Orientation and mobility of actin in different intermediate states of the ATP hydrolysis cycle.

Using polarization fluorimetry, we have investigated conformational changes of FITC-phalloidin-labeled F-actin in ghost muscle fibers. These changes were induced by myosin subfragment-1 (S1) in the absence and presence of MgADP, MgAMP-PNP, MgATPgammaS, or MgATP. Modeling of various intermediate states was accompanied by discrete changes in actomyosin orientation and mobility of fluorescent dye dipoles. This suggests multistep changes of orientation and mobility of actin monomers during the ATPase cycle. The most pronounced differences in orientation (~4 degrees ) and in mobility (~43%) of actin were found between the actomyosin states induced by MgADP and MgATP.

Effect of nucleotides on the orientation and mobility of myosin subfragment-1 in ghost muscle fiber.

Using polarization fluorimetry, the orientation and mobility of 1,5-IAEDANS specifically bound to Cys707 of myosin subfragment-1 (S1) were studied in ghost muscle tropomyosin-containing fibers in the absence and in the presence of MgADP, MgAMP-PNP, MgATPgammaS, or MgATP. Modeling of various intermediate states was accompanied by discrete changes in actomyosin orientation and mobility of fluorescent dye dipoles. This suggests multistep changes in the structural state of the myosin head during the ATPase cycle. Maximal differences in the probe orientation by 4 degrees and its mobility by 30% were found between actomyosin states in the presence of MgADP and MgATP. It is suggested that interaction of S1 with F-actin induces nucleotide-dependent rotation of the whole motor domain of the myosin head or only the dye-binding site and also change in the head mobility.

Nonmuscle caldesmon: its distribution and involvement in various cellular processes. Review article.

Smooth muscle caldesmon is a thin-filament constituent which takes part in the Ca2+-dependent regulation of actomyosin motor activity which converts chemical energy of ATP into force. The molecular anatomy of its counterpart found in a variety of nonmuscle cells is similar. Both contain about 20 nm long terminal domains responsible for functionally important multisite interactions with filamentous actin, tropomyosin, Ca2+/calmodulin, and myosin and differ by a 35 nm long central, alpha-helical fragment which is lacking in nonmuscle caldesmon. The different structural organisation of nonmuscle cells and thus distinct distribution of caldesmon implicates its different physiological functions. Due to direct interaction with globular and filamentous actin as well as with tropomyosin, nonmuscle caldesmon is involved in the assembly, dynamics, or stability of microfilaments, whereas the indirect inhibitory effect on interaction of the microfilaments with myosin causes its participation in the regulation of cell contraction and intracellular motional processes. These functions of nonmuscle caldesmon of vertebrates are controlled by Ca2+/calmodulin (or other Ca2+-binding proteins) or caldesmon phosphorylation catalysed by various protein kinases. Examples of nonmuscle caldesmon involvement in functions of higher and lower eukaryote, animal and plant cells are presented.

The effect of Ca2+ on the structure of synthetic filaments of smooth muscle myosin.

Using electron microscopy and negative staining we have studied the effect of Ca2+ on the structure of synthetic filaments of chicken gizzard smooth muscle myosin under conditions applied by Frado and Craig (1989) for demonstration of the influence of Ca2+ on the structure of synthetic filaments of scallop striated muscle myosin. The results show that Ca2+ induces the transition of compact, ordered structure of filaments with a 14.5 nm axial repeat of the myosin heads close to the filament backbone (characteristic of the relaxing conditions) to a disordered structure with randomly arranged myosin heads together with subfragments-2 (S-2) seen at a distance of up to 50 nm from the filament backbone. This order/disorder transition is much more pronounced in filaments formed of unphosphorylated myosin, since a substantial fraction of phosphorylated filaments in the relaxing solution is already disordered due to phosphorylation. Under rigor conditions some of the filaments of unphosphorylated and phosphorylated myosin retain a certain degree of order resembling those under relaxing conditions, while most of them have a substantially disordered appearance. The results indicate that Ca2+-induced movement of myosin heads away from the filament backbone is an inherent property of smooth muscle myosin, like molluscan muscle myosin regulated exclusively by Ca2+ binding, and can play a modulatory role in smooth muscle contraction.

