The C-terminus of troponin T is essential for maintaining the inactive state of regulated actin

Striated muscle contraction is regulated by the actin binding proteins tropomyosin and troponin. Defects in these proteins lead to myopathies and cardiomyopathies. Deletion of the 14 C-terminal residues of cardiac troponin T leads to hypertrophic cardiomyopathy. We showed earlier that regulated actin containing Δ14 TnT was more readily activated than wild-type regulated actin. We suggested that the equilibria among the inactive (blocked), intermediate (closed or calcium), and active (open or myosin) states was shifted to the active state. We now show that, in addition, such regulated actin filaments cannot enter the inactive or blocked state. Regulated actin containing Δ14 TnT had ATPase activities in the absence of Ca2+ that were higher than wild-type filaments but far below the fully active rate. The rapid dissociation of S1-ATP from regulated actin filaments containing Δ14 TnT and acrylodan-labeled tropomyosin did not show the fluorescence increase characteristic of moving to the inactive state. Replacing wild-type TnI with S45E TnI, that favors the inactive state, did not restore the fluorescence change. We conclude that TnT has a previously unrecognized role in forming the inactive state of regulated actin.     

X-ray diffraction studies on oriented gels of vertebrate smooth muscle thin filaments.

The ATPase activity of acto-myosin subfragment 1 (S1) at low ratios of S1 to actin in the presence of tropomyosin is dependent on the tropomyosin source and ionic conditions. Whereas skeletal muscle tropomyosin causes a 60% inhibitory effect at all ionic strengths, the effect of smooth muscle tropomyosin was found to be dependent on the ionic strength. At low ionic strength (20 mM) smooth muscle tropomyosin inhibits the ATPase activity by 60%, while at high ionic strength (120 mM) it potentiates the ATPase activity three- to five-fold. Therefore, the difference in the effect of smooth muscle and skeletal muscle tropomyosin on the acto-S1 ATPase activity was due to a greater fraction of the tropomyosin-actin complex being turned on in the absence of S1 with smooth muscle tropomyosin than with skeletal muscle tropomyosin. Using well-oriented gels of actin and of reconstituted specimens from vertebrate smooth muscle thin filament proteins suitable for X-ray diffraction, we localized the position of tropomyosin on actin under different levels of acto-S1 ATPase activity. By analysing the equatorial X-ray pattern of the oriented specimens in combination with solution scattering experiments, we conclude that tropomyosin is located at a binding radius of about 3.5 nm on the f-actin helix under all conditions studied. Furthermore, we find no evidence that the azimuthal position of tropomyosin is different for smooth muscle tropomyosin at various ionic strengths, or vertebrate tropomyosin, since the second actin layer-line intensity (at 17.9 nm axial and 4.3 nm radial spacing), which was shown in skeletal muscle to be a sensitive measure of this parameter, remains strong and unchanged. Differences in the ATPase activity are not necessarily correlated with different positions of tropomyosin on f-actin. The same conclusion is drawn from our observations that, although the regulatory protein caldesmon inhibits the ATPase activity in native and reconstituted vertebrate smooth muscle thin filaments at a molar ratio of actin/tropomyosin/caldesmon of 28:7:1, the second actin layer-line remains strong. Only adding caldesmon in excess reduces the intensity of the second actin layer-line, from which the binding radius of caldesmon can be estimated to be about 4 nm. The lack of predominant meridional reflections in oriented specimens, with caldesmon present, suggests that caldesmon does not project away from the thin filament as troponin molecules in vertebrate striated muscle in agreement with electron micrographs of smooth muscle thin filaments. In freshly prepared native smooth muscle thin filaments we observed a Ca(2+)-sensitive reversible bundling effect.(ABSTRACT TRUNCATED AT 400 WORDS)     

Comparison of Ca2+-dependent effects of caldesmon-tropomyosin-calmodulin and troponin-tropomyosin complexes on the structure of F-actin in ghost fibers and its interaction with myosin heads

