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.     

Ca2+-calmodulin binding to caldesmon and the caldesmon-actin-tropomyosin complex. Its role in Ca2+ regulation of the activity of synthetic smooth-muscle thin filaments.

We measured the concentration of calmodulin required to reverse inhibition by caldesmon of actin-activated myosin MgATPase activity, in a model smooth-muscle thin-filament system, reconstituted in vitro from purified vascular smooth-muscle actin, tropomyosin and caldesmon. At 37 degrees C in buffer containing 120 mM-KCl, 4 microM-Ca2+-calmodulin produced a half-maximal reversal of caldesmon inhibition, but more than 300 microM-Ca2+-calmodulin was necessary at 25 degrees C in buffer containing 60 mM-KCl. The binding affinity (K) of caldesmon for Ca2+-calmodulin was measured by a fluorescence-polarization method: K = 2.7 x 10(6) M-1 at 25 degrees C (60 mM-KCl); K = 1.4 x 10(6) M-1 at 37 degrees C in 70 mM-KCl-containing buffer; K = 0.35 x 10(6) M-1 at 37 degrees C in 120 mM-KCl- containing buffer (pH 7.0). At 37 degrees C/120 mM-KCl, but not at 25 degrees C/60 mM-KCl, Ca2+-calmodulin bound to caldesmon bound to actin-tropomyosin (K = 2.9 x 10(6) M-1). Ca2+ regulation in this system does not depend on a simple competition between Ca2+-calmodulin and actin for binding to caldesmon. Under conditions (37 degrees C/120 mM-KCl) where physiologically realistic concentrations of calmodulin can Ca2+-regulate synthetic thin filaments, Ca2+-calmodulin reverses caldesmon inhibition of actomyosin ATPase by forming a non-inhibited complex of Ca2+-calmodulin-caldesmon-(actin-tropomyosin).     

Bundling of actin filaments by aorta caldesmon is not related to its regulatory function

Ca2+-sensitive thin filaments from vascular smooth muscle were disassembled into their constituent proteins, actin, tropomyosin and caldesmon. Caldesmon bound to both actin and to actin-tropomyosin and inhibited actin-tropomyosin activation of skeletal muscle myosin MgATPase. It also promoted the aggregation of actin or actin-tropomyosin into parallel aligned bundles. Quantitative electron microscopy measurements showed that with 1.1 microM actin-tropomyosin, 1.6 +/- 0.5% (n = 3) of the filaments were in bundles. At 0.073 microM, caldesmon inhibited MgATPase activity by 50%, whereas bundling was 3.0 +/- 1.3% (n = 4). At 0.37 microM caldesmon, MgATPase inhibition was 83% while 28.1 +/- 6.9% (n = 4) of filaments were in bundles. Experiments at 4.4 microM in which MgATPase and bundling were measured in the same samples gave similar results. Small bundles of 2-3 filaments showed the most frequent occurrence at 1.1 microM actin. At 4.4 microM actin the most common bundle size was 3-5 filaments, with the occasional occurrence of large bundles consisting of up to 120 filaments. The incidence of bundling was the same in the presence and absence of tropomyosin. Thus caldesmon can induce the formation of actin bundles but this property bears no relationship to its inhibition of MgATPase activity.     

The functional properties of full length and mutant chicken gizzard smooth muscle caldesmon expressed in Escherichia coli

Wild type chicken gizzard caldesmon (756 amino acids) was expressed in a T7 RNA polymerase-based bacterial expression system at a yield of 1 mg pure caldesmon per litre bacterial culture. A mutant composed of amino acids 1-578 was also constructed and expressed. The wild type and mutant caldesmon were purified and compared with native chicken gizzard caldesmon. Native and wild type expressed caldesmon were indistinguishable in assays for inhibition of actin-tropomyosin activation of myosin ATPase, reversal of inhibition by Ca²⁺-calmodulin and binding to actin, actin-tropomyosin, Ca²⁺-calmodulin, tropomyosin and myosin. The mutant missing the C-terminal 178 amino acids had no inhibitory effect and did not bind to actin or Ca²⁺-calmodulin. It bound to tropomyosin with a 5-fold reduced affinity and to myosin with a greater than 10-fold reduced affinity.     

