Interaction of myosin subfragment-1 with actin. III. Effect of cleavage of the subfragment-1 heavy chain on its interaction with actin.

To determine the reason why the Mg2+-ATPase activity of subfragment-1 prepared with chymotrypsin was activated more by actin than that of subfragment-1 prepared with trypsin was and the reason why the former could enhance the polymerization of actin and the latter could not, we digested subfragment-1, prepared with chymotrypsin, with trypsin and examined the actin activated Mg2+-ATPase activity and the ability to polymerize actin. It was found that cleavage of the heavy chain decreased the actin activated Mg2+-ATPase activity of subfragment-1 prepared with chymotrypsin but did not affect its ability to polymerize actin. Trypsin attacked the subfragment-1 heavy chain at two sites and produced 26 K, 50 K, and 21 K fragments. From the comparison of the time course of tryptic digestion with that of the decrease in actin activation, it was deduced that cleavage of the 50 K-21 K junction was mainly responsible for the decrease in actin activation. We also measured the length and the amount of F-actin polymerized by the addition of different amounts of subfragment-1. It was found that the amount of F-actin increased with the increase in the amount of subfragment-1 added and that the length of F-actin also increased though slightly. We concluded from the results that subfragment-1 enhanced the polymerization not only by facilitating the nucleus formation but also by strengthening the bond between actin monomers in forming F-actin.     

Interaction of myosin subfragment-1 with actin. I. Effect of actin binding on the susceptibility of subfragment-1 to trypsin

The heavy chain of myosin subfragment-1 prepared by chymotrypsin treatment had a molecular weight of about 96 K. It was split into 26 K, 50K, and 21 K fragments on trypsin treatment. The effect of actin binding on the susceptibilities of the junctions between 26 K and 50 K and between 50 K and 21 K, and on that of alkali light chain 1 to trypsin was studied. The addition of actin increased the viscosity of the solution, and the apparent activity of trypsin decreased. We estimated this decrease as 35% by measuring the degradation of gamma-globin heavy chain, which is known not to interact with actin and subfragment-1 but is known to be susceptible to trypsin, in actin-subfragment-1 solution. Taking this value into consideration, we concluded that the 26 K-50 K junction became 5 times more and the 50 K-21 K junction became 3 times less susceptible to tryptic attack upon the binding of actin. We also observed that alkali light chain 1 became resistant to trypsin upon the binding of actin to subfragment-1. The relation between this conformational change in subfragment-1 and the cyclic interaction of subfragment-1 with actin and ATP is discussed.     

Interaction of alkali light chain 1 with the isolated 20-kilodalton fragment of myosin subfragment-1 heavy chain and F-actin

The binding of one of the alkali light chains of myosin, A1, with the isolated renatured 20-kDa fragment of myosin subfragment-1 heavy chain was demonstrated by means of difference UV absorption spectroscopy. The difference spectrum with either rabbit or chicken A1 showed two characteristic peaks at 279 and 287 nm indicating a perturbation of tyrosyl chromophores by the association with the 20-kDa fragment. The delta epsilon at 287 nm increased with an increase in the molar ratio of A1/20-kDa fragment and reached a maximum value at around equimolar ratio. The maximum delta epsilon value was approximately three times larger with rabbit A1 than with chicken A1. Based on the positions of Tyr residues in the amino acid sequences, the contact surface of A1 with myosin heavy chain was concluded to be spread over a large area of A1. The binding of 20-kDa fragment with F-actin was measured by following the increase in turbidity. The affinity appeared to increase several times in the presence of A1. A1 may possibly control the affinity of myosin for actin.     

Spatial relationship between SH1 and the actin binding site on myosin subfragment-1 surface

To examine the spatial relationship between SH1 thiol and actin binding site on subfragment-1 surface, we studied the interaction with actin of subfragment-1 whose SH1 was labeled with an iodoacetate derivative of biotin and covered with avidin. Subfragment-1--avidin complex bound F-actin and its Mg2+ ATPase activity was activated by actin. Considering the size and the location of biotin binding site on avidin, our results suggest that SH1 is separated from the actin binding site on subfragment-1 surface by at least 17-20 A.     