Comparison of two types of Ca2+-regulated thin filament, reconstructed in ghost fibers by incorporating either caldesmon-gizzard tropomyosin-calmodulin or skeletal muscle troponin-tropomyosin complex, was performed by polarized microphotometry. The changes in actin structure under the influence of these regulatory complexes, as well as those upon the binding of the myosin heads, were followed by measurements of F-actin intrinsic tryptophan fluorescence and the fluorescence of phalloidin-rhodamine complex attached to F-actin. The results show that in the presence of smooth muscle tropomyosin and calmodulin, caldesmon causes Ca2+-dependent alterations of actin conformation and flexibility similar to those induced by skeletal muscle troponin-tropomyosin complex. In both cases, transferring of the fiber from '-Ca2+' to '+Ca2+' solution increases the number of turned-on actin monomers. However, whereas troponin in the absence of Ca2+ potentiates the effect of skeletal muscle tropomyosin, caldesmon-calmodulin complex inhibits the effect of smooth muscle tropomyosin. This difference seems to be due to the qualitatively different alterations in the structure and flexibility of F-actin in ghost fibers evoked by smooth and skeletal muscle tropomyosins. Troponin can bind to F-actin-smooth muscle tropomyosin-caldesmon complex and, in the presence of Ca2+, release the restraint by caldesmon for S-1-induced alterations of conformation, and reduce that for flexibility of actin in ghost fibers. This effect seems to be related to the abolishment by troponin of the potentiating effect of tropomyosin on caldesmon-induced inhibition of actomyosin ATPase activity.     

Three-dimensional image reconstruction of reconstituted smooth muscle thin filaments: effects of caldesmon

Caldesmon inhibits actomyosin ATPase and filament sliding in vitro, and therefore may play a role in modulating smooth and non-muscle motile activities. A bacterially expressed caldesmon fragment, 606C, which consists of the C-terminal 150 amino acids of the intact molecule, possesses the same inhibitory properties as full-length caldesmon and was used in our structural studies to examine caldesmon function. Three-dimensional image reconstruction was carried out from electron micrographs of negatively stained, reconstituted thin filaments consisting of actin and smooth muscle tropomyosin both with and without added 606C. Helically arranged actin monomers and tropomyosin strands were observed in both cases. In the absence of 606C, tropomyosin adopted a position on the inner edge of the outer domain of actin monomers, with an apparent connection to sub-domain 1 of actin. In 606C-containing filaments that inhibited acto-HMM ATPase activity, tropomyosin was found in a different position, in association with the inner domain of actin, away from the majority of strong myosin binding sites. The effect of caldesmon on tropomyosin position therefore differs from that of troponin on skeletal muscle filaments, implying that caldesmon and troponin act by different structural mechanisms.     

The mechanism of smooth muscle caldesmon-tropomyosin inhibition of the elementary steps of the actomyosin ATPase

Caldesmon is a component of smooth muscle thin filaments that inhibits the actomyosin ATPase via its interaction with actin-tropomyosin. We have performed a comprehensive transient kinetic characterization of the actomyosin ATPase in the presence of smooth muscle caldesmon and tropomyosin. At physiological ratios of caldesmon to actin (1 caldesmon/7 actin monomers) actomyosin ATPase is inhibited by about 75%. Inhibitory caldesmon concentrations had little effect upon the rate of S1 binding to actin, actin-S1 dissociation by ATP, and dissociation of ADP from actin-S1 x ADP; however the rate of phosphate release from the actin-S1 x ADP x P(i) complex was decreased by more than 80%. In addition the transient of phosphate release displayed a lag of up to 200 ms. The presence of a lag phase indicates that a step on the pathway prior to phosphate release has become rate-limiting. Premixing the actin-tropomyosin filaments with myosin heads resulted in the disappearance of the lag phase. We conclude that caldesmon inhibition of the rate of phosphate release is caused by the thin filament being switched by caldesmon to an inactive state. The active and inactive states correspond to the open and closed states observed in skeletal muscle thin filaments with no evidence for the existence of a third, blocked state. Taken together these data suggest that at physiological concentrations, caldesmon controls the isomerization of the weak binding complex to the strong binding complex, and this causes the inhibition of the rate of phosphate release. This inhibition is sufficient to account for the inhibition of the steady state actomyosin ATPase by caldesmon and tropomyosin.     

