Calponin-Like Protein from Mussel Smooth Muscle Is a Competitive Inhibitor of Actomyosin ATPase.

The goal of this work was to elucidate the mechanism of inhibition of the actin-activated ATPase of myosin subfragment-1 (S1) by the calponin-like protein from mussel bivalve muscle. The calponin-like protein (Cap) is a 40-kDa actin-binding protein from the bivalve muscle of the mussel Crenomytilus grayanus. Kinetic parameters Vmax and KATPase of actomyosin ATPase in the absence and the presence of Cap were determined to investigate the mechanism of inhibition. It was found that Cap mainly causes increase in KATPase value and to a lesser extent the decrease in Vmax, which indicates that it is most likely a competitive inhibitor of actomyosin ATPase. Analysis of Vmax and KATPase parameters in the presence of tropomyosin revealed that the latter is a noncompetitive inhibitor of the actomyosin ATPase.

40-kDa protein from thin filaments of the mussel Crenomytilus grayanus changes the conformation of F-actin during the ATPase cycle.

Polarized fluorimetry was used to study in ghost muscle fibers the influence of a 40-kDa protein from the thin filaments of the mussel Crenomytilus grayanus on conformational changes of F-actin modified by the fluorescent probes 1,5-IAEDANS and FITC-phalloidin during myosin subfragment (S1) binding in the absence of nucleotides and in the presence of MgADP or MgATP. The fluorescence probes were rigidly bound with actin, which made the absorption and emission dipoles of the probes sensitive to changes in the orientation and mobility of both actin monomer and its subdomain-1 in thin filaments of the muscle fiber. On modeling different intermediate states of actomyosin, the orientation and mobility of oscillators of the dyes were changed discretely, which suggests multistep changes in the actin conformation during the cycle of ATP hydrolysis. The 40-kDa protein influenced the orientation and mobility of the fluorescent probes markedly, suppressing changes in their orientation and mobility in the absence of nucleotides and in the presence of MgADP, but enhancing these changes in the presence of MgATP. The calponin-like 40-kDa protein is supposed to prevent formation of the strong binding state of actomyosin in the absence of nucleotides and in the presence of MgADP but to activate formation of this state in the presence of MgATP.

40-kDa actin-binding protein of thin filaments of the mussel Crenomytilus grayanus inhibits the strong bond formation between actin and myosin head during the ATPase cycle.

Mobility and spatial orientation of a novel 40-kDa actin-binding protein from the smooth muscle of the mussel Crenomytilus grayanus was studied by polarized fluorometry. The influence of this protein on orientation and mobility of the myosin heads was investigated during modeling the different stages of the ATPase cycle. The 40-kDa actin-binding protein affected the strong actin-myosin binding. We suggest that the 40-kDa actin-binding protein is involved in regulation of the actin-myosin interaction in the smooth muscle of the mussel.

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 Phi(A), Phi(E), Theta(1/2) and Nu were calculated. Phi(A) and Phi(E) are angles between the fiber axis and the absorption and emission dipoles, respectively; Theta(1/2) is the angle between the F-actin filament axis and the fiber axis; Nu 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 Ca(2+)) diminished the effect of S-1, whereas CaDH2 (as well as troponin in the presence of Ca(2+)) 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.

Proteolytic cleavage of actin within the DNase-I-binding loop changes the conformation of F-actin and its sensitivity to myosin binding.

Effects of subtilisin cleavage of actin between residues 47 and 48 on the conformation of F-actin and on its changes occurring upon binding of myosin subfragment-1 (S1) were investigated by measuring polarized fluorescence from rhodamine-phalloidin- or 1, 5-IAEDANS-labeled actin filaments reconstructed from intact or subtilisin-cleaved actin in myosin-free muscle fibers (ghost fibers). In separate experiments, polarized fluorescence from 1, 5-IAEDANS-labeled S1 bound to non-labeled actin filaments in ghost fibers was measured. The measurements revealed differences between the filaments of cleaved and intact actin in the orientation of rhodamine probe on the rhodamine-phalloidin-labeled filaments, orientation and mobility of the C-terminus of actin, filament flexibility, and orientation and mobility of the myosin heads bound to F-actin. The changes in the filament flexibility and orientation of the actin-bound fluorophores produced by S1 binding to actin in the absence of ATP were substantially diminished by subtilisin cleavage of actin. The results suggest that loop 38-52 plays an important role, not only in maintaining the F-actin structure, but also in the conformational transitions in actin accompanying the strong binding of the myosin heads that may be essential for the generation of force and movement during actin-myosin interaction.

