Smooth muscle α-actinin binds tightly to fesselin and attenuates its activity toward actin polymerization

Fesselin is an actin binding protein from smooth muscle that nucleates actin polymerization in a Ca⁺⁺-calmodulin dependent manner, bundles actin and inhibits the actin-activated ATPase activity of myosin S1. We now report that fesselin binds to smooth muscle α-actinin. Binding was measured by blot overlay, affinity chromatography and sedimentation methods. Binding was moderate with an association constant of 1–4×10⁷ M⁻¹ assuming a 1:1 association of fesselin with α-actinin. Fesselin binds to the central spectrin domain repeat region of α-actinin but not to the CH1–CH2 domain. Fesselin accelerates the polymerization of actin. This activity of fesselin was attenuated by α-actinin. These observations support the role of fesselin in organizing the cytoskeleton.     

Filamin and gelsolin influence Ca2+-sensitivity of smooth muscle thin filaments

Summary Sheep aorta thin filaments were prepared by ultracentrifugation of an ATP-containing extract in the presence of different concentrations of ethanediol. Thin filaments prepared without ethanediol contained small quantities of tropomyosin (0.027 Tm:actin) and caldesmon (0.017 CD:actin) and activated the MgATPase of skeletal myosin independently of Ca²⁺. Ultracentrifugation in the presence of 10–20% ethanediol resulted in preparation of thin filaments with increased content of tropomyosin (0.17 Tm:actin) and caldesmon (0.04 CD:actin). These thin filaments possessed high Ca²⁺-sensitivity in activation of skeletal muscle myosin ATPase. Besides actin, tropomyosin and caldesmon, thin filaments contained gelsolin and filamin. Gelsolin content (0.007 gelsolin:actin) was independent of the presence of ethanediol. The filamin content decreased from 0.015 to 0.007 mol:mol actin when the ethanediol concentration was increased from 0 to 20%, and was negatively correlated with the Ca²⁺ sensitivity of thin filaments. In a reconstituted system, pure filamin or gelsolin affected caldesmon regulation of actomyosin ATPase. Gelsolin (0.01:actin) reduced the inhibition of actomyosin ATPase caused by caldesmon and increased the potency of Ca²⁺-calmodulin in reversing this inhibition. Filamin (0.007:actin) also decreased the inhibitory action of caldesmon on actin-activated myosin ATPase and also potentiated the reversal of this inhibition by calmodulin. We conclude that minor components of smooth muscle thin filaments (gelsolin and filamin) significantly modify caldesmon mediated regulation of actomyosin ATPase. We suggest a tropomyosin-mediated mechanism by which filamin or gelsolin could exert similar effects.     

Visualization of caldesmon binding to synthetic filaments of smooth muscle myosin

We have used synthetic filaments of unphosphorylated chicken gizzard myosin with a compact, highly ordered structure under relaxing conditions (in the absence of Ca²⁺ and in the presence of ATP) to visualize the mode of caldesmon binding to myosin filaments by negative staining and immunogold electron microscopy. We demonstrate that the addition of caldesmon to preformed myosin filaments leads to the appearance of numerous smooth projections curving out from the filament surface. The addition of caldesmon or its N-terminal fragment resulted in the partial masking of myosin filament periodicity. However, it did not change the inner structure of the filaments. It is demonstrated that most caldesmon molecules bind to myosin filaments through the N-terminal part, while the C-terminal parts protrude from the filament surface, as confirmed by immunoelectron microscopy visualization. Together with the available biochemical data on caldesmon binding to both actin and myosin and electron microscopic observations on the mode of caldesmon attachment to actin filaments with the C-termini of the molecules curving out from the filaments, the visualization of caldesmon attachment to myosin filaments completes the scenario of actin to myosin tethering by caldesmon. This revised version was published online in July 2006 with corrections to the Cover Date.     