Caldesmon inhibits skeletal actomyosin subfragment-1 ATPase activity and the binding of myosin subfragment-1 to actin

Smooth muscle contraction is controlled in part by the state of phosphorylation of myosin. A recently discovered actin and calmodulin-binding protein, named caldesmon, may also be involved in regulation of smooth muscle contraction. Caldesmon cross-links actin filaments and also inhibits actin-activated ATP hydrolysis by myosin, particularly in the presence of tropomyosin. We have studied the effect of caldesmon on the rate of hydrolysis of ATP by skeletal muscle myosin subfragment-1, a system in which phosphorylation of the myosin is not important in regulation. Caldesmon is a very effective inhibitor of ATP hydrolysis giving up to 95% inhibition. At low ionic strength (approximately 20 mM) this effect does not require smooth muscle tropomyosin, whereas at high ionic strength (approximately 120 mM) tropomyosin enhances the inhibitory activity of caldesmon at low caldesmon concentrations. Cross-linking of actin is not essential for inhibition of ATP hydrolysis to occur since at high ionic strength there is very little cross-linking as determined by a low speed sedimentation assay. Under all conditions examined, the decrease in the rate of ATP hydrolysis is accompanied by a decrease in the binding of myosin subfragment-1 to actin. Furthermore, caldesmon weakens the equilibrium binding of myosin subfragment-1 to actin in the presence of pyrophosphate. We conclude that caldesmon has a general weakening effect on the binding of skeletal muscle myosin subfragment-1 to actin and that this weakening in binding may be responsible for inhibition of ATP hydrolysis.     

Phosphorylation of smooth muscle myosin heads regulates the head-induced movement of tropomyosin

It has been shown that skeletal and smooth muscle myosin heads binding to actin results in the movement of smooth muscle tropomyosin, as revealed by a change in fluorescence resonance energy transfer between a fluorescence donor on tropomyosin and an acceptor on actin (Graceffa, P. (1999) Biochemistry 38, 11984-11992). In this work, tropomyosin movement was similarly monitored as a function of unphosphorylated and phosphorylated smooth muscle myosin double-headed fragment smHMM. In the absence of nucleotide and at low myosin head/actin ratios, only phosphorylated heads induced a change in energy transfer. In the presence of ADP, the effect of head phosphorylation was even more dramatic, in that at all levels of myosin head/actin, phosphorylation was necessary to affect energy transfer. It is proposed that the regulation of tropomyosin position on actin by phosphorylation of myosin heads plays a key role in the regulation of smooth muscle contraction. In contrast, actin-bound caldesmon was not moved by myosin heads at low head/actin ratios, as uncovered by fluorescence resonance energy transfer and disulfide cross-linking between caldesmon and actin. At higher head concentration caldesmon was dissociated from actin, consistent with the multiple binding model for the binding of caldesmon and myosin heads to actin (Chen, Y., and Chalovich, J. M. (1992) Biophys. J. 63, 1063-1070).     

Caldesmon freezes the structure of actin filaments during the actomyosin ATPase cycle

Hybrid contractile apparatus was reconstituted in skeletal muscle ghost fibers by incorporation of skeletal muscle myosin subfragment 1 (S1), smooth muscle tropomyosin and caldesmon. The spatial orientation of FITC-phalloidin-labeled actin and IAEDANS-labeled S1 during sequential steps of the acto-S1 ATPase cycle was studied by measurement of polarized fluorescence in the absence or presence of nucleotides conditioning the binding affinity of both proteins. In the fibers devoid of caldesmon addition of nucleotides evoked unidirectional synchronous changes in the orientation of the fluorescent probes attached to F-actin or S1. The results support the suggestion on the multistep rotation of the cross-bridge (myosin head and actin monomers) during the ATPase cycle. The maximal cross-bridge rotation by 7 degrees relative to the fiber axis and the increase in its rigidity by 30% were observed at transition between A**.M**.ADP.Pi (weak binding) and A--.M--.ADP (strong binding) states. When caldesmon was present in the fibers (OFF-state of the thin filament) the unidirectional changes in the orientation of actin monomers and S1 were uncoupled. The tilting of the myosin head and of the actin monomer decreased by 29% and 90%, respectively. It is suggested that in the "closed" position caldesmon "freezes" the actin filament structure and induces the transition of the intermediate state of actomyosin towards the weak-binding states, thereby inhibiting the ATPase activity of the actomyosin.     