The first caldesmon-like protein in higher plants.

Using anti-caldesmon polyclonal and monoclonal (raised against the N-terminal fragment of chicken gizzard caldesmon) antibodies, a plant caldesmon-like protein, 107 kDa as determined by SDS-gel electrophoresis, has been identified based on Western blotting of total extracts of Ornithogalum virens pollen tubes. Biochemical investigations showed common properties of this protein with animal caldesmon--it binds to actin and, in a Ca(2+)-dependent manner, to calmodulin. In contrast to animal caldesmon, this plant cell counterpart is relatively resistant to proteolysis by endogenous proteases and sensitive to heat treatment. Our results show the presence of a caldesmon-like protein in higher plants for the first time.

Phosphatidylserine liposomes can be tethered by caldesmon to actin filaments.

Rotary shadowing electron microscopy revealed that attachment of caldesmon to phosphatidylserine (PS) liposomes was mainly through its C-terminal end. To determine the PS-binding sites of caldesmon, we have made use of synthetic peptides covering the two C-terminal calmodulin binding sites and a recombinant fragment corresponding to the N-terminal end of the C-terminal domain that contains an amphipathic helix. Interactions of these peptides with the PS liposomes were studied by nondenaturing gel electrophoresis and fluorescence spectroscopy. The results showed that both calmodulin-binding sites of caldesmon were able to interact with PS. The affinity (Kd) of PS for these sites was in the range of 1.8-14.3 x 10(-5) M, compared to 0.69 x 10(-5) M for the whole caldesmon molecule. Fragments located outside of calmodulin-binding sites bound PS weakly (3.85 x 10(-4) M) and thus may contain a second class of lipid-binding sites. Binding of PS induced conformational changes in regions other than the C-terminal PS-binding sites, as evidenced by the changes in the susceptibility to proteolytic cleavages. Most significantly, the presence of caldesmon greatly increased binding of PS to F-actin, suggesting that caldesmon may tether PS liposomes to actin filaments. These results raise the possibility that caldesmon-lipid interactions could play a functionally important role in the assembly of contractile filaments near the membranes.

The size and shape of caldesmon and its fragments in solution studied by dynamic light scattering and hydrodynamic model calculations.

The size and the shape of caldesmon as well as its 50-kDa central and 19-kDa C-terminal fragments were investigated by photon correlation spectroscopy. The hydrodynamic radii, which have been calculated from the experimentally obtained translational diffusion coefficients, are 9.8 nm, 6.0 nm, and 2.9 nm, respectively. Moreover, the experimental values for the translational diffusion coefficients are compared with results obtained from hydrodynamic model calculations. Detailed models for the structure of caldesmon in solution are derived. The contour length is about 64 nm for all of the models used for caldesmon.

Calponin inhibits actin-activated MgATPase of myosin subfragment 1 (S1) without displacing S1 from its binding site on actin.

Calponin is a smooth-muscle thin-filament protein implicated in the regulation of contraction. Its binding to actin is a prerequisite for inhibition of actin-activated myosin MgATPase. Investigating the molecular mechanism of this inhibition, it was found that titration of acto-myosin subfragment 1 with calponin in the presence of either ADP or ATP does not displace weakly or strongly bound myosin subfragment 1 (S1) from actin. S1.ADP, however, is able to release about two-thirds of the calponin from saturated (equimolar) complexes of actin-calponin. The remaining calponin is sufficient for almost full inhibition of acto-S1 MgATPase activity. Bunding of actin filaments by calponin takes place at a higher ratio calponin/actin (above 1:3) and, therefore, is not responsible for inhibition of the ATPase. Bundle formation is inhibited by S1.ADP. These results suggest the existence of two calponin-binding sites on actin; one, that is insensitive to S1, which is responsible for inhibition of the ATPase, the other, from which calponin is readily displaced by S1.

Interaction of caldesmon with endoplasmic reticulum membrane: effects on the mobility of phospholipids in the membrane and on the phosphatidylserine base-exchange reaction.