Caldesmon binds to smooth muscle myosin and myosin rod and crosslinks thick filaments to actin filaments

Summary It is well established that caldesmon binds to actin (K _b–10⁷-10⁸ m ^–1) and to tropomyosin (K _b10⁶ m ^–1) and that it is a potent inhibitor of actomyosin ATPase. Caldesmon can also bind tightly to myosin. We investigated the binding of smooth muscle and nonmuscle caldesmon isoforms (CDh and CDl respectively) to myosin using proteins from sheep aorta. Both caldesmon isoforms bind to myosin with indistinguishable affinity. The affinity is about 10⁶ m ^–1 in low salt buffer, but is weakened by increasing [KCl] reaching 10⁵ mM^–1 in 100mm KCl. The stoichiometry of binding is about three caldesmon per myosin molecule. Stoichiometry and affinity are not dependent on whether myosin is phosphorylated nor on the presence of Mg²⁺ and ATP, provided the ionic strength is maintained constant. The caldesmon binding site of smooth muscle myosin is located in the S-2 region, consequently both HMM and myosin rod bind to caldesmon. Over a range of conditions myosin and myosin rod binding to caldesmon were indistinguishable. Skeletal muscle myosin has no caldesmon binding site. Smooth muscle myosin rods form side-polar filaments in low salt buffer in which the backbone packing of LMM into the filament shaft is clearly visible in negatively-stained electron microscopic images. Sometimes the S-2 portions can be seen frayed from the filament shaft. When caldesmon is bound the filament shaft appears to be about 20% thicker and the frayed effect is dramatically increased; long filamentous whiskers are often seen curving out from the filament shaft. Similar structures are observed with smooth muscle and with non-muscle caldesmon. Myosin also binds to caldesmon when it is incorporated into the thin filament; however, this interaction is qualitatively different. Measurements of smooth muscle HMM binding to native thin filaments in the presence of 3mm MgATP shows there is a high affinity binding (K_b=10⁶ m ^–1) which is independent of [Ca²⁺] and of the level of myosin phosphorylation. The stoichiometry is one HMM molecule per actin monomer which is equivalent to up to 14 HMM bound at high affinity per caldesmon. Negatively stained electron microscopic images of the HMM.ADP.Pi-thin filament complex have failed to show any attachment of HMM to the thin filaments. When rod filaments are added to actin plus caldesmon or to native thin filaments the rod filaments are strongly associated with the actin filament bundles. The majority of rod filaments are lined up parallel and in close proximity to actin filaments. Similar crosslinking is observed with non-muscle caldesmon. In the smooth muscle cell, caldesmon-containing thin filaments are found together with myosin filaments in the contractile domain in parallel arrays not unlike those shown in our synthetic systems. Thus caldesmon ought to be able to crosslink thick and thin filamentsin vivo.     

Smooth muscle myosin filament assembly under control of a kinase-related protein (KRP) and caldesmon

Kinase-related protein (KRP) and caldesmon are abundant myosin-binding proteins of smooth muscle. KRP induces the assembly of unphosphorylated smooth muscle myosin filaments in the presence of ATP by promoting the unfolded state of myosin. Based upon electron microscopy data, it was suggested that caldesmon also possessed a KRP-like activity (Katayama et al., 1995, J Biol Chem 270: 3919–3925). However, the nature of its activity remains obscure since caldesmon does not affect the equilibrium between the folded and unfolded state of myosin. Therefore, to gain some insight into this problem we compared the effects of KRP and caldesmon, separately, and together on myosin filaments using turbidity measurements, protein sedimentation and electron microscopy. Turbidity assays demonstrated that KRP reduced myosin filament aggregation, while caldesmon had no effect. Additionally, neither caldesmon nor its N-terminal myosin binding domain (N152) induced myosin polymerization at subthreshold Mg²⁺ concentrations in the presence of ATP, whereas the filament promoting action of KRP was enhanced by Mg²⁺. Moreover, the amino-terminal myosin binding fragment of caldesmon, like the whole protein, antagonizes Mg²⁺-induced myosin filament formation. In electron microscopy experiments, caldesmon shortened myosin filaments in the presence of Mg²⁺ and KRP, but N152 failed to change their appearance from control. Therefore, the primary distinction between caldesmon and KRP appears to be that caldesmon interacts with myosin to limit filament extension, while KRP induces filament propagation into defined polymers. Transfection of tagged-KRP into fibroblasts and overlay of fibroblast cytoskeletons with Cy3KRP demonstrated that KRP colocalizes with myosin structures in vivo. We propose a new model that through their independent binding to myosin and differential effects on myosin dynamics, caldesmon and KRP can, in concert, control the length and polymerization state of myosin filaments. This revised version was published online in July 2006 with corrections to the Cover Date.     