Kinetics of the structural transition of muscle thin filaments observed by fluorescence resonance energy transfer

Fluorescence resonance energy transfer showed that troponin-I changes the position on an actin filament corresponding to three states (relaxed, closed, and open) of the thin filament (Hai et al. (2002) J. Biochem. 131, 407-418). In combination with the stopped-flow method, fluorescence resonance energy transfer between probes attached to position 1, 133, or 181 of troponin-I and Cys-374 of actin on reconstituted thin filaments was measured to follow the transition between three states of the thin filament. When the free Ca(2+) concentration was increased, the transition from relaxed to closed states occurred with a rate constant of approximately 500 s(-1). For the reverse transition, the rate constant was approximately 60 s(-1). When myosin subfragment-1 was dissociated from thin filaments in the presence of Ca(2+) by rapid mixing with ATP, the transition from open to closed states occurred with a single rate constant of approximately 300 s(-1). Light-scattering measurements showed that the ATP-induced myosin subfragment-1 dissociation occurred with a rate constant of approximately 900 s(-1). In the absence of Ca(2+), the transition from open to relaxed states occurred with two rate constants of approximately 400 and approximately 80 s(-1). These transition rates are fast enough to allow the spatial rearrangement of thin filaments to be involved in the regulation mechanism of muscle contraction.     

Dual regulatory functions of the thin filament revealed by replacement of the troponin I inhibitory peptide with a linker

Striated muscles are relaxed under low Ca(2+) concentration conditions due to actions of the thin filament protein troponin. To investigate this regulatory mechanism, an 11-residue segment of cardiac troponin I previously termed the inhibitory peptide region was studied by mutagenesis. Several mutant troponin complexes were characterized in which specific effects of the inhibitory peptide region were abrogated by replacements of 4-10 residues with Gly-Ala linkers. The mutations greatly impaired two of troponin's actions under low Ca(2+) concentration conditions: inhibition of myosin subfragment 1 (S1)-thin filament MgATPase activity and cooperative suppression of myosin S1-ADP binding to thin filaments with low myosin saturation. Inhibitory peptide replacement diminished but did not abolish the Ca(2+) dependence of the ATPase rate; ATPase rates were at least 2-fold greater when Ca(2+) rather than EGTA was present. This residual regulation was highly cooperative as a function of Ca(2+) concentration, similar to the degree of cooperativity observed with WT troponin present. Other effects of the mutations included 2-fold or less increases in the apparent affinity of the thin filament regulatory Ca(2+) sites, similar decreases in the affinity of troponin for actin-tropomyosin regardless of Ca(2+), and increases in myosin S1-thin filament ATPase rates in the presence of saturating Ca(2+). The overall results indicate that cooperative myosin binding to Ca(2+)-free thin filaments depends upon the inhibitory peptide region but that a cooperatively activating effect of Ca(2+) binding does not. The findings suggest that these two processes are separable and involve different conformational changes in the thin filament.     

Activation of smooth muscle myosin Mg2+-ATPase by native thin filaments and actin/tropomyosin

Application of the myosin competition test (Lehman, W., and Szent-Gy?rgyi, A. G. (1975) J. Gen. Physiol. 66, 1-30) to chicken gizzard actomyosin indicated that this smooth muscle contains a thin filament-linked regulatory mechanism. Chicken gizzard thin filaments, isolated as described previously (Marston, S. B., and Lehman, W. (1985) Biochem. J. 231, 517-522), consisted almost exclusively of actin, tropomyosin, caldesmon, and an unidentified 32-kilodalton polypeptide in molar ratios of 1:1/6:1/26:1/17, respectively. When reconstituted with phosphorylated gizzard myosin, these thin filaments conferred Ca2+ sensitivity (67.8 +/- 2.1%; n = 5) on the myosin Mg2+-ATPase. On the other hand, no Ca2+ sensitivity of the myosin Mg2+-ATPase was observed when purified gizzard actin or actin plus tropomyosin was reconstituted with phosphorylated gizzard myosin. Native thin filaments were rendered essentially free of caldesmon and the 32-kilodalton polypeptide by extraction with 25 mM MgCl2. When reconstituted with phosphorylated gizzard myosin, caldesmon-free thin filaments and native thin filaments exhibited approximately the same Ca2+ sensitivity (45.1 and 42.7%, respectively). The observed Ca2+ sensitivity appears, therefore, not to be due to caldesmon. Only trace amounts of two Ca2+-binding proteins could be detected in native thin filaments. These were identified as calmodulin (present at a molar ratio to actin of 1:733) and the 20-kilodalton light chain of myosin (present at a molar ratio to actin of 1:270). The Ca2+ sensitivity observed in an in vitro system reconstituted from gizzard thin filaments and either skeletal myosin or phosphorylated gizzard myosin is due, therefore, to calmodulin and/or an unidentified minor protein component of the thin filaments which may be an actin-binding protein involved in regulating actin filament structure in a Ca2+-dependent manner.     