Competitive binding of the troponin T-specific pool of caldesmon antibodies and tropomyosin to skeletal troponin T and smooth muscle caldesmon.

The fraction of polyclonal caldesmon antibodies cross-reacting with rabbit skeletal troponin T are shown to compete with smooth muscle tropomyosin for caldesmon and troponin T, as revealed by ELISA method. The epitope recognized by these antibodies was also found in Mr 77 kDa non-muscle caldesmon. These results provide functional confirmation for the suggestion that the regions of amino acid sequence homology in caldesmon isoforms and troponin T belong to the tropomyosin binding sites.     

The effect of caldesmon on actin-myosin interaction in skeletal muscle fibers

The effects of caldesmon on structural and dynamic properties of phalloidin-rhodamine-labeled F-actin in single skeletal muscle fibers were investigated by polarized microphotometry. The binding of caldesmon to F-actin in glycerinated fibers reduced the alterations of thin filaments structure and dynamics that occur upon the transition of the fibers from rigor to relaxing conditions. In fibers devoid of myosin and regulatory proteins (ghost fibers) the binding of caldesmon to F-actin precluded structural changes in actin filaments induced by skeletal muscle myosin subfragment 1 and smooth muscle tropomyosin. These results suggest that the restraint for the alteration of actin structure and dynamics upon binding of myosin heads and/or tropomyosin evoked by caldesmon can be related to its inhibitory effect on actin-myosin interaction.     

Caldesmon reduces the apparent rate of binding of myosin S1 to actin-tropomyosin

Equilibrium measurements of the rate of binding of caldesmon and myosin S1 to actin-tropomyosin from different laboratories have yielded different results and have led to different models of caldesmon function. An alternate approach to answering these questions is to study the kinetics of binding of both caldesmon and S1 to actin. We observed that caldesmon decreased the rate of binding of S1 to actin in a concentration-dependent manner. The inhibition of the rate of S1 binding was enhanced by tropomyosin, but the effect of tropomyosin on the binding was small. Premixing actin with S1 reduced the amplitude (extent) of caldesmon binding in proportion to the fraction of actin that contained bound S1, but the rate of binding of caldesmon to free sites was not greatly altered. No evidence for a stable caldesmon-actin-tropomyosin-S1 complex was observed, although S1 did apparently bind to gaps between caldesmon molecules. These results indicate that experiments involving caldesmon, actin, tropomyosin, and myosin are inherently complex. When the concentration of either S1 or caldesmon is varied, the amount of the other component bound to actin-tropomyosin cannot be assumed to remain fixed. The results are not readily explained by a mechanism in which caldesmon acts only by stabilizing an inactive state of actin-tropomyosin. The results support regulatory mechanisms that involve changes in the actin-S1 interaction.     

Role of caldesmon in the Ca2+ regulation of smooth muscle thin filaments: evidence for a cooperative switching mechanism

Smooth muscle thin filaments are made up of actin, tropomyosin, caldesmon, and a Ca(2+)-binding protein and their interaction with myosin is Ca(2+)-regulated. We suggested that Ca(2+) regulation by caldesmon and Ca(2+)-calmodulin is achieved by controlling the state of thin filament through a cooperative-allosteric mechanism homologous to troponin-tropomyosin in striated muscles. In the present work, we have tested this hypothesis. We monitored directly the thin filament transition between the ON and OFF state using the excimer fluorescence of pyrene iodoacetamide (PIA)-labeled smooth muscle alphaalpha-tropomyosin homodimers. In steady state fluorescence measurements, myosin subfragment 1 (S1) cooperatively switches the thin filaments to the ON state, and this is exhibited as an increase in the excimer fluorescence. In contrast, caldesmon decreases the excimer fluorescence, indicating a switch of the thin filament to the OFF state. Addition of Ca(2+)-calmodulin increases the excimer fluorescence, indicating a switch of the thin filament to the ON state. The excimer fluorescence was also used to monitor the kinetics of the ON-OFF transition in a stopped-flow apparatus. When ATP induces S1 dissociation from actin-PIA-tropomyosin, the transition to the OFF state is delayed until all S1 molecules are dissociated actin. In contrast, caldesmon switches the thin filament to the OFF state in a cooperative way, and no lag is displayed in the time course of the caldesmon-induced fluorescence decrease. We have also studied caldesmon and Ca(2+)-calmodulin-caldesmon binding to actin-tropomyosin in the ON and OFF states. The results are used to discuss both caldesmon inhibition and Ca(2+)-calmodulin-caldesmon activation of actin-tropomyosin.     

