of myosin heads to AED190{alpha}{beta}Tm/DAB-actin (38). On the contrary, CaD has no effect on FRET between AED36{alpha}{beta}Tm and DAB-actin (Fig. 5B). These findings indicate that the CaD binding to actin/Tm results in a movement of some part of the COOH-terminal half of Tm, which includes Cys-190, to a new position on actin, whereas there is no movement of at least part of the NH2-terminal half of the molecule that includes Cys-36.



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  		FIG. 5.
CaD effect on FRET between AED-labeled Tm and DAB-actin. FRET between AED donor on Tm {alpha}{beta}-heterodimer at Cys-190 (A) of the {alpha}-chain or Cys-36 (B) of the {beta}-chain and DAB acceptor attached to actin Cys-374, in the presence or absence of CaD, is shown. Protein ratios and solution conditions are as in Fig. 1. AED excitation is at 340 nm. CaD increases the energy transfer between AED (on Tm) and DAB (on actin) in A but not in B.

 
Conversely, Tm changes the position of actin-bound CaD. FRET between an AED donor on Cys-595 of CaD and a DDP acceptor on Cys-374 of actin (50), as evidenced by a drop in AED fluorescence upon the addition of DDP-actin, increased further upon binding of Tm (Fig. 6). This change in FRET corresponded to a decreased distance between the probes by about 2 Å. This finding indicates that the binding of Tm to actin/CaD results in a movement of the region of CaD which includes Cys-595 in the COOH-terminal domain. Thus CaD and Tm alter each other's binding position on actin.



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  		FIG. 6.
Tm effect on FRET between AED-labeled CaD and DDP-actin. FRET between AED donor on Cys-595 of CaD and DDP acceptor attached to actin Cys-374, in the presence or absence of Tm, is shown. AED excitation is at 340 nm. Actin/Tm and actin/CaD molar ratios are 8/1 and 14/1, respectively. Solution conditions are: 40 mM NaCl, 5 mM MOPS, 2.5 mM MgCl2, pH 7.5, 20 °C. Tm increases the energy transfer between AED (on CaD) and DDP (on actin). AED(595)CaD, CaD labeled with AED at Cys-595.

 
Effect of Caldesmon on the Myosin Head-induced Movement of Tropomyosin—In our previous work (38, 39), it was shown by FRET that myosin heads binding to actin resulted in the movement of Tm. The movement was such that the AED donor on Cys-36 of Tm moved closer to the DAB acceptor(s) on actin Cys-374, i.e. a decrease in donor fluorescence, and that the donor on Cys-190 of Tm moved farther from the acceptor(s) (on different actin protomers), i.e. an increase in donor fluorescence (38, 39). In the present work, it can be seen that CaD had little effect on the ability of the heads to move the AED donor on Tm Cys-36 (Fig. 7), whereas the presence of CaD severely limited the head-induced movement of the AED donor on Tm Cys-190 (Fig. 8). Over the range of added S1/actin of 0 – 4/7, at least 80% of the added caldesmon was bound to DAB-actin/AED-Tm, as well as close to 100% of the S1. In this binding assay, performed under the same exact conditions of the FRET measurements, S1 was added last to simulate the FRET experiments.



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  		FIG. 7.
CaD effect on the S1-induced change in FRET between AED36{alpha}{beta}Tm and DAB-actin. The S1 effect on FRET between AED donor on Tm {alpha}{beta}-heterodimer at Cys-36 of the {beta}-chain and DAB acceptor attached to actin Cys-374, in the absence (A) or presence (B) of CaD, is shown. S1/A = 4/7, and other protein ratios and solution conditions are as in Fig. 1. AED excitation is at 340 nm. CaD has almost no effect on the ability of S1 to increase the energy transfer between AED (on Cys-36 of Tm) and DAB (on actin).