Conformational changes of contractile proteins and their role in muscle contraction.

The review summarizes the results of studies on conformational changes in contractile proteins that occur during muscle contraction. Polarized fluorescence of tryptophan residues in actin and of fluorescent probes bound specifically to different sites on actin, myosin, or tropomyosin in muscle fibers was measured. The results show that the transition of actomyosin complex from the weak to the strong-binding state is accompanied by a change in the orientation of F-actin subunits with the C and N termini moving opposite to a large part of the subunit. Myosin light chains and some areas in the 20-kDa domain of myosin head move in the same direction as the C- and N-terminal regions of actin. It is established that troponin, caldesmon, calponin, and myosin systems of regulation of muscle contraction modify intramolecular actomyosin rearrangements in a Ca(2+)-dependent manner. The role of intramolecular movements of contractile proteins in muscle contraction is discussed.

Calcium ions modulate regulation of smooth muscle contraction mediated by phosphorylation of myosin regulatory light chains.

The effect of calcium ions on conformational changes of F-actin initiated by decoration of thin filaments with phosphorylated and dephosphorylated heavy meromyosin from smooth muscles was studied by fluorescence polarization spectroscopy. It is shown that heavy meromyosin with phosphorylated regulatory light chains (pHMM) promotes structural changes of F-actin which are typical for the "strong" binding of actin to the myosin heads. Heavy meromyosin with dephosphorylated regulatory light chains (dpHMM) causes conformational changes of F-actin which are typical for the "weak" binding of actin to the myosin heads. The presence of calcium enhances the pHMM effect and attenuates the dpHMM effect. We propose that a Ca2+-dependent mechanism exists in smooth muscles which modulates the regulation of actin--myosin interaction occurring via phosphorylation of myosin regulatory light chains.

Calcium modulates conformational changes in F-actin induced by smooth muscle heavy meromyosin.

The effect of Ca2+ on conformational changes in rhodamine-phalloidin-labeled F-actin induced by binding of smooth muscle heavy meromyosin (HMM) with either phosphorylated or dephosphorylated regulatory light chains (LC20) was studied by polarized fluorimetry. LC20 phosphorylation caused alterations in the F-actin structure typical of the force-producing (strong-binding) state, while dephosphorylation of the chains led to alterations typical of the formation of non-force-producing (weak-binding) state of the actomyosin complex. The presence of Ca2+ enhanced the effect of LC20 phosphorylation and weakened the effect of LC20 dephosphorylation. These data suggest that Ca2+ modulates actin-myosin interaction in smooth muscle by promoting formation of the strong-binding state.

Effects of denervation and muscle inactivity on the organization of F-actin.

To discriminate between the influences of a motoneuron and muscle activity on the conformation of actin filaments, the extrinsic polarized fluorescence [of rhodamine-phalloidin and N-(iodoacetylamine)-1-naphthylamine-5-sulfonic acid attached to F-actin] was measured in "ghost" fibers from intact rat soleus muscles and atrophying muscles after denervation, immobilization, or tenotomy. The results show that the conformation of F-actin changed in all the atrophying muscles, but differently. In the denervated muscle, the flexibility of the actin filaments decreased, whereas in the other experimental muscles it remained as in the intact muscle. In the denervated muscle, the mobility of the C-terminus of the actin polypeptide increased. Attachment of myosin subfragment-1 influenced the F-actin conformation differently in the denervated muscle than in the other muscles studied. These results suggest that changes in the conformation of the actin filament are induced by the lack of connection with the motoneuron rather than by muscle inactivity.

The shortening of the N-terminus of myosin essential light chain A1 influences the interaction of heavy meromyosin with actin.

The effects resulting from the removal of the N-terminus of heavy meromyosin (HMM) A1 light chain by papain digestion are investigated. The fluorometry of TRITC-phalloidin labelled actin in ghost fibers is used as a tool for sensing conformational changes of rigor complex of phosphorylated and dephosphorylated HMM with actin filament. The experiments were performed both under conditions assuring saturation of RLC with magnesium cation (4 mM EGTA) or calcium cation (0.1 mM CaCl2), and in constant presence of 1 mM magnesium chloride. HMM native and with A1 shortened from the N-terminus is used. As it was observed previously rigor complex of actin filament and native HMM shows sensitivity to the kind of cation saturating RLC and to the phosphorylation status of RLC. In particular, the sin2 theta parameter of actin bound rhodamine-phalloidin fluorescence polarization representing roughly the flexibility of actin filament HMM complex changes significantly with the changes of RLC phosphorylation and cation saturation. Removal of the N-terminus of A1 reduces this sensitivity to cation and phosphorylation both in the case of dephosphorylated and phosphorylated HMM. Our results suggest that the N-terminus of A1 plays significant role in the rigor interaction of myosin heads with actin and is involved in modulatory function of RLC in this interaction.