We have previously demonstrated by tryptophan fluorescence the interaction of caldesmon with anionic phospholipid vesicles [Czurylo, Zborowski and Dabrowska (1993) Biochem. J. 291, 403-408]. In the present work we investigated the interaction of caldesmon with natural-membrane (rat liver endoplasmic reticulum) phospholipids by co-sedimentation assay. The results indicate that 1 mol of caldesmon binds approx. 170 mol of membrane phospholipids with a binding affinity constant of 7.3 x 10(6) M-1. The caldesmon-membrane phospholipid complex dissociates with increasing salt concentration and in the presence of Ca2+/calmodulin. As indicated by EPR measurements of membrane lipids labelled with 5-doxyl stearate and TEMPO-phosphatidylethanolamine, binding of caldesmon results in an increase in mobility of the acyl chains (in the region of carbon 5) and a decrease in polar headgroup mobility of phospholipids. Interaction of caldesmon with phospholipids is accompanied by inhibition of phosphatidylethanolamine synthesis via a phospholipid base-exchange reaction, with phosphatidylserine as substrate. This shows that, of the endoplasmic reticulum membrane phospholipids, the main target of caldesmon is phosphatidylserine.

The influence of caldesmon on papain proteolysis of monomeric smooth muscle myosin.

The influence of caldesmon on papain digestion of chicken gizzard monomeric myosin in folded (10S) conformation depends on its phosphorylation. Caldesmon exposes the head/rod junction of myosin in phosphorylated form to proteolytic attack (particularly in the presence of Ca2+) and slightly screens it in unphosphorylated form. In both folded forms RLCs are protected by caldesmon, more in unphosphorylated than in phosphorylated myosin. The results indicate that the conformations of folded unphosphorylated and phosphorylated myosin are distinct and suggest that caldesmon destabilizes the regulatory domain in folded conformation of phosphorylated myosin.

The effects of caldesmon extraction on mechanical properties of skinned smooth muscle fibre preparations.

The role of caldesmon in the regulation of smooth muscle contraction was investigated in chemically skinned smooth muscle fibres from the guinea-pig taenia coli. A 19-kDa C-terminal fragment of caldesmon gave a minor (<5%) reduction of force in fully thiophosphorylated fibres, but reduced force by about 50% at intermediate activation levels without affecting the level of light chain phosphorylation. An extraction procedure was developed using incubation in solutions containing high Mg2+ concentrations. Protein analysis revealed a selective decrease in the amount of caldesmon in the fibres. Maximal active force per cross-sectional area was unaffected. The Ca2+ dependence of active force was shifted towards lower Ca2+ concentrations and became less steep. The effects of extraction of caldesmon could in part be reversed by incubation in a solution containing purified caldesmon. The results are consistent with the hypothesis that caldesmon in smooth muscle thin filaments inhibits force generation and plays a role in regulating cooperative attachment of cross-bridges at sub-maximal levels of activation in smooth muscle.

Modulation of gelsolin-induced actin-filament severing by caldesmon and tropomyosin and the effect of these proteins on the actin activation of myosin Mg(2+)-ATPase activity.

We have investigated the cumulative effects of three smooth-muscle actin-binding proteins, gelsolin, caldesmon and tropomyosin, on actin activation of myosin Mg(2+)-ATPase activity under low-ionic-strength conditions. A combination of tropomyosin (at a stoicheiometric ratio to actin) and gelsolin (at a molar ratio to actin of up to 1:100) showed essentially additive stimulatory effects that were counteracted by caldesmon. Suppression of the gelsolin-induced activation of the ATPase by caldesmon was higher in the presence of tropomyosin although it was not complete even at stoicheiometric amounts of both proteins to actin. Since activation of actin-activated ATPase activity of myosin by gelsolin is related to its severing action, it is concluded that caldesmon and tropomyosin cannot fully protect actin filaments against the severing activity of gelsolin. Direct analysis of the actin-severing activity of gelsolin by a fluorimetric assay using pyrene-labelled actin confirmed this conclusion. Tropomyosin and caldesmon in saturating amounts relative to actin inhibited the activity of gelsolin by between 21 and 40% and 25 and 48% respectively, depending on the molar ratio of gelsolin to actin. The inhibitory effect was increased with a combination of both (up to 67%) although it was evident that even under these conditions the actin filaments were not fully protected from being severed by gelsolin. These findings were corroborated by electron-microscopic investigation of actin filaments with or without tropomyosin and caldesmon after the addition of gelsolin.

