Search

Caldesmon (CaD) is known as an actin binding protein. In this study, we proposed that a trace amount of caldesmon (TACD) could highly, efficiently, interact with myosin by producing a 'domino-like cascade' and characterized that TACD (lowest caldesmon/myosin molar ratio: 1/10,000) significantly increased precipitations and intrinsic tryptophan fluorescence intensity of myosin in both phosphorylated and unphosphorylated states compared to the base controls (P < 0.01). Actin-blocked TACD-myosin interaction, suggesting that it functioned as a negative regulator. Since CaD is not an enzyme, the in vivo significance of the highly efficient TACD-myosin interaction needs further investigation.

Orientation and mobility of acrylodan fluorescent probe specifically bound to caldesmon Cys580 incorporated into muscle ghost fibers decorated with myosin S1 and containing tropomyosin was studied in the presence or absence of MgADP, MgAMP-PNP, MgATPgammaS or MgATP. Modeling of various intermediate states of actomyosin has shown discrete changes in orientation and mobility of the dye dipoles which is the evidence for multistep changes in the structural changes of caldesmon during the ATPase hydrolysis cycle. It is suggested that S1 interaction with actin results in nucleotide-dependent displacement of the C-terminal part of caldesmon molecule and changes in its mobility. Thus inhibition of the actomyosin ATPase activity may be due to changes in caldesmon position on the thin filament and its interaction with actin. Our new findings described in the present paper as well as those published recently elsewhere might conciliate the two existing models of molecular mechanism of inhibition of the actomyosin ATPase by caldesmon.

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.

New data on the movements of tropomyosin singly labeled at alpha- or beta-chain during the ATP hydrolysis cycle in reconstituted ghost fibers have been obtained by using the polarized fluorescence technique which allowed us following the azimuthal movements of tropomyosin on actin filaments. Pronounced structural changes in tropomyosin evoked by myosin heads suggested the "rolling" of the tropomyosin molecule on F-actin surface during the ATP hydrolysis cycle. The movements of actin-bound tropomyosin correlated to the strength of S1 to actin binding. Weak binding of myosin to actin led to an increase in the affinity of the tropomyosin N-terminus to actin with simultaneous decrease in the affinity of the C-terminus. On the contrary, strong binding of myosin to actin resulted in the opposite changes of the affinity to actin of both ends of the tropomyosin molecule. Caldesmon inhibited the "rolling" of tropomyosin on the surface of the thin filament during the ATP hydrolysis cycle, drastically decreased the affinity of the whole tropomyosin molecule to actin, and "freezed" tropomyosin in the position characteristic of the weak binding of myosin to actin.

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.

The effect of caldesmon (CaD) on conformational changes in F-actin modified by fluorescent probe TRITC-phalloidin was investigated by polarized fluorimetry. Changes were induced by a subfragment-1 (S-1) of myosin in the absence or presence of CaD in ghost muscle fibers obtained from intact and denervated slow (SOL) and fast (EDL) skeletal muscles of rats. S-1 binding to actin of both SOL and EDL muscles was shown to cause changes in polarized parameters of TRITC-phalloidin typical for a strong actin-myosin binding as well as of transition ofactin subunits from "off" to "on" state. CaD inhibits this significantly. Denervation atrophy inhibits the effect of S-1 as well but does not affect the capability of CaD decreasing the formation of strong binding in actomyosin complex. It is supposed that CaD "freezes" F-actin structure in "off" state. The denervation atrophy has no effect on CaD responsibility to bind thin filaments and to switch "off" actin monomers.