The primary structure of skeletal muscle myosin heavy chain: II. Sequence of the 50 kDa fragment of subfragment-1

The complete amino acid sequence of the 50 kDa fragment of subfragment-1 from adult chicken pectoralis muscle myosin was determined. It contained 431 residues including an epsilon-N-trimethyllysine at position 346. The 431-residue sequence corresponds to the sequence of residues 206 to 639 of chicken embryonic breast muscle myosin heavy chain which was predicted from the nucleotide sequence of the cDNA by Molina et al. [Molina, M. I., Kropp, K.E., Gulick, J., & Robbins, J. (1987) J. Biol. Chem. 262, 6478-6488]. Comparing the two sequences, 23 amino acid substitutions and three deletions/insertions are recognized.     

Interference with myosin subfragment-1 binding by site-directed mutagenesis of actin

Three N-terminal double mutants of beta-actin expressed in the yeast Saccharomyces cerevisiae have been characterized with respect to DNase-I interaction, N-terminal post-translational modification, polymerizability and myosin subfragment-1 binding. The results strongly support earlier suggestions that the acidic residues at the N-terminus of actin are part of the myosin-binding site, while they seem to be of no importance for the other aspects of actin biochemistry tested. The suitability of this expression system for production of recombinant actin in general is discussed.     

Interaction between stretch of residues 633-642 (actin binding site) and nucleotide binding site on skeletal myosin subfragment 1 heavy chain

Using a complementary sequence or antipeptide to selectively neutralize the stretch of residues 633-642 of skeletal myosin heavy chain, we recently demonstrated that this segment is an actin binding site operating in the absence as in the presence of nucleotide and that this stretch 633-642 is not part of the nucleotide binding site [Chaussepied & Morales (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7471-7475]. In the present study, we determined that the covalent cross-linking of the antipeptide to the stretch 633-642 [induced by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide] does not alter the overall polypeptide conformation since no changes were observed on the far-ultraviolet CD spectra and thiol reactivity measurements. The presence of the antipeptide did not influence significantly the enhancement of tryptophan fluorescence induced by ATP.Mg2+ or ADP.Mg2+ binding to the myosin head (S1) nor did it on the ATP.Mg2+-induced tryptic proteolysis of S1 heavy chain. Moreover, fluorescence quenching studies, using acrylamide and the analogue, 1,N6-ethenoadenosine 5'-triphosphate, indicated that the nucleotide bound to antipeptide-S1 complex has an accessibility to the solute quencher close to that observed when it is bound to native S1. Additionally, neutralization of the stretch 633-642 of the S1 heavy chain by the antipeptide did not influence the stabilization of the Mg2+.ADP.sodium vanadate-S1 complex. On the other hand, experiments using antipeptide-induced protection against the cleavage of the S1 heavy chain by Arg-C protease demonstrated that the presence of Mg2+.ADP.sodium vanadate in the S1 nucleotide site did not affect the interaction of the antipeptide with the stretch of residues 633-642.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on the heterogeneity of subfragment-1 preparations. Isolation of a new proteolytic fragment of the heavy chain of myosin

1. The physical, chemical and enzymic properties of subfragment 1 prepared from myosin of rabbit skeletal muscle by using two different concentrations of insoluble papain were compared. 2. Subfragment 1 prepared by using a myosin/papain ratio of 2000: 1 (by wt.) migrated on electrophoresis in non-dissociating conditions as a single enzymically active band. When prepared with a myosin/papain ratio of 200: 1 the preparation consisted of two enzymically active components of slightly different electrophoretic mobility. 3. The two types of preparation were obtained in similar yield and possessed similar specific adenosine triphosphatase activities when determined in the presence of Ca(2+). 4. Gel electrophoresis in the presence of 8m-urea showed that both preparations contained three light components. The component of molecular weight 15500 was apparently identical with one of the light-chain components of myosin (Ml(1)). The other two light-chain components of subfragment 1 were not identical with any of the light-chain components of myosin. 5. The heavy-chain fraction of subfragment 1 prepared by using low concentrations of papain dissociated into components with molecular weights of 87000, 69000 and 26000 on electrophoresis in sodium dodecyl sulphate. The heavy-chain fraction of subfragment 1 prepared by using higher concentrations of papain contained components with molecular weights of 69000 and 53000 and relatively increased amounts of the component of molecular weight 26000. 6. The isolated 26000 dalton component had an amino acid composition similar to that of the heavy-chain fraction of subfragment 1 and contained 3-methylhistidine and mono-and tri-N(epsilon)-methyl-lysine. It was homogeneous on electrophoresis in the presence of sodium dodecyl sulphate but gave two bands on electrophoresis in 8m-urea.     