Caldesmon: binding to actin and myosin and effects on elementary steps in the ATPase cycle

The actin binding protein caldesmon inhibits the actin-activation of myosin ATPase activity. The steps in the cycle of ATP hydrolysis that caldesmon could inhibit include: (1) the binding of myosin to actin, (2) the transition between any two actin–myosin states and (3) the distribution between inactive and active states of actin. The analysis of these possibilities is complicated because caldesmon binds to both myosin and actin and because each caldesmon molecule binds to several actin monomers. This paper reviews procedures for analysing these interactions and summarizes current information on the stability and dynamics of the interaction of caldesmon with actin and myosin. Possible effects of caldesmon on transitions within the ATPase cycle of actomyosin are also discussed.     

Identification of functioning regulatory sites and a new myosin binding site in the C-terminal 288 amino acids of caldesmon expressed from a human clone

Summary 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 Mg²⁺-ATPase activity by 90% with half maximal inhibition at 0.03–0.04 mol H1 per mol actin. The inhibition could be reversed by Ca²⁺-calmodulin. H1 bound actin, Ca²⁺-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.Abbreviations DTT dithiothreitol - H1 expressed C-terminal 288 amino acids of human caldesmon - PMSF phenylmethanesulfonyl fluoride - TA 2,5-dichloro-triazinyl-(N,N-diethylamino-)benzene - TLCK 1 chloro-3-tosylamido-7-amino-L-2-heptanone     

Caldesmon Tethers Myosin V to Actin and Facilitates in vitro Motility

The current study examines the hypothesis that caldesmon can facilitate the interaction of myosin V with actin filaments by tethering myosin V to actin. Myosin V, purified from bovine brain stem, translocated actin filaments in an in vitro motility assay at a velocity of 0.30+/-0.05 microm/s in the absence of caldesmon at a myosin concentration of 50 microg/ml (ionic strength=110 mM). Filament binding and motility was absent when the myosin concentration applied to the coverslip was reduced to 5 microg/ml, however, the addition of 0.4 microM caldesmon restored binding and motility (0.28+/-0.06 microm/s). This restoration of motility in the presence of caldesmon was blocked by an N-terminal fragment of caldesmon that competitively inhibits the binding of intact caldesmon to myosin. Similar to previous findings with both smooth muscle myosin and platelets (Haeberle et al., 1992; Hemric et al., 1994), these results demonstrate that caldesmon can form a mobile tether that maintains the proximity of myosin V with actin filaments without restricting filament sliding.     

Myosin light chain kinase: functional domains and structural motifs

Conventional myosin light chain kinase found in differentiated smooth and non-muscle cells is a dedicated Ca²⁺/calmodulin-dependent protein kinase which phosphorylates the regulatory light chain of myosin II. This phosphorylation increases the actin-activated myosin ATPase activity and is thought to play major roles in a number of biological processes, including smooth muscle contraction. The catalytic domain contains residues on its surface that bind a regulatory segment resulting in autoinhibition through an intrasteric mechanism. When Ca²⁺/calmodulin binds, there is a marked displacement of the regulatory segment from the catalytic cleft allowing phosphorylation of myosin regulatory light chain. Kinase activity depends upon Ca²⁺/calmodulin binding not only to the canonical calmodulin-binding sequence but also to additional interactions between Ca²⁺/calmodulin and the catalytic core. Previous biochemical evidence shows myosin light chain kinase binds tightly to actomyosin containing filaments. The kinase has low-affinity myosin and actin binding sites in Ig-like motifs at the N- and C-terminus, respectively. Recent results show the N-terminus of myosin light chain kinase is responsible for filament binding in vivo. However, the apparent binding affinity is greater for smooth muscle myofilaments, purified thin filaments, or actin-containing filaments in permeable cells than for purified smooth muscle F-actin or actomyosin filaments from skeletal muscle. These results suggest a protein on actin thin filaments that may facilitate kinase binding. Myosin light chain kinase does not dissociate from filaments in the presence of Ca²⁺/calmodulin raising the interesting question as to how the kinase phosphorylates myosin in thick filaments if it is bound to actin-containing thin filaments.     