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

High velocity cytoplasmic streaming is found in various plant cells from algae to angiosperms. We characterized mechanical and enzymatic properties of a higher plant myosin purified from tobacco bright yellow-2 cells, responsible for cytoplasmic streaming, having a 175 kDa heavy chain and calmodulin light chains. Sequence analysis shows it to be a class XI myosin and a dimer with six IQ motifs in the light chain-binding domains of each heavy chain. Electron microscopy confirmed these predictions. We measured its ATPase characteristics, in vitro motility and, using optical trap nanometry, forces and movement developed by individual myosin XI molecules. Single myosin XI molecules move processively along actin with 35 nm steps at 7 micro m/s, the fastest known processive motion. Processivity was confirmed by actin landing rate assays. Mean maximal force was approximately 0.5 pN, smaller than for myosin IIs. Dwell time analysis of beads carrying single myosin XI molecules fitted the ATPase kinetics, with ADP release being rate limiting. These results indicate that myosin XI is highly specialized for generation of fast processive movement with concomitantly low forces.

Myosin X is a member of the diverse myosin superfamily that is ubiquitously expressed in various mammalian tissues. Although its association with actin in cells has been shown, little is known about its biochemical and mechanoenzymatic function at the molecular level. We expressed bovine myosin X containing the entire head, neck, and coiled-coil domain and purified bovine myosin X in Sf9 cells. The Mg(2+)-ATPase activity of myosin X was significantly activated by actin with low K(ATP). The actin-activated ATPase activity was reduced at Ca(2+) concentrations above pCa 5 in which 1 mol of calmodulin light chain dissociates from the heavy chain. Myosin X translocates F-actin filaments with the velocity of 0.3 microm/s with the direction toward the barbed end. The actin translocating activity was inhibited at concentrations of Ca(2+) at pCa 6 in which no calmodulin dissociation takes place, suggesting that the calmodulin dissociation is not required for the inhibition of the motility. Unlike class V myosin, which shows a high affinity for F-actin in the presence of ATP, the K(actin) of the myosin X ATPase was much higher than that of myosin V. Consistently nearly all actin dissociated from myosin X in the presence of ATP. ADP did not significantly inhibit the actin-activated ATPase activity of myosin X, suggesting that the ADP release step is not rate-limiting. These results suggest that myosin X is a nonprocessive motor. Consistently myosin X failed to support the actin translocation at low density in an in vitro motility assay where myosin V, a processive motor, supports the actin filament movement.

This minireview will cover current concepts on the identity and mechanistic function of smooth muscle actin binding proteins that may regulate actin-myosin interactions. The potential roles of tropomyosin, caldesmon, calponin, and SM22 will be discussed. The review, for purposes of brevity, will be nonexhaustive but will give an overview of available information on the in vitro biochemistry and potential in vivo function of these proteins. Preterm labor is discussed as a possible example of where thin filament regulation may be relevant. Considerable controversy surrounds the putative physiological significance of these proteins, and emphasis will be placed on the need for more experimental work to determine the degree to which tissue- and species-specific effects have clouded the interpretation of functional data.

High-resolution structures of the motor domain of myosin II and lower resolution actin-myosin structures have led to the "swinging lever arm" model for myosin force generation. The available kinetic data are not all easily reconciled with this model and understanding the final details of the myosin motor mechanism must await actin-myosin co-crystals. The observation that myosin can populate multiple states in the absence of actin has nonetheless led to significant insights. The currently known myosin structures correspond to defined kinetic states that bind weakly (K(d)>microM) to actin. It is possible that the myosin lever arm could complete its swing before strong binding to actin and force generation--a process that would correspond, in the absence of load, to a Brownian ratchet. We further suggest that, under load, internal springs within the myosin head could decouple force generation and lever arm movement.