Interaction of Lys-61 labeled actin with myosin subfragment-1 and the regulatory proteins

Actin modified at Lys-61 with fluorescein 5-isothiocyanate (FITC) recovers the ability to polymerize following the binding of phalloidin. The resulting polymer (FITC-P-actin) activates the S1-Mg2+-ATPase activity to the same extent as non-labeled F-actin. However, in the absence of phalloidin, FITC-actin (0.5 mg/ml) neither polymerized nor activated the S1-Mg2+-ATPase activity effectively even when it was preincubated with S1 for 3 h in 0.1 mM ATP, 0.1 mM CaCl2, and 1 mM Tris/HCl (pH 8.0), in contrast to the previous report [Miller, L., Phillips, M., & Reisler, E. (1988) Eur. J. Biochem. 174, 23-29]. The modification of Lys-61 did not impair the ability to bind tropomyosin or tropomyosin-troponin. On the other hand, the fluorescence polarization of FITC-P-actin increased when tropomyosin or troponin-tropomyosin was added. Moreover, the modification of Lys-61 affected the regulation of the actin activation of the S1-Mg2+-ATPase activity by the tropomyosin and troponin complex. In 30 mM KCl, 2.5 mM ATP, and 5 mM MgCl2, tropomyosin alone has been shown to inhibit the actin-activated S1-Mg2+-ATPase. This inhibition did not occur with FITC-P-actin even though tropomyosin was tightly bound. When troponin-tropomyosin was added, the FITC-P-actin activation of S1-Mg2+-ATPase activity was regulated in response to micromolar Ca2+ concentrations. On the other hand, in 30 mM KCl, 2.5 mM ATP, and 2 mM MgCl2, tropomyosin alone did not inhibit the actin-activated S1-Mg2+-ATPase activity with either non-labeled F-actin or FITC-actin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Caldesmon inhibits skeletal actomyosin subfragment-1 ATPase activity and the binding of myosin subfragment-1 to actin

Smooth muscle contraction is controlled in part by the state of phosphorylation of myosin. A recently discovered actin and calmodulin-binding protein, named caldesmon, may also be involved in regulation of smooth muscle contraction. Caldesmon cross-links actin filaments and also inhibits actin-activated ATP hydrolysis by myosin, particularly in the presence of tropomyosin. We have studied the effect of caldesmon on the rate of hydrolysis of ATP by skeletal muscle myosin subfragment-1, a system in which phosphorylation of the myosin is not important in regulation. Caldesmon is a very effective inhibitor of ATP hydrolysis giving up to 95% inhibition. At low ionic strength (approximately 20 mM) this effect does not require smooth muscle tropomyosin, whereas at high ionic strength (approximately 120 mM) tropomyosin enhances the inhibitory activity of caldesmon at low caldesmon concentrations. Cross-linking of actin is not essential for inhibition of ATP hydrolysis to occur since at high ionic strength there is very little cross-linking as determined by a low speed sedimentation assay. Under all conditions examined, the decrease in the rate of ATP hydrolysis is accompanied by a decrease in the binding of myosin subfragment-1 to actin. Furthermore, caldesmon weakens the equilibrium binding of myosin subfragment-1 to actin in the presence of pyrophosphate. We conclude that caldesmon has a general weakening effect on the binding of skeletal muscle myosin subfragment-1 to actin and that this weakening in binding may be responsible for inhibition of ATP hydrolysis.     

Interaction of actin with myosin A and heavy meromyosin.