Three-dimensional reconstruction of thin filaments containing mutant tropomyosin

Interactions of the components of reconstituted thin filaments were investigated using a tropomyosin internal deletion mutant, D234, in which actin-binding pseudo-repeats 2, 3, and 4 are missing. D234 retains regions of tropomyosin that bind troponin and form end-to-end tropomyosin bonds, but has a length to span only four instead of seven actin monomers. It inhibits acto-myosin subfragment 1 ATPase (acto-S-1 ATPase) and filament sliding in vitro in both the presence and absence of Ca(2+) (, J. Biol. Chem. 272:14051-14056) and lowers the affinity of S-1.ADP for actin while increasing its cooperative binding. Electron microscopy and three-dimensional reconstruction of reconstituted thin filaments containing actin, troponin, and wild-type or D234 tropomyosin were carried out to determine if Ca(2+)-induced movement of D234 occurred in the filaments. In the presence and absence of Ca(2+), the D234 position was indistinguishable from that of the wild-type tropomyosin, demonstrating that the mutation did not affect normal tropomyosin movement induced by Ca(2+) and troponin. These results suggested that, in the presence of Ca(2+) and troponin, D234 tropomyosin was trapped on filaments in the Ca(2+)-induced position and was unable to undergo a transition to a completely activated position. By adding small amounts of rigor-bonded N-ethyl-maleimide-treated S-1 to mutant thin filaments, thus mimicking the myosin-induced "open" state, inhibition could be overcome and full activation restored. This myosin requirement for full activation provides support for the existence of three functionally distinct thin filament states (off, Ca(2+)-induced, myosin-induced; cf.;, J. Mol. Biol. 266:8-14). We propose a further refinement of the three-state model in which the binding of myosin to actin causes allosteric changes in actin that promote the binding of tropomyosin in an otherwise energetically unfavorable "open" state.     

Disassembly and reconstitution of the Ca2+-sensitive thin filaments of vascular smooth muscle

The Ca2+-sensitive thin filaments of aorta smooth muscle have been, disassembled into their constituent proteins, actin, tropomyosin and a 120-kDa protein. The 120-kDa protein bound to aorta actin-tropomyosin and inhibited its ability to activate myosin MgATPase. This inhibition correlated with the binding of one 120-kDa protein molecule per 29 actin monomers. Upon the addition of calmodulin to the actin-tropomyosin-120-kDa protein complex, the inhibition was relieved in 10(-4) M Ca2+ but not 10(-9) M Ca2+. The full release of inhibition was not accompanied by a full release of 120-kDa protein binding to actin-tropomyosin. A fully active, Ca2+-sensitive aorta thin filament has thus been reconstituted from just four components: actin, tropomyosin, 120-kDa protein and calmodulin.     

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.     

Inhibition of actin-tropomyosin activation of myosin MgATPase activity by the smooth muscle regulatory protein caldesmon.