The thin filaments of smooth muscles

Summary Contraction in vertebrate smooth and striated muscles results from the interaction of the actin filaments with crossbridges arising from the myosin filaments. The functions of the actin based thin filaments are (1) interaction with myosin to produce force; (2) regulation of force generation in response to Ca²⁺ concentration; and (3) transmission of the force to the ends of the cell. The major protein components of smooth muscle thin filaments are actin, tropomyosin and caldesmon, present in molar ratios of 28:4:1 respectively. Other smooth muscle proteins which may be associated with the thin filaments in the cell are filamin, vinculin, -actinin, myosin light chain kinase and calmodulin. We have reviewed the structural and functional properties of these proteins and where possible we have suggested what their function and mechanism of action may be. We propose that actin and tropomyosin are involved in the force producing interaction with myosin, and that this interaction is controlled by a Ca²⁺-dependent mechanism involving caldesmon, tropomyosin and calmodulin. Vinculin, -actinin and filamin appear to be involved in the attachment of the thin filaments to the cell membrane and their spatial organization within the cell. We conclude that the filaments of smooth muscles share many common properties with those from skeletal muscle, but that they are also quite distinct in terms of both their caldesmon based regulatory mechanism and their mode of organization into a contractile apparatus.     

A novel Ca2+ binding protein associated with caldesmon in Ca2+-regulated smooth muscle thin filaments: evidence for a structurally altered form of calmodulin

Smooth muscle thin filaments are made up of actin, tropomyosin, the inhibitory protein caldesmon and a Ca²⁺-binding protein. Thin filament activation of myosin MgATPase is Ca²⁺-regulated but thin filaments assembled from smooth muscle actin, tropomyosin and caldesmon plus brain or aorta calmodulin are not Ca²⁺-regulated at 25°C/50 mM KCl. We isolated the Ca²⁺-binding protein (CaBP) from smooth muscle thin filaments by DEAE fast-flow chromatography in 6 M urea and phenyl sepharose chromatography using sheep aorta as our starting material. CaBP combines with smooth muscle actin, tropomyosin and caldesmon to reconstitute a normally regulated thin filament at 25°C/50 mM KCl. It reverses caldesmon inhibition at pCa5 under conditions where CaM is largely inactive, it binds to caldesmon when complexed with actin and tropomyosin rather than displacing it and it binds to caldesmon independently of [Ca²⁺]. Amino acid sequencing, and electrospray mass spectrometry show the CaBP is identical to CaM. Structural probes indicate it is different: calmodulin increases caldesmon tryptophan fluorescence but CaBP does not. The distribution of charged species in electrospray mass spectrometry and nozzle skimmer fragmentation patterns are different indicating a less stable N-terminal lobe for CaBP. Brief heating abolishes these special properties of the CaBP. Mass spectrometry in aqueous buffer showed no evidence for the presence of any covalent or non-covalently bound adduct. The only remaining conclusion is that CaBP is calmodulin locked in a metastable altered state.     

Structural interactions between actin,tropomyosin, caldesmon and calcium binding protein and the regulation of smooth muscle thin filaments

The basic structure and functional properties of smooth muscle thin filaments were established about 10 years ago. Since then we and others have been working on the details of how tropomyosin, caldesmon and the Ca²⁺-binding protein regulate actin interaction with myosin. Our work has tended to emphasize the similarities between caldesmon and troponin function whilst others have been more concerned with the differences. The need to resolve the resulting differences has stimulated us to find new and more direct ways of investigating the mechanism of thin filament regulation. In recent years an apparent divergence has opened up between functional measurements, which indicate an allosteric-cooperative regulatory mechanism in which caldesmon and Ca²⁺-binding protein control actin—tropomyosin state in the same way as troponin, and structural measurements which show thin filament structures unlike striated muscle thin filaments. The challenge is to interpret function in terms of structure. We have combined functional studies with expression and mutagenesis of caldesmon and with structural methods including X-ray crystalography of tropomyosin—caldesmon crystals, electron microscopy and helical reconstruction of actin—tropomyosincaldesmon complexes and high resolution nuclear magnetic resonance spectroscopy of the C-terminus of caldesmon in interaction with actin and calmodulin. We have used this information to propose a structural mechanism for caldesmon regulation of the smooth muscle thin filament.     