Ca2+ "free" actomyosin suspensions as well as actin heavy meromyosin (HMM) solutions in the presence of Ca2+ showed no contractile response (superprecipitation) and had low steady-state Mg2+-ATPase activity. Under the same experimental conditions both the enzymatic activity increased and contractile response was restored if the solubility of the proteins was depressed by the addition of polyethylene glycol 4000 (PEG-4000). The stability of the enzymatically active actomyosin or actin HMM complexes was 10 times lower in cleared solutions than in the insoluble actomyosin or actin HMM suspensions. It was concluded that soluble actomyosin or actin HMM solutions are inadequate test tube models for studying muscular contraction.     

A mosaic multiple-binding model for the binding of caldesmon and myosin subfragment-1 to actin.

Binding of caldesmon to actin causes a decrease in the quantity of bound myosin and results in a reduction in the rate of actin-activated adenosine triphosphate hydrolysis. It is generally assumed that the binding of caldesmon and myosin to actin is a pure competitive interaction. However, recent binding studies of enzyme digested caldesmon subfragments directed at mapping the actin binding site of caldesmon have shown that a small 8-kD fragment around the COOH-terminal can compete directly with the myosin subfragment 1 (S-1) binding to actin; at least one other fragment that binds to actin does not inhibit the actin-activated adenosine triphosphate activity of myosin. That is, only a part of the caldesmon sequence may be responsible for directly blocking the binding of S-1 to actin. This prompts us to question the actual mode of binding of intact caldesmon and myosin S-1 to actin: whether the entire intact caldesmon molecule is competing with S-1 binding (pure competitive model) or just a small part of it (mosaic multiple-binding model). To answer this question, we measured the amount of myosin S-1 and caldesmon bound per actin monomer as a function of the total concentration of S-1 added to the system at constant concentrations of actin and caldesmon. A formalism for calculating the titration data based on the pure competitive model and a mosaic multiple-binding model was then developed. When compared with theoretical calculations, it is found that the binding of caldesmon and S-1 to actin cannot be pure competitive if no cooperativity exists between S-1 and caldesmon.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bundling of myosin subfragment-1-decorated actin filaments

We have reported previously that rabbit skeletal myosin subfragment-1 (S-1) assembles actin filaments into bundles. The rate of this reaction can be estimated roughly from the initial rate (Vo) of the accompanying turbidity increase ("super-opalescence") of the acto-S-1 solution. Vo is a function of the molar ratio (r) of S-1 to actin, with a peak at r = 1/6 to 1/7 and minimum around r = 1. In the present paper we report a different type of opalescence (we call it "hyper-opalescence") of acto-S-1 solutions, which also resulted from bundle formation. Adjacent filaments in the bundles had a distance of approximately 180 A. Hyper-opalescence occurred at r approximately equal to 1 when KCOOCH3 was used instead of KCl. By comparing the effects of ADP, epsilon-ADP, tropomyosin or ionic strength upon the super- and hyper-opalescence, we concluded that the two types of S-1-induced actin bundling had different molecular mechanisms. The hyper-opalescence type of bundling seemed to be induced by S-1, which was not complexed with actin in the manner of conventional rigor binding. The presence of the regulatory light chain did not affect hyper-opalescence (or super-opalescence), since there were no significant differences between papain S-1 and chymotryptic S-1 with respect to these phenomena.     

Conformational changes of actin induced by strong or weak myosin subfragment-1 binding

Movements of different areas of polypeptide chains within F-actin monomers induced by S1 or pPDM-S1 binding were studied by polarized fluorimetry. Thin filaments of ghost muscle were reconstructed by adding G-actin labeled with fluorescent probes attached alternatively to different sites of actin molecule. These sites were: Cys-374 labeled with 1,5-IAEDANS, TMRIA or 5-IAF; Lys-373 labeled with NBD-Cl; Lys-113 labeled with Alexa-488; Lys-61 labeled with FITC; Gln-41 labeled with DED and Cys-10 labeled with 1,5-IAEDANS, 5-IAF or fluorescein-maleimid. In addition, we used TRITC-, FITC-falloidin and e-ADP that were located, respectively, in filament groove and interdomain cleft. The data were analysed by model-dependent and model-independent methods (see appendixes). The orientation and mobility of fluorescent probes were significantly changed when actin and myosin interacted, depending on fluorophore location and binding site of actomyosin. Strong binding of S with actin leads to 1) a decrease in the orientation of oscillators of derivatives of falloidin (TRITC-falloidin, FITC-falloidin) and actin-bound nucleotide (e-ADP); 2) an increase in the orientation of dye oscillators located in the "front' surface of the small domain (where actin is viewed in the standard orientation with subdomains 1/2 and 3/4 oriented to the right and to the left, respectively); 3) a decrease in the angles of dye oscillators located on the "back" surface of subdomain-1. In contrast, a weak binding of S1 to actin induces the opposite effects in orientation of these probes. These data suggest that during the ATP hydrolysis cycle myosin heads induce a change in actin monomer (a tilt and twisting of its small domain). Presumably, these alterations in F-actin conformation play an important role in muscle contraction.     