The primary myosin-binding site of caldesmon was thought to be in the N-terminal region of the molecule, but the exact nature of the caldesmon-myosin interaction has not been well characterized. A caldesmon fragment that encompasses residues 1-240 (N240) was found to bind full-length smooth muscle myosin on the basis of co-sedimentation experiments. The interaction between myosin and N240 was not affected by phosphorylation of myosin, but it was weakened by the presence of Ca(2+)/calmodulin. To locate the myosin-binding site, we have designed several synthetic peptides based on the N-terminal caldesmon sequence. We found that a peptide stretch corresponding to the first 27 residues (Met-1 to Tyr-27), but not that of the first 22 residues (Met-1 to Ala-22), exhibited a moderate affinity toward myosin. We also found that a peptide containing the segment from Ile/Leu-25 to Lys-53 bound both myosin and heavy meromyosin more strongly and was capable of displacing caldesmon from myosin. Our results demonstrate that the sequence near the N-terminal extreme of caldesmon harbors a major myosin-binding site of caldesmon, in which both the nonpolar residues and clusters of positively and negatively charged residues confer the specificity and affinity of the caldesmon-myosin interaction.

To assess the functional significance of tethering actin to myosin by caldesmon in the regulation of smooth muscle contraction, we investigated the effects of synthetic peptides, containing the myosin-binding sequences in the N-terminal region of caldesmon, on force directly recorded from single permeabilized smooth muscle cells of ferret portal vein. Two peptides were used, IK29C and MY27C, containing residues from Ile(25) to Lys(53) and from Met(1) to Tyr(27) of the human and chicken caldesmon sequence, respectively, plus an added cysteine at the C terminus. In cells clamped at pCa 6. 7, both peptides increased basal tone. Pretreatment of cells at pCa 6.7 with IK29C or MY27C decreased the amplitude of subsequent phenylephrine-induced contractions but not microcystin-racemic mixture-induced contractions. In all cases the effects of the peptides were concentration-dependent, and IK29C was more potent than MY27C, in agreement with their relative affinity toward myosin. The peptides were ineffective after the phenylephrine contraction was established. MY27C did not further increase the magnitude of contraction caused by a maximally effective concentration of IK29C, consistent with the two peptides having the same mechanism of action. Neither polylysine nor two control peptides containing scrambled sequences of IK29C, which do not bind myosin, had any effect on basal or phenylephrine-induced force. Our results suggest that IK29C and MY27C induce contraction by competing with the myosin-binding domain of endogenous caldesmon. Digital imaging of fluoroisothiocyanate-tagged IK29C confirmed the association of the peptide with intracellular filamentous structures. The results are consistent with a model whereby tethering of actin to myosin by caldesmon may play a role in regulating vascular tone by positioning the C-terminal domain of caldesmon so that it is capable of blocking the actomyosin interaction.

The effect of caldesmon and its actin-binding C-terminal 35 kDa fragment on conformational alterations of actin in a muscle fiber at relaxation, rigor and at simulation of strong and weak binding of myosin heads to actin was studied by polarizational fluorimetry technique. The strong and weak binding forms were mimicked during binding of F-actin of ghost muscle fibers to myosin subfragment-1 modified with NEM (NEM-S1) or pPDM (pPDM-S1), respectively. As a test for alterations in actin conformation, changes in orientation and mobility of a fluorescent probe, TRITC-phalloidin, bound specifically to F-actin were used. The results obtained have shown that during transition of the muscle fiber from the relaxed state into the rigor and during binding of actin filaments to NEM-S1, changes of polarization parameters take place, which are characteristic of formation between actin and myosin of the strong binding and of transformation of actin subunits from the "turned-off" (inactive) to the "turned-on" (active) conformation. Binding of pPDM-S1 to actin and relaxation of the muscle fiber are accompanied, on the contrary, by the changes of orientation and of the fluorescent probe mobility, which are typical of formation of the weak ("non-force-producing") form of actin-myosin binding and of transformation of actin subunits from the active conformation into the inactive one. Caldesmon and its C-terminal fragment markedly inhibit formation of the strong binding at rigor and activate transition of actin monomers to the switched off conformation at relaxation of muscle fiber. In parallel experiments, these regulatory proteins have been shown to inhibit an active force developed at the transition of a muscle fiber from relaxation to rigor. Besides, caldesmon and its fragment decrease the rate of actin filament sliding over myosin in an in vitro motility assay. Caldesmon is suggested to regulate the smooth muscle contraction in an allosterical manner. The alterations in actin conformation inhibit formation of strong binding of myosin cross bridges to actin and activate the ability of weakly bound cross bridges to switch actin monomers from the "on" to the "off" conformation.