Overexpression,purification, and characterization of full-length and mutant caldesmons using a baculovirus expression system

Summary Three recombinant chicken gizzard caldesmon (CaD) baculovirus vectors that contained the full-length CaD codon sequence (Pv1CaD), the full-length CaD codon sequence and a six-histidine tag at the 5-end (pBlueBacHisCaD), or the full-length CaD codon sequence and an extra six-histidine codon sequence at the 3-end (PvlHisCaD) were constructed. Spodoptera frugiperda (Sf9) cells transfected with these constructs overexpressed full-length CaD, yielding 2, 20, and 50 g per 10⁶ cells for pBlueBacHisCaD, PvlHisCaD, and PvlCaD, respectively. Time course assays for the expressed proteins demonstrated that the optimum harvest time was 36 h postinfection. Immunofluorescence microscopy revealed PvlCaD localized on the plasma membrane of Sf9 cells at 24 h postinfection and distributed throughout the cytoplasm at 36–48 h postinfection. Analysis of the purified recombinant full-length CaD revealed most of the characteristics of the authentic CaD, including (a) an electrophoretic mobility corresponding to 125 kDa, (b) heat stability, (c) binding to actin, tropomyosin-actin, myosin, and calmodulin, (d) ability to inhibit actin-activated ATP hydrolysis by smooth muscle myosin, and (e) ability of Ca²⁺-calmodulin to reverse the inhibition. A CaD mutant with a deletion of 159 amino acids from the carboxyl terminus of the full-length CaD was also expressed at high levels in Sf9 cells. However, this mutant showed a decreased ability to bind to actin, tropomyosin-actin, and calmodulin, whereas the myosin binding was unaffected; actin-activated ATP hydrolysis by smooth muscle myosin was not inhibited by this mutant.     

Sarcomeric binding pattern of exogenously added intact caldesmon and its C-terminal 20-kDa fragment in skinned fibers of skeletal muscle

Intact caldesmon and particularly the actin-binding C-terminal fragment (20-kDa) of caldesmon have been shown in skeletal muscle fibers to selectively displace low affinity, weakly bound cross-bridges from actin without significantly altering the actin attachment of force producing, strong binding cross-bridges (Brenner et al., 1991; Kraft et al., 1995a). However, the sarcomeric distribution and the specific binding of externally added caldesmon to the myofilaments of skeletal muscle fibers was not known. It was e.g., unclear whether caldesmon binds along actin in a manner similar to tropomyosin or whether it also binds to myosin. In this study, we determined the binding pattern of exogenously added intact caldesmon and its C-terminal 20-kDa fragment, respectively, in MgATP-relaxed rabbit skeletal muscle fibers using electron (EM) and confocal fluorescence microscopy (CFM). EM showed that similar to what has been demonstrated earlier for smooth muscle thin filaments (Lehman et al., 1989), intact caldesmon binds periodically every 38nm along the thin filaments. CFM revealed that rhodamine-labeled intact caldesmon and the 20-kDa caldesmon fragment bind along nearly the entire length of the thin filaments. A portion of the I-band near the Z-line appears unlabeled, both when equilibrated at normal and long sarcomere lengths. The width of the unlabeled region seems to depend on ionic strength. The 20-kDa C-terminal caldesmon fragment binds in essentially the same pattern as intact caldesmon. This indicates that the high fluorescence intensity in the overlap region seen with intact caldesmon does not depend on caldesmon binding to myosin. X-ray diffraction was used to monitor the effects on filament lattice. Intact caldesmon at >0.3mg/ml induced disorder in the myofilament lattice. No such disordering was observed, however, when fibers were equilibrated with up to 0.8mg/ml of the 20-kDa caldesmon fragment.     