Comparison of the effects of calponin and a 38-kDa caldesmon fragment on formation of the "strong-binding" state in ghost muscle fibers.

We studied the conformational changes in actin filaments induced by the binding of calponin or a 38-kDa fragment of caldesmon, two actin-binding proteins known to inhibit actin-activated ATP hydrolysis by phosphorylated smooth muscle myosin. The F-actinin myosin-free muscle fibers (ghost fibers) was labeled with fluorescein-5-maleimide and the conformational change in actin was determined by polarized fluorimetry. Data show that both calponin and the 38-kDa caldesmon fragment inhibit the conformational changes in F-actin that are compatible with the "strong-binding" state between myosin heads and actin. Tropomyosin slightly reduced the effect produced by calponin, but enhances the effect produced by the 38-kDa caldesmon fragment.

Significance of the N-terminal fragment of myosin regulatory light chain for myosin-actin interaction.

The influence of myosin regulatory light chains (LC2s) lacking the 2kD N-terminal portions of these chains, on internal organization of myosin heads and on actin-myosin interaction was studied with limited proteolysis and polarized fluorescence methods. For these studies heavy meromyosin (HMM) preparations were used: HMM containing intact LC2s (phosphorylated or dephosphorylated) and HMM containing LC2s lacking the 2kD N-terminal portions (including serine which can be phosphorylated). It was found that the susceptibility of the heavy chain cleavage site to trypsin and the alkali light chain (LC1) site to papain of myosin containing shortened regulatory LC2s, is not dependent on saturation of LC2s with Ca2+ or Mg2+ ions. This is in contrast to the myosin containing intact LC2s where Ca2+ or Mg2+ ion saturation does demonstrate a dependence. Similarly, in spectroscopic experiments, dephosphorylated HMM containing intact LC2s causes decrease or increase of actin filament flexibility depending on whether Mg2+ or Ca2+ are bound to LC2s. Correspondingly, HMM with shortened LC2s induces only increase of actin flexibility despite cations being bound. We conclude that the N-terminal fragment of LC2 is important for ensuring a proper Ca2+ dependent conformation of myosin head in the course of its actin-activated ATP hydrolysis.

Secretion from permeabilised mast cells is enhanced by addition of gelsolin: contrasting effects of endogenous gelsolin.

Permeabilised rat mast cells were exposed to gelsolin and its N-terminal half (S1-3), proteins that sever actin filaments in a calcium-dependent and independent manner, respectively. Gelsolin and S1-3 induced a decrease in cellular F-actin content and an increase in the extent of the secretory response. The calcium sensitivities of both these effects were consistent with the differential calcium requirements of the two proteins. Segment 1 (S1), which binds G-actin and caps filaments but does not sever them, did not show these effects. Thus, secretion of mast cells is promoted as a consequence of the severing activity of exogenous gelsolin or S1-3. Most of the endogenous gelsolin remained within permeabilised, washed mast cells and its distribution in resting state was predominantly cortical. Addition of calcium in the absence of MgATP did not reduce the F-actin content; by contrast, calcium with MgATP induced F-actin loss that was unaffected by the presence of anti-gelsolin. Because this antibody inhibits the severing activity of gelsolin, these results indicate that in permeabilised mast cells the severing activity of the remaining endogenous gelsolin is not involved in cortical actin filaments disassembly. Upon exposure to GTP-gamma-S in the absence of calcium, the content of cortical gelsolin was reduced. This parallels our previous observation of a GTP-gamma-S induced reduction of cortical actin filaments followed by their relocation to the cell's interior (Norman et al. (1994) J. Cell Biol. 126, 1005-1015) and suggests that actin redistribution may be a consequence of dissociation of gelsolin caps brought about by activation of a GTP-binding protein.

The effect of Ca2+ on the conformation of tropomyosin and actin in regulated actin filaments with or without bound myosin subfragment 1.