Caldesmon and myosin subfragment-1 act differently on the structural state of 1,5-IAEDANS-modified tropomyosin in ghost muscle fibers

The effect of caldesmon (CD) and subfragment 1 of myosin (S1) on the structural state of tropomyosin (TM) modified with N-(iodoacetyl)-N-(1-naphthyl-5-sulfo)-ethylene-diamine (1.5-IAEDANS) in single myosin-free skeletal muscle fibers was studied using polarized microfluorimetry. S1 was performed from skeletal muscles of rabbits, whereas CD and TM were prepared from the smooth muscle of chicken gizzards. An analysis of experimental data revealed that CD initiates and increases the motility of 1.5-IAEDANS-TM, while S1 decreases it. In the presence of CD S1 binding to actin is accompanied by significant changes in the fluorescent label motility. It is supposed that CD and S1 induce in TM conformational changes which interfere with the protein interaction with F-actin.     

C-terminal actin-binding sites of smooth muscle caldesmon switch actin between conformational states

Caldesmon is a component of the thin filaments of smooth muscles where it is believed to play an essential role in regulating the thin filaments’ interaction with myosin and hence contractility. We studied the effects of caldesmon and two recombinant fragments CaDH1 (residues 506–793) and CaDH2 (residues 683–767) on the structure of actin–tropomyosin by making measurements of the fluorescence polarisation of probes specifically attached to actin. CaDH1, like the parent molecule caldesmon, is an inhibitor of actin–tropomyosin interaction with myosin whilst CaDH2 is an activator. The F-actin in permeabilised and myosin free rabbit skeletal muscle ‘ghost’ fibres was labelled by tetramethyl rhodamine-isothiocyanate (TRITC)–phalloidin or fluorescein-5′-isothiocyanate (FITC) at lysine 61. Fluorescence polarisation measurements were made and the parameters Φ_A, Φ_E, Θ_1/2 and N were calculated. Φ_A and Φ_E are angles between the fiber axis and the absorption and emission dipoles, respectively; Θ_1/2 is the angle between the F-actin filament axis and the fiber axis; N is the relative number of randomly oriented fluorophores. Actin–tropomyosin interaction with myosin subfragment-1 induced changes in the parameters of the polarised fluorescence that are typical of strong binding of myosin to actin and of the ‘on’ conformational state of actin. Caldesmon and CaDH1 (as well as troponin in the absence of Ca²⁺) diminished the effect of S-1, whereas CaDH2 (as well as troponin in the presence of Ca²⁺) enhanced the effect of S1. Thus the structural evidence correlates with biochemical evidence that C-terminal actin-binding sites of caldesmon can modulate the structural transition of actin monomers between ‘off’ (caldesmon and CaDH1) and ‘on’ (S-1 and CaDH2) states in a manner analogous to troponin.     

The troponin tail domain promotes a conformational state of the thin filament that suppresses myosin activity

In cardiac and skeletal muscles tropomyosin binds to the actin outer domain in the absence of Ca(2+), and in this position tropomyosin inhibits muscle contraction by interfering sterically with myosin-actin binding. The globular domain of troponin is believed to produce this B-state of the thin filament (Lehman, W., Hatch, V., Korman, V. L., Rosol, M., Thomas, L. T., Maytum, R., Geeves, M. A., Van Eyk, J. E., Tobacman, L. S., and Craig, R. (2000) J. Mol. Biol. 302, 593-606) via troponin I-actin interactions that constrain the tropomyosin. The present study shows that the B-state can be promoted independently by the elongated tail region of troponin (the NH(2) terminus (TnT-(1-153)) of cardiac troponin T). In the absence of the troponin globular domain, TnT-(1-153) markedly inhibited both myosin S1-actin-tropomyosin MgATPase activity and (at low S1 concentrations) myosin S1-ADP binding to the thin filament. Similarly, TnT-(1-153) increased the concentration of heavy meromyosin required to support in vitro sliding of thin filaments. Electron microscopy and three-dimensional reconstruction of thin filaments containing TnT-(1-153) and either cardiac or skeletal muscle tropomyosin showed that tropomyosin was in the B-state in the complete absence of troponin I. All of these results indicate that portions of the troponin tail domain, and not only troponin I, contribute to the positioning of tropomyosin on the actin outer domain, thereby inhibiting muscle contraction in the absence of Ca(2+).     

