Proteolysis and binding of myosin subfragment 1 to actin

Rates of proteolytic cleavage of myosin subfragment 1 were measured in the absence and presence of different amounts of actin. The rates of tryptic digestion at the 50K/20K junction and papain digestion at the 25K/50K junction of the myosin head were progressively inhibited with increasing substoichiometric molar ratios of actin to myosin subfragment 1. The percentage inhibitions of digestion reactions corresponded precisely to the molar compositions of actin-subfragment 1 solutions and demonstrated that equimolar complexes of these proteins were responsible for the observed changes in the proteolysis of myosin heads.     

Kinetic model for the interaction of myosin subfragment 1 with regulated actin

A one-dimensional kinetic Ising model is developed to describe the binding of myosin subfragment 1 (SF-1) to regulated actin. The model allows for cooperative interactions between individual actin sites with bound SF-1 ligands rather than assuming that groups of actin monomer sites change their state in a cooperative fashion. With the triplet closure approximation, the model yields a set of 16 independent differential (master) equations which may be solved numerically to yield the extent of binding as a function of time. The predictions of the model are compared with experiments on the transient binding of SF-1 to regulated actin in the presence of Ca2+ and in the absence of Ca2+ with varying amounts of SF-1 prebound to the actin filament and on the equilibrium binding of SF-1 X ADP to regulated actin in the absence of Ca2+. In all cases, the calculations fit the data to within the experimental errors. In the case of SF-1 X ADP, the results suggest that a repulsive interaction exists between adjacently bound SF-1 at the ends of two neighboring seven-site actin units.     

Relationship between regulated actomyosin ATPase activity and cooperative binding of myosin to regulated actin

The protein complex, troponin-tropomyosin, which is bound to the thin actin filament, regulates muscle contraction and relaxation. In the absence of Ca²⁺ the troponin-tropomyosin complex causes muscle to relax, whereas in the presence of Ca²⁺, contraction occurs. Biochemical studies have shown that the troponin-tropomyosin complex has a dual effect on the interaction of the myosin cross-bridge with actin. In the presence of ATP, troponin-tropomyosin strongly inhibits the actomyosin ATPase activity, whereas in the absence of ATP, troponin-tropomyosin confers positive cooperativity on the binding of myosin to actin. We have proposed a simple model [Hill, T. L., Greene, L. E., and Eisenberg, E. (1980)Proc. Natl. Acad. Sci. USA 77, 3186–3190] that accounts for these biochemical observations by postulating that the troponin-tropomyosin-actin complex (regulated actin) can occur in two forms, a turned-on form and a turned-off form. This model defines several cooperativity parameters that describe the behavior of regulated actin. In previous studies we have determined the values of these parameters by studying the cooperative binding of myosin to regulated actin in the absence of ATP. In the present study we also used ATPase and fluorescence measurements to determine these cooperativity parameters. Assuming that the fluorescence change occurs only when two adjacent tropomyosin units shift into the turned-on form, our results show that all three methods give the same values for the cooperativity parameters. These results confirm the prediction of our model that a regulated actin unit that is turned off not only binds S-1 weakly but is also unable to activate the actomyosin ATPase activity.     

Parking problem and negative cooperativity of binding of myosin subfragment 1 to F-actin

Previously we provided evidence that myosin subfragment 1 (S1) can bind either one (state 1) or two actin monomers (state 2) in solution and in muscle fiber. Here we present results of the kinetics study of binding of S1 to F-actin labeled with fluorescent dye pyrene. A transition from state 1 to state 2 depends on probability that the second actin is free, which is high when molar ratio of S1/actin (R) is less than 0.5, and it decreases dramatically when R>2.0 due to the parking problem. The kinetics data obtained at different molar ratios were well fitted by two binding states model. The sequential binding of myosin head initially with one actin monomer and then with the second actin monomer in F-actin can play a key role in force generation by actin-myosin and their directed movement.     

The binding of heat-treated myosin subfragment 1 to actin and substructure considerations

Heat treatment of myosin subfragment 1 at 35 degrees C caused about 95% inactivation of the catalytic function but did not block its binding to actin. Heat-treated subfragment 1 showed specific, strong, and close to stoichiometric binding to actin. MgATP but not MgADP dissociated these complexes. However, in contrast to intact subfragment 1, the heat-treated protein did not polymerize G-actin and was not protected from trypsin by the binding to actin. Tryptic degradation of the 50K fragment abolished, or reduced greatly, the binding of heat-treated subfragment 1 to actin in solution but not on nitrocellulose overlays. These results are discussed in the context of subfragment 1 substructure.     