The effects of Ca2+ and myosin subfragment 1 on the conformation of tropomyosin and actin in regulated actin filaments in ghost fibers were investigated by means of the polarized fluorescence technique. Regulated thin filaments were reconstituted in skeletal muscle ghost fibers by incorporation into the fibers of either skeletal muscle troponin-tropomyosin or smooth-muscle caldesmon-calmodulin-tropomyosin complexes. Tropomyosin and actin were specifically labeled with fluorescent probes, 1,5-IAEDANS and phalloidin-rhodamine, respectively. Analysis of the fluorescence parameters indicated that the binding of Ca2+ to regulated actin filaments induces conformational changes in tropomyosin and actin that lead to the strengthening of the interaction between these two proteins and weakening of the binding of actin monomers in the filament. These changes become larger when regulated actin forms rigor links with myosin subfragment 1. No notable alterations in the position of tropomyosin relative to actin in the frontal plane of the fiber were detected either upon binding of Ca2+ or upon the additional binding of myosin subfragment 1 to regulated actin.

Troponin I and caldesmon restrict alterations in actin structure occurring on binding of myosin subfragment 1.

The effect of troponin I and caldesmon on phalloidin-rhodamine- and 1,5-IAEDANS-labelled actin in skeletal muscle ghost fibers was investigated by polarized fluorescence. Both these proteins inhibited the structural alterations in the actin monomer and the increase of flexibility of actin filaments occurring on binding of myosin heads, and their effects were potentiated by tropomyosin. This immobilization of the actin filament through troponin I and caldesmon seems to originate from restriction of the relative motions of the two domains within the monomer.

Caldesmon weakens the bonding between myosin heads and actin in ghost fibers.

Earlier studies using polarized microphotometry have shown that caldesmon inhibits the alterations in structure and flexibility of actin in ghost fibers that take place upon the binding of myosin heads (Galazkiewicz et al. (1987) Biochim. Biophys. Acta 916, 368-375). The present investigations, performed with an IAEDANS label attached to myosin subfragment 1 (S-1), revealed that this inhibition results from the weakening of the binding between myosin heads and actin as indicated by the caldesmon-induced increase in the random movement of S-1. Parallel experiments with actin labeled at Cys-374 demonstrated that this effect of caldesmon is transmitted to the C-terminus of the actin molecule resulting in a conformational adjustment in this region of the molecule.

The effect of myosin light chain phosphorylation and Mg2+ on the conformation of myosin in thick filaments of glycerinated fibers of rabbit skeletal muscle.

It has been shown by polarization microfluorimetry that phosphorylation of myosin light chain 2, in stretched single glycerinated fibers of rabbit skeletal muscle, results in changes in polarized fluorescence anisotropy of both the tryptophan residues of myosin molecules and the fluorescent label, N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine, associated with the fast-reacting thiol group in myosin heads. These changes are also dependent on the presence or absence of Mg2+ in the medium: they are most pronounced in the presence of 5 mM MgCl2. It is assumed that both Mg2+ binding to myosin and phosphorylation of light chain 2 associated with myosin heads induce structural changes in myosin filaments of muscle fibres which are expressed as changes in the orientation of myosin heads and in the conformation of myosin rods.

Interaction of tropomyosin with F-actin-heavy meromyosin complex.

The effect of phosphorylated and dephosphorylated heavy meromyosins (HMMs) saturated with Ca2+ or Mg2+ on the binding of tropomyosin to F-actin and on the conformational changes of tropomyosin on actin was investigated. The experimental data were analysed on the basis of th emodel of cooperative binding of tropomyosin to F-actin with overlapping binding sites. In general, attachment of both HMMs to F-actin increased around 100-fold the tropomyosin-binding affinity but concomittantly reduced the cooperatively of binding. In the presence of Ca2+ and in the absence of ATP the binding of tropomyosin to F-actin in a "doubly contiguous" manner was three-fold stronger for F-actin saturated with dephosphorylated HMM as compared to phosphorylated HMM. Under the same rigor conditions but in the absence of Ca2+ the reverse was true but the difference was about 1.5-fold. The binding stoichiometry of tropomyosin to actin was 7:1 in the presence of dephosphorylated HMM saturated with Ca2+ or phosphorylated-saturated with Mg2+ and tended to be about 6:1 for both after the exchange of the cation bound to myosin heads. Bound HMM was also found to influence the fluorescence polarization of 1,5-IAEDANS-labelled tropomyosin complexed with F-actin in muscle ghost fibres. In the presence of Ca2+, the amount of randomly arranged tropomyosin fluorophores decreased when dephosphorylated HMM was bound to ghost fibres, in contrast to an observed increase in the case of bound phosphorylated HMM. Thus HMM induced conformational changes of tropomyosin in the actin-tropomyosin complex that was reflected in an alteration of the geometrical arrangement between tropomyosin and actin.(ABSTRACT TRUNCATED AT 250 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.

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.