Functional characterization of the secondary actin binding site of myosin II

The role of the interaction between actin and the secondary actin binding site of myosin (segment 565-579 of rabbit skeletal muscle myosin, referred to as loop 3 in this work) has been studied with proteolytically generated smooth and skeletal muscle myosin subfragment 1 and recombinant Dictyostelium discoideum myosin II motor domain constructs. Carbodiimide-induced cross-linking between filamentous actin and myosin loop 3 took place only with the motor domain of skeletal muscle myosin and not with those of smooth muscle or D. discoideum myosin II. Chimeric constructs of the D. discoideum myosin motor domain containing loop 3 of either human skeletal muscle or nonmuscle myosin were generated. Significant actin cross-linking to the loop 3 region was obtained only with the skeletal muscle chimera both in the rigor and in the weak binding states, i.e., in the absence and in the presence of ATP analogues. Thrombin degradation of the cross-linked products was used to confirm the cross-linking site of myosin loop 3 within the actin segment 1-28. The skeletal muscle and nonmuscle myosin chimera showed a 4-6-fold increase in their actin dissociation constant, due to a significant increase in the rate for actin dissociation (k(-)(A)) with no significant change in the rate for actin binding (k(+A)). The actin-activated ATPase activity was not affected by the substitutions in the chimeric constructs. These results suggest that actin interaction with the secondary actin binding site of myosin is specific for the loop 3 sequence of striated muscle myosin isoforms but is apparently not essential either for the formation of a high affinity actin-myosin interface or for the modulation of actomyosin ATPase activity.     

Interaction of myosin subfragment-1 with F- and G-actin in equilibrium.

The structural changes of the F-actin-myosin head (S1) complex during the cross-bridge cycle are essential in muscle contraction. Although a large body of evidence has accumulated showing that the actin: S1 stoichiometry in the decorated F-actin-S1 filament is 1:1 at saturation by S1, a recent report by Andreev and Borejdo (1991, Biochem. Biophys. Res. Comm. 177, 350-356) indicated that under some conditions, the actin: S1 stoichiometry could be 2:1 at saturation by S1. Because of the important implications of this result in the mechanism of acto-myosin motility, we have re-investigated this issue. It is shown here that evidence for the 2:1 stoichiometry was circumstantial and was only observed under conditions where 50% of the actin was F-actin, i.e. at a total actin concentration twice as large as the critical concentration. The interaction of S1 with both F- and G-actin in dynamic equilibrium is studied in detail. The present data fully support the 1:1 actin: S1 stoichiometry in the decorated filament at saturation by S1.     

Interaction of 75-106 actin peptide with myosin subfragment-1 and its trypsin modified derivative

To explore the role of a hydrophobic domain of actin in the interaction with a myosin chain we have synthesized a peptide corresponding to residues 75-106 of native actin monomer and studied by fluorescence and ELISA the interaction (13+/-2.6x10(-6) M) with both S-1 and (27 kDa-50 kDa-20 kDa) S-1 trypsin derivative of myosin. The loop corresponding to 96-103 actin residues binds to the S-1 only in the absence of Mg-ATP and under similar conditions but not to the trypsin derivative S-1. Biotinylated C74-K95 and I85-K95 peptide fragments were purified after actin proteolysis with trypsin. The C74-K95 peptide interacted with both S-1 and the S-1 trypsin derivative with an apparent Kd(app) of 6+/-1.2x10(-6) M in the presence or absence of nucleotides. Although peptide fragment I85-K95 binds to S-1 with a Kd(app) of 12+/-2.4x10(-6) M, this fragment did not bind to the trypsin S-1 derivative. We concluded that the actin 85-95 sequence should be a potential binding site to S-1 depending of the conformational state of the intact 70 kDa segment of S-1.     