TRITC-phalloidin or FITC-labeled F-actin of ghost muscle fibers was bound to tropomyosin and C-terminal recombinant fragments of caldesmon CaDH1 (residues 506-793) or CaDH2 (residues 683-767). After that the fibers were decorated with myosin subfragment 1. In the absence of caldesmon fragments, subfragment 1 interaction with F-actin caused changes in parameters of polarized fluorescence, that were typical of "strong" binding of myosin heads to F-actin and of the "switched on" conformational state of actin. CaDH1 inhibited, whereas CaDH2 activated the effect of subfragment 1. It is suggested that C-terminal part of caldesmon may modulate the transition of F-actin subunits from the "switched on" to the "switched off" state.

Multiple functional domains responsible for calmodulin (CaM) binding and actin-binding/actomyosin ATPase inhibition are present in the region between residues 598-756 of the chicken gizzard smooth muscle caldesmon (CaD) molecule. To precisely localize these functional domains and to further elucidate the structural basis of these domains, we analyzed a series of purified mutants of chicken gizzard smooth muscle CaD generated by internal deletions of amino acid sequences and expression in a baculovirus expression system. Our results demonstrate that, in addition to a strong actin-binding site sequence between residues 718-723 (Wang, Z., and Chacko, S. (1996) J. Biol. Chem. 271, 25707-25714), two weak actin-binding motifs are present in the regions between residues 690-699 and 650-666. These weak actin-binding regions function independently and are associated with weak actomyosin inhibitory activity. Analysis of the CaM-binding sites A (residues 658-666) and B (residues 690-695), the major CaM-binding sites in the C-terminal region of CaD, provided direct evidence for the involvement of both CaM-binding sites in the CaM-mediated reversal of the inhibition of actomyosin ATPase activity by CaD and for the functional independence of the two CaM-binding sites. Furthermore, the sequences between residues 598-649, upstream of CaM-binding site A, and 700-717, downstream of CaM-binding site B, appear to have no effect on either actin-binding or CaM-binding. The data also suggest that both CaM-binding sites A and B structurally overlap or lie in close proximity to the adjacent weak actin-binding sites and weak actomyosin ATPase inhibitory determinants.

We have designed a series of recombinant peptides derived from the C-terminus of human caldesmon (amino acids 663-793, domain 4) to determine the structural basis of the multiple-sited caldesmon-actin-tropomyosin interaction. All the recombinant peptides are able to bind to actin and inhibit actin-activated myosin ATPase activity; 1 mol of peptide is bound per actin for >90% inhibition. However, equivalent inhibition of actin-tropomyosin activation of myosin ATPase requires less than one peptide per seven actin to be bound. We have found two sequences, H2 (amino acids 683-767) and H2+12 (amino acids 683-779), from the center of domain 4 which potentiate actin-tropomyosin filament activity; i.e., their effect is opposite to caldesmon. Maximum potentiation correlates with one H2 or H2+12 bound per four actin. This effect is completely dependent upon the presence of tropomyosin on the actin filament. H2 and H2+12 also increase actin-tropomyosin filament velocity in the in vitro motility assay. If the H2 sequence is extended by 20 amino acids at the N-terminal end to the N-terminus of domain 4, the peptide becomes an inhibitor. If H2 is extended by 19 amino acids at its C terminus, it becomes a tropomyosin-dependent inhibitor, and with a further extension of 7 amino acids to reach the C-terminus of human caldesmon (H2+26), inhibition is more potent. We conclude that three regions in domain 4 of caldesmon contribute to tropomyosin-dependent inhibition of actomyosin ATPase: a central segment [747-767 (690-710 in the chicken sequence)], which is essential but not sufficient for tropomyosin-dependent inhibition of actomyosin ATPase; and two actin binding segments N-terminal and C-terminal to this segment, 663-682 (606-625) and 770-793 (713-737). If only the central segment is present (H2, H2+12), the actin-tropomyosin-caldesmon peptide complex is not inhibitory, and its properties resemble actin-tropomyosin-caldesmon-Ca2+ x calmodulin.