 



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  		FIG. 8.
CaD effect on the S1-induced change in FRET between AED190{alpha}{beta}Tm and DAB-actin. The S1 effect on FRET between AED donor on Tm {alpha}{beta}-heterodimer at Cys-190 of the {alpha}-chain and DAB acceptor attached to actin Cys-374, in the absence (A) or presence (B) of CaD, is shown. S1/A = 3/7, and other protein ratios and solution conditions are as in Fig. 1. AED excitation is at 340 nm. CaD inhibits the ability of S1 to decrease the energy transfer between AED (on Cys-190 of Tm) and DAB (on actin).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Tropomyosin-Caldesmon Interaction—The present work concluded that the Cys-190 region of tropomyosin interacts with the COOH-terminal domain of caldesmon when both are bound to actin. The distance between FRET probes on tropomyosin Cys-190 and caldesmon Cys-595 was about 51 Å in the presence of actin and 55 Å in its absence. That the distance is roughly the same regardless of the presence of actin indicates that caldesmon and tropomyosin are interacting when bound to actin and not just close to each other. Although these two cysteines are not very close, they are close enough to indicate that a region of tropomyosin that includes Cys-190 in the COOH-terminal half of the molecule interacts with a region of caldesmon that contains Cys-595 in the COOH-terminal domain. This finding is further supported by our results that the fluorescence of a probe on tropomyosin Cys-190 is enhanced and blue-shifted upon interaction with caldesmon.

Previous published works on the interaction of tropomyosin and caldesmon and their fragments are consistent with our conclusions. It was concluded from the interaction of tropomyosin fragments with caldesmon that the main (i.e. strongest) caldesmon-binding site on tropomyosin is in the region encompassed by residues 142–227, with cleavage at Cys-190 being particularly detrimental to the interaction (59, 60). Weaker binding was detected in the NH2-terminal half of tropomyosin (59, 61). Our inability to detect energy transfer between tropomyosin Cys-36 and either Cys-153 or Cys-595 of caldesmon or to detect any effect of caldesmon on the fluorescence of probes attached to Cys-36 is consistent with this latter finding. Studies on the interaction of caldesmon fragments with tropomyosin indicated that the strongest tropomyosin-binding site on caldesmon resides in the COOH-terminal domain (61–65). There also appears to be weaker binding to the NH2-terminal domain of caldesmon (61, 66), but such binding is even weaker in the presence of actin (50, 51, 61). In summary, the present study, together with other published work, concludes that the main interaction interface between tropomyosin and caldesmon, bound to actin, involves the COOH-terminal domain of caldesmon and, at least, the part of the COOH-terminal half of tropomyosin that includes Cys-190.

Caldesmon-induced Movement of Tropomyosin Bound to Actin—Caldesmon binding to actin/tropomyosin resulted in the movement of the Cys-190 region of tropomyosin without any effect on the position of the Cys-36 region of the molecule. Our results lead us to conclude that caldesmon binds to the Cys-190 region of tropomyosin and moves it to a new position on actin, whereas caldesmon does not interact, or only weakly interacts, with the Cys-36 region of tropomyosin and thus has no effect on the position of this region of the molecule. These conclusions are generally consistent with electron microscopy studies, which have shown a movement of tropomyosin on actin in response to caldesmon binding to the thin filament (46, 47). However, since the electron microscopy studies average over the entire tropomyosin molecule, they are "blind" to the kind of heterogeneous movement along the molecule that we have observed. Conversely, our work demonstrated that the presence of tropomyosin altered the position of caldesmon on actin.

It is interesting to note that caldesmon induced a movement of the Cys-190 region of tropomyosin on actin that is opposite to the movement promoted by the actin binding of myosin heads (38, 39). That is, the donor (on tropomyosin) and acceptor (on actin) probes move closer together in the former case and farther apart in the latter situation. This movement correlates, in a very general sense, with the opposite effects that caldesmon and myosin binding have on the tropomyosin-actomyosin ATPase activity, i.e. inhibition and activation, respectively (see the Introduction). This suggests that myosin heads move tropomyosin to a position on actin where activation of the ATPase activity results, as suggested previously (39), and caldesmon moves tropomyosin in the opposite direction to a position on actin such that this activation is inhibited.

Effect of Caldesmon on the Myosin-induced Movement of Tropomyosin—Previous work has shown that myosin skeletal S1 head binding to actin resulted in the movement of smooth muscle tropomyosin {alpha}{beta}-heterodimer (38). This was evidenced by an S1-dependent change in FRET between an AED donor attached to tropomyosin, at either Cys-36 of the {beta}-chain or Cys-190 of the {alpha}-chain, and a DAB acceptor attached to Cys-374 of actin. Although the maximum S1-induced change in the AED-DAB distance was negative (–6 Å) for AED at Cys-36 and positive (+5 Å) for AED at Cys-190, both changes showed the same S1 concentration dependence. Thus the movement of both halves of the tropomyosin were responding similarly to myosin heads, indicating that the whole molecule appeared to be moving in some concerted manner.