Amino acids 519-524 of Dictyostelium myosin II form a surface loop that aids actin binding by facilitating a conformational change

Residues 519-524 of Dictyostelium myosin II form a small surface loop on the actin binding face, and have been suggested to bind directly to actin through high affinity hydrophobic interactions. To test this hypothesis, we have characterized mutant myosins that lack this loop in vivo and in vitro. A mutant myosin in which this loop was replaced by an Ala residue (delta519-524/+A) was non-functional in vivo. Replacement with a single Gly residue instead of Ala yielded partial function, suggesting that structural flexibility, rather than hydrophobicity, is the key feature of the loop. The in vivo phenotype of the mutant enabled us to identify a number of additional amino acid changes that restore function to the delta519-524/+A mutation. Intriguingly, many of these, including L596S, were located at some distances away from the 519-524 loop. We have also isolated suppressors for the L596S mutant myosin, which was not functional in vivo. The suppressors for delta519-524/+A and those for L596S showed complementary charge patterns. In ATPase assays, delta519-524/+A S1 showed very low activity and little enhancement by actin, whereas L596S S1 was hyper active and displayed enhanced affinity for actin. In motility assays, delta519-524/+A myosin released actin filaments upon addition of ATP and was unable to support movements. L596S myosin was also inactive, but in this case actin filaments stayed immobile even after the addition of ATP. Transient kinetic measurements demonstrated that delta519-524/+A S1 is not only slower than wild type to bind actin filaments, but also slower to dissociate from actin filaments. Based on these results, we concluded that the 519-524 loop is not a major actin binding site but aids actin binding by facilitating a critical conformational change.     

Effect of unphosphorylated smooth muscle myosin on caldesmon-mediated regulation of actin filament velocity

Summary The effect of smooth muscle myosin at different levels of light chain phosphorylation on caldesmon-mediated movement of actin filaments was investigated using an in vitro motility assay. Myosin at different levels of phosphorylation was obtained by mixing different proportions of fully phosphorylated and unphosphorylated myosin in monomeric form, while keeping the total myosin concentration constant. The average velocity of actin filaments containing tropomyosin was 1.20±0.046 m s^–1 at 30°C with fully phosphorylated myosin. This velocity was not altered when the percentage of unphosphorylated myosin coated on the nitrocellulose surface was increased to 80%; further increases lowered the velocity. When the actin filaments with caldesmon bound at stoichiometric levels were used, filament velocity was unaffected until 50% of the myosin was unphosphorylated, but further increases in the percentage of unphosphorylated myosin induced a decrease in the velocity, and at 95% unphosphorylated myosin, filament movement had ceased. The decreased filament velocity in the presence of caldesmon was also observed when phosphorylated myosin was mixed with myosin rod instead of unphosphorylated myosin, but was not observed when the 38 kDa caldesmon C-terminal fragment, which lacks the myosin-binding domain, was used instead of intact caldesmon. These data indicate that the decreased filament velocity in the presence of caldesmon reflects the mechanical load produced by the tethering of actin to myosin through the interaction of the caldesmon N-terminal domain and the myosin S-2 region. The tethering effect mediated by caldesmon may play a role in smooth muscle contraction when a large number of myosin heads are dephosphorylated, as in force maintenance.     

Control of shortening speed in single guinea-pig taenia coli smooth muscle cells by Ca2+, phosphorylation and caldesmon

 We studied the effect of caldesmon peptides on the regulation of shortening of single guinea-pig taenia coli cells permeabilised with saponin. When contraction was initiated by Ca²⁺ and MgATP shortening rate at pCa 4.5 was 0.17±0.04 cell lengths s^–1 and half-maximal rate was at pCa 5.6. Following thiophosphorylation with 1 mM adenosine 5′-O-(3-thiotriphosphate) (ATP[γ-S]) at pCa 4.5 for 10 min, on addition of ATP these cells contracted at of 0.25±0.04 cell lengths s^–1 independently of pCa. If thiophosphorylated cells were preincubated with H1 (domains 3 and 4 of caldesmon), shortening speed was reduced (ID₅₀=2 μM). Shortening speed was also reduced by 658C (domain 4b) at higher concentrations (ID₅₀=400 μM). H13 (domain 4a), which does not block weak binding but inhibits actin-tropomyosin, inhibited cell shortening (ID₅₀=6 μM). H2, which blocks weak binding but does not inhibit actin-tropomyosin, did not inhibit shortening. Western blots of the cells showed that the peptides were tightly bound within the cell but the native caldesmon was not displaced. These results indicate that exogenous caldesmon peptides added to smooth muscle cells may be incorporated into the thin filaments and produce effects on shortening, as expected if it were involved in tropomyosin-dependent inhibition of the actin filament in the cell. Received: 3 July 1998 / Received after revision and accepted: 1 September 1998     