Radial equilibrium lengths of the weakly attached, force-generating, and rigor cross-bridges are determined by recording their resistance to osmotic compression. Radial equilibrium length is the surface-to-surface distance between myosin and actin filaments at which attached cross-bridges are, on average, radially undistorted. We previously proposed that differences in the radial equilibrium length represent differences in the structure of the actomyosin cross-bridge. Until now the radial equilibrium length had only been determined for various strongly attached cross-bridge states and was found to be distinct for each state examined. In the present work, we demonstrate that weakly attached cross-bridges, in spite of their low affinity for actin, also exert elastic forces opposing osmotic compression, and they are characterized by a distinct radial equilibrium length (12.0 nm vs. 10.5 nm for force-generating and 13.0 nm for rigor cross-bridge). This suggests significant differences in the molecular structure of the attached cross-bridges under these conditions, e.g., differences in the shape of the myosin head or in the docking of the myosin to actin. Thus, the present finding supports our earlier conclusion that there is a structural change in the attached cross-bridge associated with the transition from a weakly bound configuration to the force-generating configuration. The implications for imposing spatial constraints on modeling actomyosin interaction in the filament lattice are discussed.

The ability of chicken gizzard smooth muscle caldesmon (CaD) to inhibit actomyosin ATPase activity is due mainly to an inhibitory domain that resides within the C-terminal 67 amino acid residues of the CaD molecule. In the present study, a series of C-terminal truncation and internal deletion mutants of chicken gizzard smooth muscle CaD were systematically designed using a site-directed mutagenesis approach, and these mutant proteins were overexpressed in a baculovirus expression system. Analysis of actin binding and inhibition of actomyosin ATPase activity using these mutants identified a strong actin-binding motif of 6 amino acid residues (from Lys718 to Glu723), which also form the core sequence for CaD-induced inhibition of actomyosin ATPase. However, maximal inhibition by CaD requires the presence of residues 728-731, which are not associated with actin binding. Our data provide direct evidence for the requirement of actin binding to a specific region in CaD for CaD-induced inhibition of actin activation of smooth muscle myosin ATPase. Furthermore, our findings also show that the region between residues 690 and 717 is responsible for the weak inhibition of actomyosin ATPase and reveal that the inhibitory determinants located in the regions between residues 690 and 717 and residues 718 and 756 can function independently.

The actin binding and ATPase properties, as well as the functional domain structure of chick brain myosin-V, a two-headed, unconventional myosin, is reported here. Compared to conventional myosin from skeletal muscle, brain myosin-V exhibits low K-EDTA- and Ca-ATPase activities (1.8 and 0.8 ATP/s per head). The physiologically relevant Mg-ATPase is also low (approximately 0.3 ATP/s), unless activated by the presence of both F-actin and Ca2+ (Vmax of 27 ATP/s). Ca2+ stimulates the actin-activated Mg-ATPase over a narrow concentration range between 1 and 3 microM. In the presence of saturating Ca2+ and 75 mM KCl, surprisingly low concentrations of F-actin activate the Mg-ATPase in a hyperbolic manner (KATPase of 1.3 microM). Brain myosin-V also binds with relatively high affinity (compared to other known myosins) to F-actin in the presence of ATP, as assayed by cosedimentation. Digestion of brain myosin-V with calpain yielded a 65-kDa head domain fragment that cosediments with actin in an ATP-sensitive manner and a 80-kDa tail fragment that does not interact with F-actin. The 80-kDa fragment results from cleavage one residue beyond the proline-, glutamate-, serine-, threonine-rich region. Our data indicate that the Mg-ATPase cycle of brain myosin-V is tightly regulated by Ca2+, probably via direct binding to the calmodulin light chains in the neck domain, which like brush border myosin-I, results in partial (approximately 30%) dissociation of the calmodulin associated with brain myosin-V. The effect of Ca2+ binding, which appears to relieve suppression by the neck domain, can be mimicked by calpain cleavage near the head/neck junction.