Caldesmon inhibition of actin-tropomyosin activation of myosin MgATPase activity was investigated. greater than 90% inhibition of ATPase activation correlated with 0.035-0.1 caldesmon bound per actin monomer over a wide range of conditions. Caldesmon inhibited sheep aorta actin-tropomyosin activation of skeletal muscle heavy meromyosin (HMM) by 85%, but had no effect on the binding affinity of HMM.ADP.Pi to actin. At ratios of 2 and 0.12 subfragment 1 (S1):1 actin, addition of caldesmon inhibited the ATPase activation by up to 95%, but did not alter the fraction of S1.ADP.Pi associated with actin-tropomyosin. We concluded that caldesmon inhibited actomyosin ATPase by slowing the rate-limiting step of the activation pathway. At concentrations comparable to the ATPase measurements, S1 displaced caldesmon from native thin filaments both in the absence (rigor) and the presence of MgATP. We therefore concluded that caldesmon could displace S1.ADP.Pi from actin-tropomyosin only under exceptional circumstances. An expressed mutant of caldesmon comprising just the C-terminal 99 amino acids bound actin 10 times weaker than whole caldesmon but otherwise inhibited actin-tropomyosin activation with the same potency and same mechanism as intact caldesmon. Thus, the entire inhibitory function of caldesmon resides in its extreme C terminus.     

The effects of smooth muscle caldesmon on actin filament motility.

The movement of reconstituted thin filaments over an immobilized surface of thiophosphorylated smooth muscle myosin was examined using an in vitro motility assay. Reconstituted thin filaments contained actin, tropomyosin, and either purified chicken gizzard caldesmon or the purified COOH-terminal actin-binding fragment of caldesmon. Control actin-tropomyosin filaments moved at a velocity of 2.3 +/- 0.5 microns/s. Neither intact caldesmon nor the COOH-terminal fragment, when maintained in the monomeric form by treatment with 10 mM dithiothreitol, had any effect on filament velocity; and yet both were potent inhibitors of actin-activated myosin ATPase activity, indicating that caldesmon primarily inhibits myosin binding as reported by Chalovich et al. (Chalovich, J. M., Hemric, M. E., and Velaz, L. (1990) Ann. N. Y. Acad. Sci. 599, 85-99). Inhibition of filament motion was, however, observed under conditions where cross-linking of caldesmon via disulfide bridges was present. To determine if monomeric caldesmon could "tether" actin filaments to the myosin surface by forming an actin-caldesmon-myosin complex as suggested by Chalovich et al., we looked for caldesmon-dependent filament binding and motility under conditions (80 mM KCl) where filament binding to myosin is weak and motility is not normally seen. At caldesmon concentrations > or = 0.26 microM, actin filament binding was increased and filament motion (2.6 +/- 0.6 microns/s) was observed. The enhanced motility seen with intact caldesmon was not observed with the addition of up to 26 microM COOH-terminal fragment. Moreover, a molar excess of the COOH-terminal fragment competitively reversed the enhanced binding seen with intact caldesmon. These results show that tethering of actin filaments to myosin by the formation of an actin-caldesmon-myosin complex enhanced productive acto-myosin interaction without placing a significant mechanical load on the moving filaments.     

Caldesmon: A calmodulin — Binding actin — Regulatory protein

The protein caldesmon, originally isolated from smooth muscle tissue where it is the most abundant calmodulin-binding protein, has since been shown to have a wide distribution in actin- and myosin- containing cells where it is localized in sub-cellular structures concerned with motility, shape changes and exo- or endo-cytosis. Caldesmon is believed to be an actin- regulatory protein, and binds with high affinity to actin or actin-tropomyosin. Caldesmon inhibits the activation by actin-tropomyosin of myosin MgATPase activity, and the inhibition can be reversed by Ca2+.calmodulin. The binding of caldesmon to smooth muscle proteins has been studied in detail, enabling a model to be constructed which could account for the observed Ca2+ regulation of smooth muscle thin filaments. The abundance of caldesmon, and the Ca2+-regulation of its activity via calmodulin, mean that it is potentially an important intracellular regulator of processes such as smooth muscle contraction, cell motility and secretion.     