Inhibition of actin filament movement by monoclonal antibodies against the motor domain of myosin

Summary Conformational transitions in defined regions of the motor domain of skeletal muscle myosin involved in ATP hydrolysis, actin binding and motility were probed with monoclonal antibodies. Competition binding assays demonstrate that three different monoclonal antibodies react with spatially related sites on the myosin heavy chain. One recognizes a sequential epitope between residues 65 and 80 and has no effect on actin filament movement in an in vitro motility assay despite tight binding to myosin and acto-S1. The other two monoclonal antibodies react with sequential epitopes between residues 29 and 60 and both inhibit actin filament movement. A fourth monoclonal antibody reacts with the N-terminus of the heavy chain (residues 1–12) at a spatially distinct site on the myosin head and also inhibits actin filament movement. These four monoclonal antibodies have been mapped by immunoelectron microscopy to the large, actin binding region of the myosin head; however, the antibody binding sites remain accessible on rigor complexes of acto-S1. Thus, this group of monoclonal antibodies identify sequential epitopes in a mobile segment of the myosin heavy chain. In addition, two conformation-sensitive monoclonal antibodies are described that are affected by ATP and actin binding to myosin S1, and display distinct and marked inhibitory effects on actin filament movement. In contrast, an anti-light chain monoclonal antibody that binds near the myosin head-rod junction has little effect on the number and velocity of moving actin filaments. These results identify mobile regions on the myosin head that are perturbed by antibody binding and that may be linked to force production and motion.     

Purification and properties of Ca2+-regulated thin filaments and F-actin from sheep aorta smooth muscle

Summary We have investigated the conditions for isolation of Ca²⁺-regulated thin filaments from sheep aorta. Inhibition of proteolysis by 2 µg ml^–1 leupeptin and chymostatin and of oxidation with 5mm dithiothreitol were essential. Washed homogenates were extracted in 10mm ATP of low ionic strength at pH 6.1 to minimize coextraction of myosin with thin filaments. Thin filaments were separated from myosin by high speed sedimentation; 20% glycol was added to prevent loss of regulatory factors and tropomyosin. The resulting thin filaments (yield 2.5 mg protein g^–1 artery wet weight) were made up of actin, tropomyosin and a 120 000M _r protein (molar ratio 1:1/5:1/29) and were up to 4 µm long. They activated skeletal muscle myosin at least 50 times in presence of Ca²⁺. Up to 80% inhibition was observed in the absence of Ca²⁺. We also prepared pure arterial F-actin, which activated skeletal myosin more than the thin filaments, but was similar to skeletal F-actin. We conclude that Ca²⁺ regulation is negative, involves cooperative interactions between actin, myosin and tropomyosin and suggest that it is mediated by the 120 000M _r protein.     

Functional sequences of the myosin head

Summary Muscle contraction originates from the sliding of myosin filaments on actin filaments, the energy for which is supplied by the hydrolysis of adenosine-5-triphosphate (ATP) by myosin. The nucleotide first binds to the acto-myosin complex in the myosin head (or subfragment-1), producing a conformational change which induces actin dissociation. The release of phosphate (Pi) then allows a return to the strong actin-myosin association, corresponding to the rigor state.We discuss here certain controversial points arising from current concepts of the actin and nucleotide binding regions at the amino acid sequence level within the subfragment-1 heavy chain. We consider the actin and nucleotide binding regions to be two distinct sites (for each of these regions) one of which is shared competitively between actin and the nucleotide. In our model the cyclical actin-S1 association-dissociation steps correspond to different ATP, actin and ADP affinities for the same amino acid sequence of the S1 heavy chain, contributing alternatively to a single hydrolytic nucleotide site or a strong actin site.We propose the existence of a flexible segment that forms or dismantles the nucleotide or actin sites. The large region (amino acids 540–707) overlapping the actin-myosin interface appears to be the main flexible region of the S1 molecule and we propose that this particular sequence plays a key role in the dissociation pathway of the actin-myosin complex and in the conversion of chemical energy into the mechanical energy of contraction.