Molecular dynamics simulation of a myosin subfragment-1 docking with an actin filament

Myosins are typical molecular motor proteins, which convert the chemical energy of ATP into mechanical work. The fundamental mechanism of this energy conversion is still unknown. To explain the experimental results observed in molecular motors, Masuda has proposed a theory called the “Driven by Detachment (DbD)” mechanism for the working principle of myosins. Based on this theory, the energy used during the power stroke of the myosins originates from the attractive force between a detached myosin head and an actin filament, and does not directly arise from the energy of ATP. According to this theory, every step in the myosin working process may be reproduced by molecular dynamics (MD) simulations, except for the ATP hydrolysis step. Therefore, MD simulations were conducted to reproduce the docking process of a myosin subfragment-1 (S1) against an actin filament. A myosin S1 directed toward the barbed end of an actin filament was placed at three different positions by shifting it away from the filament axis. After 30 ns of MD simulations, in three cases out of ten trials on average, the myosin made a close contact with two actin monomers by changing the positions and the orientation of both the myosin and the actin as predicted in previous studies. Once the docking was achieved, the distance between the myosin and the actin showed smaller fluctuations, indicating that the docking is stable over time. If the docking was not achieved, the myosin moved randomly around the initial position or moved away from the actin filament. MD simulations thus successfully reproduced the docking of a myosin S1 with an actin filament. By extending the similar MD simulations to the other steps of the myosin working process, the validity of the DbD theory may be computationally demonstrated.     

Spatial relationship between the nucleotide-binding site, Lys-61 and Cys-374 in actin and a conformational change induced by myosin subfragment-1 binding

The spatial relationship between Lys-61, the nucleotide binding site and Cys-374 was studied. Lys-61 was labelled with fluorescein-5-isothiocyanate as a resonance energy acceptor, the nucleotide-binding site was labelled with the fluorescent ATP analogues epsilon ATP or formycin-A 5'-triphosphate (FTP) and Cys-374 was labelled with 5-(2-[(iodoacetyl)amino]ethyl)aminonaphthalene-1-sulfonic acid (1,5-IAEDANS) as a resonance energy donor. The distances between the nucleotide binding site and Lys-61 or between Lys-61 and Cys-374 were calculated to be 3.5 +/- 0.3 nm and 4.60 +/- 0.03 nm, respectively. (The assumption has been made in calculating these distances that the energy donor and acceptor rotate rapidly relative to the fluorescence lifetime.) On the other hand, when doubly-labelled actin with 1,5-IAEDANS at Cys-374 and FITC at Lys-61 was polymerized in the presence of a twofold molar excess of phalloidin [Miki, M. (1987) Eur. J. Biochem. 164, 229-235], the fluorescence of 1,5-IAEDANS bound to actin was quenched significantly. This could be attributed to inter-monomer energy transfer. The inter-monomer distance between FITC attached to Lys-61 in a monomer and 1,5-IAEDANS attached to Cys-374 in its nearest-neighbour monomer in an F-actin filament was calculated to be 3.34 +/- 0.06 nm, assuming that the likely change in the intra-monomer distance does not change during polymerization by more than 0.4 nm. One possible spatial relationship between Lys-61, Cys-374 and the nucleotide binding site in an F-actin filament is proposed. The effect of myosin subfragment-1 (S1) binding on the energy transfer efficiency was studied. The fluorescence intensity of AEDANS-FITC-actin decreased by 30% upon interaction with S1. The fluorescence intensity of AEDANS-FITC-actin polymer in the presence of phalloidin increased by 21% upon interaction with S1. The addition of ATP led to the fluorescence intensity returning to the initial level. Assuming that the change of fluorescence intensity can be attributed to conformational change in the actin molecule induced by S1 binding, the intra-monomer distance was reduced by 0.4 nm and the inter-monomer distance was increased by 0.2 nm.