The binding of caldesmon to actin and its effect on the ATPase activity of soluble myosin subfragments in the presence and absence of tropomyosin

The binding of 125I- and 14C-caldesmon to actin and actin-tropomyosin was studied using a cosedimentation technique and was analyzed by the method of McGhee and von Hippel [1974) J. Mol. Biol. 86, 469-489) for the binding of large ligands to a homogeneous lattice. The binding was adequately described by a single class of binding sites with a stoichiometry between 1:7 and 1:10. The binding exhibited a small degree of positive cooperativity (omega = 5-6) which was the same in the presence and absence of tropomyosin. The association constant for the binding of caldesmon to an isolated binding site was enhanced, from about 6 X 10(5) to about 1.4 X 10(6) M-1, by the presence of smooth muscle tropomyosin. Caldesmon inhibited the actin-activated ATPase activity of skeletal myosin subfragment 1 in both the absence and presence of tropomyosin. Maximum inhibition of ATPase activity occurred when one caldesmon molecule bound to seven actin monomers. A greater degree of inhibition was observed in the presence of tropomyosin than in the absence. This greater inhibition cannot be explained totally by the increased strength of binding of caldesmon to actin in the presence of tropomyosin. Finally, Ca2+-calmodulin completely reversed the binding of caldesmon to actin.     

Behavior of caldesmon upon interaction of thin filaments with myosin subfragment 1 in ghost fibers

Caldesmon is a component of smooth muscle thin filaments which inhibits their interaction with myosin. We have used polarized fluorescence technique to study the behavior of caldesmon during the interaction of myosin subfragment 1 (S1) with thin filaments reconstituted in rabbit skeletal muscle ghost fibers by incorporation of smooth muscle tropomyosin and caldesmon labeled with acrylodan at cysteine residue located in the C-terminal region. Significant changes in acrylodan fluorescence intensity upon addition of skeletal muscle S1 reflected substantial displacement of caldesmon from thin filaments, while alterations in the calculated fluorescence parameters indicated the simultaneous rearrangement of the remaining caldesmon fraction. The orientation of caldesmon in the S1-thin filament complex relative to the fiber axis changes by approximately 7 degrees and the mobility of the fluorescent probe by about 9%. The alterations in caldesmon orientation were proportional to the strength of S1 binding and diminished respectively upon addition of ADP and ADP-V(i). The changes in orientation of acrylodan-caldesmon evoked by the interaction of S1 with thin filaments were more pronounced than that in AEDANS-F-actin which suggests that the spatial arrangement of caldesmon in the complex is governed not only by F-actin but also by S1. The results may indicate that the changes in spatial arrangement of caldesmon are adjusted to the conformation of F-actin and S1 characteristic for particular steps of the ATP hydrolysis cycle.     

Three-dimensional reconstruction of caldesmon-containing smooth muscle thin filaments

Caldesmon is known to inhibit actomyosin ATPase and filament sliding in vitro, and may play a role in modulating smooth muscle contraction as well as in diverse cellular processes including cytokinesis and exocytosis. However, the structural basis of caldesmon action has not previously been apparent. We have recorded electron microscope images of negatively stained thin filaments containing caldesmon and tropomyosin which were isolated from chicken gizzard smooth muscle in EGTA. Three-dimensional helical reconstructions of these filaments show actin monomers whose bilobed shape and connectivity are very similar to those previously seen in reconstructions of frozen-hydrated skeletal muscle thin filaments. In addition, a continuous thin strand of density follows the long-pitch actin helices, in contact with the inner domain of each actin monomer. Gizzard thin filaments treated with Ca2+/calmodulin, which dissociated caldesmon but not tropomyosin, have also been reconstructed. Under these conditions, reconstructions also reveal a bilobed actin monomer, as well as a continuous surface strand that appears to have moved to a position closer to the outer domain of actin. The strands seen in both EGTA- and Ca2+/calmodulin-treated filaments thus presumably represent tropomyosin. It appears that caldesmon can fix tropomyosin in a particular position on actin in the absence of calcium. An influence of caldesmon on tropomyosin position might, in principle, account for caldesmon's ability to modulate actomyosin interaction in both smooth muscles and non-muscle cells.     