Characterization of chicken gizzard calcyclin and examination of its interaction with caldesmon.

Using a procedure developed to purify calcyclin from mouse Ehrlich ascites tumor cells calcyclin was purified from smooth muscle of chicken gizzard. Chicken gizzard calcyclin bound to phenyl-Sepharose in a calcium dependent manner as did mouse EAT cells and rabbit lung calcyclin but appeared to be more acidic than its mammalian counterparts as revealed by ion exchange chromatography on Mono Q. Chicken gizzard calcyclin bound 45Ca2+ on nitrocellulose filters and exhibited a shift in electrophoretic mobility on urea-PAGE depending on Ca2+ concentration. Crosslinking experiments with BS3 showed that chicken gizzard calcyclin was able to form noncovalent dimers. As indicated by a decrease in maximum tryptophan fluorescence emission of caldesmon (about 14% at 1:1 molar ratio) and displacement of calmodulin from its complex with caldesmon, chicken gizzard calcyclin binds caldesmon. This binding was, however, much weaker than that of calmodulin and could not influence the interaction of caldesmon with actin. In consequence, calcyclin was unable to reverse the inhibitory effect of caldesmon on actin-activated Mg(2+)-ATPase activity of myosin in the presence of Ca2+.

Interaction of calponin with actin and its functional implications.

Titration of F-actin with calponin causes the formation of two types of complexes. One, at saturation, contains a lower ratio of calponin to actin (0.5:1) and is insoluble at physiological ionic strength. The another is soluble, with a higher ratio of calponin to actin (1:1). Electron microscopy revealed that the former complex consists of paracrystalline bundles of actin filaments, whereas the latter consists of separate filaments. Ca(2+)-calmodulin causes dissociation of bundles with simultaneous increase in the number of separate calponin-containing filaments. Further increase in the calmodulin concentration results in full release of calponin from actin filaments. In motility assays, calponin, when added together with ATP to actin filaments complexed with immobilized myosin, evoked a decrease in both the number and velocity of moving actin filaments. Addition of calponin to actin filaments before their binding to myosin resulted in a formation of actin filament bundles which were dissociated by ATP.

Polymerization of actin induced by actin-binding fragments of caldesmon.

Our earlier studies revealed that caldesmon causes assembly of G-actin into polymers morphologically indistinguishable from those formed in the presence of salt (Galazkiewicz, B., Belagyi, J. and Dabrowska, R. (1989) Eur. J. Biochem. 181, 607-614). In this work we have investigated the effect of actin-binding fragments of caldesmon on actin polymerization process followed by measurements of the changes in fluorescence of pyrenyl conjugated with G-actin and ATP hydrolysis. The results indicate that C-terminal 34 kDa fragment of caldesmon containing two actin-binding sites and 19 kDa containing high-affinity binding site have similar capability to polymerize actin to that of intact molecule. Binding of each of these fragments to G-actin causes bypassing of nucleation phase. The 11.5 kDa fragment comprising low affinity actin-binding site has much lower potency to polymerize actin. Conformation of actin monomers in filaments formed upon 19 kDa fragment and that formed upon 11.5 kDa fragment differs. The former fragment seems to resemble more conformation of monomers in filaments formed upon intact caldesmon than the latter one.

Studies on secondary structure of caldesmon and its C-terminal fragments.

Evaluation of the secondary structure of caldesmon from c.d. spectra revealed that it contains 51% helix, 9% beta-strand and 40% of remainder structures. These values agree well with the predicted ones from amino acid sequence, assuming an extended chain structure for caldesmon. The estimates of the secondary-structure elements in C-terminal 34 kDa and 19 kDa fragments are: 11 and 12% helix, 22 and 20% beta-strand, 13 and 17% beta-turns and loops, and 54 and 50% of remainder structure respectively. The best fit of experimental data was obtained assuming the globular state of the fragments. On the basis of structural analysis and fragmentation by proteolytic and chemical cleavages the six-domain model of caldesmon is proposed.