The mechanism of Ca2+ regulation of vascular smooth muscle thin filaments by caldesmon and calmodulin

The interactions of vascular smooth muscle caldesmon with actin, tropomyosin, and calmodulin were determined under conditions in which the four proteins can form reconstituted Ca2+-sensitive smooth muscle thin filaments. Caldesmon bound to actin in a complex fashion with high affinity sites (K = 10(7) M-1) saturating at a stoichiometry of 1 per 28 actins, and lower affinity sites at 1 per 7 actins. The affinity of binding was increased in the presence of tropomyosin, and this could be attributed to a direct interaction between caldesmon and tropomyosin which was demonstrated using caldesmon cross-linked to Sepharose. In the presence of tropomyosin, occupancy of the high affinity sites was associated with inhibition of actin-activated myosin MgATPase activity. Caldesmon was found to bind to calmodulin in the presence of Ca2+, with an affinity of 10(6) M-1. The binding of Ca2+ X calmodulin to caldesmon was associated with the neutralization of inhibition of actin-tropomyosin. Ca2+ X calmodulin binding reduced but did not abolish the binding of caldesmon to actin-tropomyosin. From this data we have proposed a model for smooth muscle thin filaments in which Ca2+ regulates activity by converting the inhibited actin-tropomyosin-caldesmon complex to the active complexes, actin-tropomyosin-caldesmon-calmodulin X Ca2+ and actin-tropomyosin.     

Stoichiometry and stability of caldesmon in native thin filaments from sheep aorta smooth muscle.

Ca2(+)-regulated native thin filaments were extracted from sheep aorta smooth muscle. The caldesmon content determined by quantitative gel electrophoresis was 0.06 caldesmon molecule/actin monomer (1 caldesmon molecule per 16.3 actin monomers). Dissociation of caldesmon and tropomyosin from the thin filament and the depolymerization of actin was measured by sedimenting diluted thin filaments. Actin critical concentration was 0.05 microM at 10.1 and 0.13 at 10.05 compared with 0.5 microM for pure F-actin. Tropomyosin was tightly bound, with half-maximal dissociation at less than 0.3 microM thin filaments (actin monomer) under all conditions. Caldesmon dissociation was independent of tropomyosin and not co-operative. The concentration of thin filaments where 50% of the caldesmon was dissociated (CD50) ranged from 0.2 microM (actin monomer) at 10.03 to 8 microM at 10.16 in a 5 mM-MgCl2, pH 7.1, buffer. Mg2+, 25 mM at constant I, increased CD50 4-fold. CD50 was 4-fold greater at 10(-4) M-Ca2+ than at 10(-9) M-Ca2+. Aorta heavy meromyosin (HMM).ADP.Pi complex (2.5 microM excess over thin filaments) strongly antagonized caldesmon dissociation, but skeletal-muscle HMM.ADP.Pi did not. The behaviour of caldesmon in native thin filaments was indistinguishable from caldesmon in reconstituted synthetic thin filaments. The variability of Ca2(+)-sensitivity with conditions observed in thin filament preparations was shown to be related to dissociation of regulatory caldesmon from the thin filament.     

Binding of caldesmon to smooth muscle myosin

Caldesmon, a major calmodulin binding protein, was found to bind smooth muscle myosin. Addition of caldesmon to smooth muscle myosin induced the formation of small aggregates of myosin in the absence of Ca2+-calmodulin, but not in the presence of Ca2+-calmodulin. The binding site of myosin was studied by using caldesmon-Sepharose 4B affinity chromatography. Subfragment 1 was not retained by the column, while heavy meromyosin and subfragment 2 were bound to the caldesmon affinity column in the absence of Ca2+-calmodulin but not in its presence. It was therefore concluded that the binding site of caldesmon on myosin molecule was the subfragment 2 region and that binding of caldesmon to myosin was abolished in the presence of Ca2+ and calmodulin. Cross-linking of actin and myosin mediated by caldesmon was studied. While actomyosin was completely dissociated in the presence of Mg2+-ATP, the addition of caldesmon caused aggregation of the actomyosin. By low speed centrifugation at which actomyosin alone was not precipitated in the presence of Mg2+-ATP, the aggregate induced by caldesmon was precipitated and the composition of the precipitate was found to be actin, caldesmon, and myosin. In the presence of Mg2+-ATP, pure actin did not bind to a myosin-Sepharose 4B affinity column, while all of the actin was retained when the actin/caldesmon mixture was applied to the column. These results indicate that caldesmon can cross-link actin and myosin.     