Interaction between caldesmon and tropomyosin in the presence and absence of smooth muscle actin

Cysteine residues of caldesmon were labeled with the fluorescent reagent N-(1-pyrenyl)maleimide. The number of sulfhydryl (SH) groups in caldesmon was around 3.5 on the basis of reactivity to 5,5'-dithiobis(2-nitrobenzoate); 80% of the SH groups were labeled with pyrene. The fluorescence spectrum from pyrene-caldesmon showed the presence of excited monomer and dimer (excimer). As the ionic strength increased, excimer fluorescence decreased, disappearing at salt concentrations higher than around 50 mM. The labeling of caldesmon with pyrene did not affect its ability to inhibit actin activation of heavy meromyosin Mg-ATPase and the release of this inhibition in the presence of Ca2+-calmodulin. Tropomyosin induced a change in the fluorescence spectrum of pyrene-caldesmon, indicating a conformational change associated with the interaction between caldesmon and tropomyosin. The affinity of caldesmon to tropomyosin was dependent on ionic strength. The binding constant was 5 x 10(6) M-1 in low salt, and the affinity was 20-fold less at ionic strengths close to physiological conditions. In the presence of actin, the affinity of caldesmon to tropomyosin was increased 5-fold. The addition of tropomyosin also changed the fluorescence spectrum of pyrene-caldesmon bound to actin filaments. The change in the conformation of tropomyosin, caused by the interaction between caldesmon and tropomyosin, was studied with pyrene-labeled tropomyosin. Fluorescence change was evident when unlabeled caldesmon was added to pyrene-tropomyosin bound to actin. These data suggest that the interaction between caldesmon and tropomyosin on the actin filament is associated with conformational changes on these thin filament associated proteins. These conformational changes may modulate the ability of thin filament to interact with myosin heads.     

Effect of caldesmon on the ATPase activity and the binding of smooth and skeletal myosin subfragments to actin

We have previously shown that inhibition of the ATPase activity of skeletal muscle myosin subfragment 1 (S1) by caldesmon is correlated with the inhibition of S1 binding in the presence of ATP or pyrophosphate (Chalovich, J., Cornelius, P., and Benson, C. (1987) J. Biol Chem. 262, 5711-5716). In contrast, Lash et al. (Lash, J., Sellers, J., and Hathaway, D. (1986) J. Biol. Chem. 261, 16155-16160) have shown that the inhibition of ATPase activity of smooth muscle heavy meromyosin (HMM) by caldesmon is correlated with an increase in the binding of HMM to actin in the presence of ATP. We now show, in agreement, that caldesmon does increase the binding of smooth muscle HMM to actin-tropomyosin while decreasing the ATPase activity. The effect of caldesmon on the binding of smooth HMM is reversed by Ca2+-calmodulin. Caldesmon strengthens the binding of smooth S1.ATP and skeletal HMM.ATP to actin-tropomyosin but to a lesser extent than smooth HMM.ATP. Furthermore, this increase in binding of smooth S1.ATP and skeletal HMM.ATP does not parallel the inhibition of ATPase activity. In contrast, in the absence of ATP, all smooth and skeletal myosin subfragments compete with caldesmon for binding to actin. Thus, the effect that caldesmon has on the binding of myosin subfragments to actin-tropomyosin depends on the source of myosin, the type of subfragment, and the nucleotide present. The inhibition of actin-activated ATP hydrolysis by caldesmon, however, is not greatly different for different smooth and skeletal myosin subfragments. Evidence is presented that caldesmon inhibits actin-activated ATP hydrolysis by attenuating the productive interaction between myosin and actin that normally accelerates ATP hydrolysis. The increased binding seen by some myosin subfragments, in the presence of ATP, may be due to binding of these subfragments to a nonproductive site on actin-caldesmon. The subfragments which show an increase in binding in the presence of ATP and caldesmon appear to bind directly to caldesmon as demonstrated by affinity chromatography.