In the present work, the presence of caldesmon altered the response of this FRET to S1 heads. For the donor at Cys-36 of tropomyosin, caldesmon had almost no effect on the ability of S1 to induce movement of this region of tropomyosin. However, caldesmon strongly limited the ability of S1 to move the Cys-190 region of tropomyosin, the distance between the donor and acceptor changing by only about +1.7 Å, as opposed to +5 Å in the absence of caldesmon (38), upon the binding of S1. Thus, together with the above results, it appears that, on actin, caldesmon interacts with the Cys-190 region of tropomyosin, thereby moving this part of tropomyosin to a new position on actin and inhibiting the ability of the myosin heads to move this region of tropomyosin. On the other hand, caldesmon does not bind, or weakly binds, to the Cys-36 region of tropomyosin and thus does not move this part of tropomyosin nor inhibit the movement of this region of the molecule by myosin heads. Just how much of the actin-bound tropomyosin molecule is "tied down" by caldesmon is unknown. However, it includes the Cys-190 region of tropomyosin and could possibly comprise a significant portion of the central region of the molecule since the 142–227 tropomyosin peptide binds strongly to caldesmon (59, 60).

Model for Caldesmon Inhibition of Tropomyosin-Actomyosin ATPase Activity—How do the results from this study further our understanding of how caldesmon and tropomyosin act to regulate or modulate actomyosin ATPase activity and possibly smooth muscle contractility? Smooth muscle tropomyosin enhances, or potentiates, actomyosin ATPase activity and in vitro motility, and thus, presumably, contractility. This potentiation is enhanced with increasing myosin concentration in the presence of ATP, ADP, or no nucleotide (see the Introduction and Refs. 24 and 41–43). In the presence of ADP or in the absence of nucleotide, myosin heads bind strongly to actin. Increasing strong-binding myosin head concentration also results in the movement of tropomyosin on actin (38), a movement that is more facile for phosphorylated as compared with unphosphorylated smooth muscle myosin heads (39). Furthermore, the myosin-induced movement of tropomyosin (38) and the myosin-induced potentiation of tropomyosin-actomyosin ATPase activity (24) both leveled off at about one (strong-binding) myosin head per seven actin monomers. Thus it was proposed that the enhanced tropomyosin potentiation by myosin is the result of the myosin-induced movement of tropomyosin to a position on actin where it maximally potentiates ATPase activity and in vitro motility (39). Since this enhanced potentiation by myosin is inhibited by caldesmon (24) and we find that the myosin-induced movement of tropomyosin is interfered with by caldesmon, we propose that caldesmon inhibits tropomyosin's potentiation of ATPase activity and in vitro motility by impairing the ability of myosin to move tropomyosin.

However, since caldesmon can inhibit actomyosin ATPase activity independently of tropomyosin, then inhibiting the myosin-induced movement of tropomyosin may not be the only mechanism by which caldesmon functions. Indeed, it has been suggested that caldesmon might exhibit dual inhibition of ATPase activity both by blocking the binding of myosin-ATP to actin and by reversing the potentiation by tropomyosin by some allosteric mechanism that inhibits a step in the actomyosin ATPase cycle (12, 67). We propose in this work that this latter mechanism involves interference with the myosin-induced tropomyosin movement. This duality includes the myosin ATP-blocking model of Chalovich and colleagues (23, 28, 68, 69) and the allosteric inhibitory mechanism of Marston and colleagues (14, 27, 70).

An important question is: can myosin binding to actin move tropomyosin in vivo? It has been determined that during active isometric contraction of skeletal muscle, 20–40% of myosin heads are bound to actin, and most of these are thought to be strong-binding heads (71–73). Furthermore, during the actomyosin ATPase hydrolytic cycle, smooth muscle myosin spends a considerably greater time attached to actin than does skeletal muscle myosin (74, 75). This is most likely due to the fact that smooth muscle myosin-ADP binds much more tightly to actin than does skeletal muscle myosin (76–78) and that ADP binds more strongly to actin-bound smooth muscle myosin than to actin-bound skeletal muscle myosin (76). These results suggest that there is a substantial fraction of strong-binding heads (i.e. with bound MgADP) attached to actin in smooth muscle, making the myosin-induced movement of tropomyosin a likely possibility. MgADP-bound and nucleotide-free (skeletal muscle or phosphorylated smooth muscle) myosin heads move tropomyosin with equal effectiveness (38, 39).