Olfactory adenylate cyclase of the rat. Stimulation by odorants and inhibition by Ca2+.

The Ca2+-dependent regulation of the activation of myosin MgATPase by vascular-smooth-muscle thin filaments involves caldesmon. This effect may be due to the direct interaction of caldesmon with a Ca2+-binding protein such as calmodulin or phosphorylation of caldesmon by a Ca2+-dependent kinase. I have found that Ca2+ switches on aorta thin filaments in less than 10 s, whereas the caldesmon in the thin filaments is phosphorylated only slowly (half-time greater than 10 min) and the maximum phosphorylation is very low (1 molecule per 7 molecules of caldesmon). I conclude that the phosphorylation of caldesmon hypothesis is untenable.     

Ca(2+)-dependent, myosin subfragment 1-induced proximity changes between actin and the inhibitory region of troponin I.

In order to help understand the spatial rearrangements of thin filament proteins during the regulation of muscle contraction, we used fluorescence resonance energy transfer (FRET) to measure Ca(2+)-dependent, myosin-induced changes in distances and fluorescence energy transfer efficiencies between actin and the inhibitory region of troponin I (TnI). We labeled the single Cys-117 of a mutant TnI with N-(iodoacetyl)-N'-(1-sulfo-5-naphthyl)ethylenediamine (IAEDANS) and Cys-374 of actin with 4-dimethylaminophenylazophenyl-4'-maleimide (DABmal). These fluorescent probes were used as donor and acceptor, respectively, for the FRET measurements. We reconstituted a troponin-tropomyosin (Tn-Tm) complex which contained the AEDANS-labeled mutant TnI, together with natural troponin T (TnT), troponin C (TnC) and tropomyosin (Tm) from rabbit fast skeletal muscle. Fluorescence titration of the AEDANS-labeled Tn-Tm complex with DABmal-labeled actin, in the presence and absence of Ca(2+), resulted in proportional, linear increases in energy transfer efficiency up to a 7:1 molar excess of actin over Tn-Tm. The distance between AEDANS on TnI Cys-117 and DABmal on actin Cys-374 increased from 37.9 A to 44.1 A when Ca(2+) bound to the regulatory sites of TnC. Titration of reconstituted thin filaments, containing AEDANS-labeled Tn-Tm and DABmal-labeled actin, with myosin subfragment 1 (S1) decreased the energy transfer efficiency, in both the presence and absence of Ca(2+). The maximum decrease occurred at well below stoichiometric levels of S1 binding to actin, showing a cooperative effect of S1 on the state of the thin filaments. S1:actin molar ratios of approximately 0.1 in the presence of Ca(2+), and approximately 0.3 in the absence of Ca(2+), were sufficient to cause a 50% reduction in normalized transfer efficiency. The distance between AEDANS on TnI Cys-117 and DABmal on actin Cys-374 increased by approximately 7 A in the presence of Ca(2+) and by approximately 2 A in the absence of Ca(2+) when S1 bound to actin. Our results suggest that TnI's interaction with actin inhibits actomyosin ATPase activity by modulating the equilibria among active and inactive states of the thin filament. Structural rearrangements caused by myosin S1 binding to the thin filament, as detected by FRET measurements, are consistent with the cooperative behavior of the thin filament proteins.     

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.     

Reversal of caldesmon function by anti-caldesmon antibodies confirms its role in the calcium regulation of vascular smooth muscle thin filaments

Direct evidence that caldesmon is the Ca2+-regulated inhibitory component of native smooth muscle thin filaments is provided by studies using caldesmon-specific antibodies as antagonists. The antibodies reverse caldesmon inhibition of actomyosin ATPase and abolish Ca2+-regulation of native aorta thin filament activation of myosin ATPase. This effect is a result of antibody binding to the caldesmon on the filament thereby inactivating it and not due to antibody-induced caldesmon dissociation from the filament. The antibodies, however, neutralise caldesmon only in systems using skeletal muscle myosin and not in those using smooth muscle myosin; this implies that smooth muscle myosin prevents appropriate antibody binding to caldesmon perhaps because smooth muscle myosin binds to caldesmon thus preventing access of antibody to antigenic sites.