Previously, we attributed the binding of F-actin to the 38-residue stretch of gizzard calponin encompassing the sequence A145-Y182 and postulated the hexapeptide motif VKYAEK, representing residues 142-147, as a putative actin-binding site [Mezgueldi, M., Fattoum, A., Derancourt, J. & Kassab, R. (1992) J. Biol. Chem. 267, 15943-15951]. Herein, the nature of the ATPase inhibitory amino acids of calponin and their relative position within the actin binding domain was investigated by expressing the following fragments of mouse calponin with or without substitution or deletion of the hexapeptide V142-K147: amino acids 1-228 (CaP1-228), 45-228 (CaP45-228), 131-228 (CaP131-228), and CaP1-228 with substitution of A145 with S (CaP1-228A145S) or deletion of V142-K147 (CaP1-228de1142-147). All the recombinant fragments displayed most of the biochemical properties of the smooth muscle purified calponin including (a) expected electrophoretic mobility, (b) heat stability, (c) binding to actin, tropomyosin and calmodulin, and (d) zero-length cross-linking to actin switched by calmodulin in a calcium-dependent fashion. However, while the wild-type recombinant fragments inhibit the acto-S-1 ATPase activity to the same extent as do the parent calponin, modulation of the hexapeptide by either substitution or deletion strongly affect the inhibitory activity with only slightly decreasing actin binding capacity. The data indicate that the stretch VKYAEK is crucial for ATPase inhibition by calponin but represents only part of the actin-binding domain. These results are discussed in terms of multiple contact sites between actin and calponin.

The smooth muscle tropomyosin isoforms beta and gamma were isolated in pure form and labeled with N-(1-pyrenyl)iodoacetamide (PIA) on the cysteine residues at either the N- or the C-terminal region (Cys-36 and Cys-190 of beta- and gamma-isoforms, respectively). The effect of caldesmon (CaD) on local conformational changes in different regions of the tropomyosin molecule was determined on the basis of changes in the excimer fluorescence (excited dimer of pyrene) formed in homodimers of tropomyosin isoforms. In the absence of actin, excimer fluorescence from the pyrene at Cys-190 of gamma-tropomyosin homodimer decreased in a simple manner on the addition of CaD, whereas the excimer from the Cys-36 of beta-tropomyosin homodimer exhibited a biphasic change, suggesting that additional weak binding sites exist near Cys-36. In the presence of actin, CaD-induced changes in the excimer fluorescence of pyrene-tropomyosin were observed only with Cys-36, and this change was associated with an inhibition of actin-activated myosin ATPase. A competition study with unlabeled tropomyosin isoforms indicated that the different excimer changes exhibited by beta- and gamma-tropomyosin in the presence of CaD were due to conformational changes in different regions of the tropomyosin molecule and not to differences in their affinities for CaD. Experiments with recombinant CaD mutants derived using the baculovirus expression system showed that the inhibition of tropomyosin potentiation of actomyosin ATPase by CaD requires the regions between residues 728-756 and 718-727 on the CaD molecule, although the latter region was sufficient for direct interaction with tropomyosin.