Movement of Tropomyosin as a Flexible Molecule—Although the nature of the movement of tropomyosin is unknown, it has been speculated that it moves as a continuous flexible strand as opposed to a discontinuous rigid rod (79, 80). Several studies have concluded that the tropomyosin molecule is a semiflexible rod (81–84), although there is little experimental data on the flexibility of tropomyosin bound to actin. An electron cryomicroscopy study concluded that the NH2-terminal and COOH-terminal regions of skeletal tropomyosin bound to actin and troponin move differently upon binding of Ca2+ to troponin (85), suggesting flexibility in the tropomyosin molecule. In another study, the cooperative binding of myosin heads to tropomyosin-actin was modeled with tropomyosin as a continuous flexible chain (86).

In the present work, experimental evidence has been provided in support of the flexibility of actin-bound tropomyosin upon its caldesmon-induced and myosin head-induced movement. In the absence of caldesmon, myosin head binding to actin moves tropomyosin such that the Cys-36 region of tropomyosin comes closer to Cys-374 of actin protomers, whereas the Cys-190 region of tropomyosin ends up farther from the Cys-374 of different actin protomers (38). In the presence of caldesmon, myosin heads move tropomyosin such that Cys-36 moves closer to actin Cys-374 by about the same amount as in its absence, whereas Cys-190 hardly moves at all. In the absence of myosin heads, caldesmon binding to actin moves tropomyosin such that Cys-36 does not appear to move at all and Cys-190 moves closer to actin Cys-374, opposite to the direction moved by myosin heads. Thus the Cys-36 and Cys-190 regions of tropomyosin, in either half of the molecule, appear to move independently of each other, indicating that tropomyosin moves as a flexible molecule while attached to actin. This analysis does not take into consideration the further complexity that tropomyosin might move about actin in a rolling motion as has been concluded recently (40, 53, 87).

In conclusion, the position of smooth muscle tropomyosin in reconstituted actin thin filaments can be changed by myosin heads binding to actin, and this position and movement appear to be controlled by the phosphorylated state of the myosin (39) and by actin-bound caldesmon (this work), both of which control the tropomyosin-actomyosin ATPase activity. Therefore we propose that, although the phosphorylation of myosin is the main contractile switch in smooth muscle, the position of tropomyosin on actin can modulate contractility by either accelerating (potentiating) the ATPase activity or not. Furthermore, other proteins, which might be candidates for control of smooth muscle contraction through their binding to the actin thin filament, might also act by influencing the position and/or movement of tropomyosin.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants HL66219 and AR41637. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Boston Biomedical Research Institute, 64 Grove St., Watertown, MA 02472. Tel.: 617-658-7813; Fax: 617-972-1753; E-mail: graceffa{at}bbri.org.

1 The abbreviations and trivial names used are: FRET, fluorescence resonance energy transfer; Tm, tropomyosin; CaD, caldesmon; S1, single-headed chymotryptic subfragment 1 of skeletal muscle myosin; DAB-maleimide, N(4-((4-(dimethylamino)phenyl)azo)-phenyl)maleimide; DDP-maleimide, N-(4-(dimethylamino)-3,5-dinitrophenyl)maleimide; IAEDANS, 5-((((iodoacetyl)amino)ethyl)-amino)naphthalene-1-sulfonic acid; AED, IAEDANS attached to a protein; acrylodan, 6-acryloyl-2-(dimethylamino)naphthalene; CPM, 7-(diethylamino)-3-(4'-maleimidylphenyl)-4-methlycoumarin; FM, fluorescein-maleimide; DAB-A, actin labeled at Cys-374 with DAB-maleimide; DDP-A, actin labeled at Cys-374 with DDP-maleimide; AED190{alpha}{beta}Tm, Tm {alpha}{beta}-heterodimer labeled with AED at Cys-190 of the {alpha}-chain; AED36{alpha}{beta}Tm, Tm {alpha}{beta}-heterodimer labeled with AED at Cys-36 of the {beta}-chain; MOPS, 4-morpholinepropanesulfonic acid. Back

2 Our previous publications referred to CaD Cys-595 as Cys-580. However, it has since been confirmed that there are an additional 15 residues in the central region of CaD (1). Back


   ACKNOWLEDGMENTS
 
We thank Miss Elena Graceffa and Dr. Sherwin Lehrer for a critical reading of the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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