We have produced nine recombinant fragments, H1 to H9, from a human cDNA that codes for the C-terminal 288 residues of caldesmon. The fragment H1, encompassing the 288 residues, is equivalent to domains 3 and 4 of caldesmon (amino acids 506-793 in human, 476-737 in the chicken gizzard sequence). It has been shown [Huber, Redwood, Avent, Tanner and Marston (1993) J. Muscle Res. Cell Motil. 14, 385-391] to bind to actin, Ca(2+)-calmodulin, tropomyosin and myosin. The fragments, H2 to H9, differ in length between 60 and 176 residues and cover the whole of domains 3 and 4 with many of the fragments overlapping. We have characterized the myosin and tropomyosin binding of these fragments. The binding of both tropomyosin and myosin is highly dependent on salt concentration, indicating the ionic nature of these interactions. The location of the myosin binding is an extended region encompassing the junction of domains 3/4 and domain 4a (residues 622-714, human; 566-657, chicken gizzard). Tropomyosin binds in a smaller region within domain 4a of caldesmon (residues 663-714, human; 606-657 chicken gizzard). We confirmed predictions based on sequence similarities of a tropomyosin binding site in domain 3 of caldesmon; however, this site bound to skeletal-muscle tropomyosin and had little affinity for the smooth-muscle tropomyosin isoform. None of the protein fragments H2-H9 retained the affinity of the parent fragment H1 for either myosin or tropomyosin. This indicates the need for several interaction sites scattered over an extended region to attain higher affinity. The regions interacting with caldesmon in both tropomyosin and myosin are coiled-coil structures. This is probably the reason for their shared interaction sites on caldesmon and their similar natures of binding.

We report the identification and characterization of myr 4 (myosin from rat), the first mammalian myosin I that is not closely related to brush border myosin I. Myr 4 contains a myosin head (motor) domain, a regulatory domain with light chain binding sites and a tail domain. Sequence analysis of myosin I head (motor) domains suggested that myr 4 defines a novel subclass of myosin I's. This subclass is clearly different from the vertebrate brush border myosin I subclass (which includes myr 1) and the myosin I subclass(es) identified from Acanthamoeba castellanii and Dictyostelium discoideum. In accordance with this notion, a detailed sequence analysis of all myosin I tail domains revealed that the myr 4 tail is unique, except for a newly identified myosin I tail homology motif detected in all myosin I tail sequences. The Ca(2+)-binding protein calmodulin was demonstrated to be associated with myr 4. Calmodulin binding activity of myr 4 was mapped by gel overlay assays to the two consecutive light chain binding motifs (IQ motifs) present in the regulatory domain. These two binding sites differed in their Ca2+ requirements for optimal calmodulin binding. The NH2-terminal IQ motif bound calmodulin in the absence of free Ca2+, whereas the COOH-terminal IQ motif bound calmodulin in the presence of free Ca2+. A further Ca(2+)-dependent calmodulin binding site was mapped to amino acids 776-874 in the myr 4 tail domain. These results demonstrate a differential Ca2+ sensitivity for calmodulin binding by IQ motifs, and they suggest that myr 4 activity might be regulated by Ca2+/calmodulin. Myr 4 was demonstrated to be expressed in many cell lines and rat tissues with the highest level of expression in adult brain tissue. Its expression was developmentally regulated during rat brain ontogeny, rising 2-3 wk postnatally, and being maximal in adult brain. Immunofluorescence localization demonstrated that myr 4 is expressed in subpopulations of neurons. In these neurons, prominent punctate staining was detected in cell bodies and apical dendrites. A punctate staining that did not obviously colocalize with the bulk of F-actin was also observed in C6 rat glioma cells. The observed punctate staining for myr 4 is reminiscent of a membranous localization.

A partial clone of caldesmon, coding for the C-terminal 288 amino acids, was isolated from a human fetal liver cDNA library and sequenced. Expression of the clone in Escherichia coli produced a peptide called H1 (M(r) 32,549), which inhibited tropomyosin-enhanced actomyosin Mg(2+)-ATPase activity by 90% with half maximal inhibition at 0.03-0.04 mol H1 per mol actin. The inhibition could be reversed by Ca(2+)-calmodulin. H1 bound actin, Ca(2+)-calmodulin and tropomyosin and smooth muscle myosin with high affinities. This latter finding shows the presence of a second myosin-binding site in caldesmon. This was confirmed in thrombic digests of native sheep aorta and chicken gizzard caldesmon.