Interactions of the actin and nucleotide binding sites on myosin subfragment 1.

The effects of selected nucleotides (N) on the binding of myosin subfragment 1 (S-1) and pure F-actin (A) were measured by time-resolved fluorescence depolarization for 0.15 M KCl, pH 7.0 at 4 degrees. The association constants K'A, KN, and K'N in the scheme (see article), were determined for the magnesium salts of ADP, adenyl-5'-yl imidodiphosphate AMP-P(NH)P, and PPi. The nucleotide binding site on S-1 was "mapped" with respect to its interaction on the actin binding site. The subsites were the beta- and gamma-phosphoryl groups of ATP bind had the largest effects. A quantitative measure of the interaction, the interaction free energy, was defined as -RT ln (KA/K'A). For ADP, K'A was 2.7 X 10(5) M-1 and the interaction free energy was -4.67 kJ M-1. For AMP-P(NH)P and PPi it was much larger. A ternary complex was shown to exist for ADP, S-1, and actin in the presence of Mg2+ and evidence from AMP-P(NH)P and PPi measurements indicated that ATP also likely forms a ternary complex. The mechanism of (S-1)-actin dissociation is discussed in light of these results.     

Nucleotide-induced states of myosin subfragment 1 cross-linked to actin

Actomyosin interactions and the properties of weakly bound states in carbodiimide-cross-linked complexes of actin and myosin subfragment 1 (S-1) were probed in tryptic digestion, fluorescence, and thiol modification experiments. Limited proteolysis showed that the 50/20K junction on S-1 was protected in cross-linked acto-S-1 from trypsin even under high-salt conditions in the presence of MgADP, MgAMPPNP, and MgPPi (mu = 0.5 M). The same junction was exposed to trypsin by MgATP and MgATP gamma S but mainly on S-1 cross-linked via its 50K fragment to actin. p-Phenylenedimaleimide-bridged S-1, when cross-linked to actin, yielded similar tryptic cleavage patterns to those of cross-linked S-1 in the presence of MgATP. By using p-nitrophenylenemaleimide, it was found that the essential thiols of cross-linked S-1 were exposed to labeling in the presence of MgATP and MgATP gamma S in a state-specific manner. In contrast to this, the reactive thiols were protected from modification in the presence of MgADP, MgAMPPNP, and MgPPi at mu = 0.5 M. These modifications were compared with similar reactions on isolated S-1. Experiments with pyrene-actin cross-linked to S-1 showed enhancement of fluorescence intensity upon additions of MgATP and MgATP gamma S, indicating the release of the pyrene probe on actin from the sphere of S-1 influence. The results of this study contrast the "open" structure of weakly bound actomyosin states to the "tight" conformation of rigor complexes.     

Alteration of the ATP hydrolysis and actin binding properties of thrombin-cut myosin subfragment 1

We have characterized various structural and enzymatic properties of the (68K-30K)-S-1 derivative obtained by thrombic cleavage [Chaussepied, P., Mornet, D., Audemard, E., Derancourt, J., & Kassab, R. (1986) Biochemistry (preceding paper in this issue)]. The far-ultraviolet CD spectra and thiol reactivity measurements indicated an unchanged overall polypeptide conformation of the enzyme whereas the CD spectra in the near-ultraviolet region suggested a local change in the environments of phenylalanine side chains; the latter finding was rationalized by considering the existence of about five of these amino acids in the vicinity of the cleavage sites. When the binding of Mg2+-ATP and Mg2+-ADP to the derivative was assessed by CD spectroscopy, distinct spectra were obtained with the two nucleotides as with native subfragment 1 (S-1), but some spectral features were unique to the nicked S-1. Stern-Volmer fluorescence quenching studies using acrylamide and the analogues 1,N6-ethenoadenosine 5'-triphosphate and 1,N6-ethenoadenosine 5'-diphosphate indicated that the complexes formed with the modified S-1 have a solute quencher accessibility close to that observed for the complexes with the normal S-1. However, in contrast to the parent enzyme, the thrombin-cut S-1 was unable to bind irreversibly Mg2+-ATP, nor did it form a stable Mg2+-ADP-sodium vanadate complex or achieve the entrapping of Mg2+-ADP after cross-linking of SH1 and SH2 with N,N'-p-phenylenedimaleimide. Additionally, the amplitude of the Pi burst was very low, indicating that the inactivation of the proteolyzed S-1 was linked to the suppression of the hydrolysis step in the ATPase cycle.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Hill model for binding myosin S1 to regulated actin is not equivalent to the McKillop-Geeves model

The Hill two-state cooperativity model and the McKillop-Geeves (McK-G) three-state model predict very similar binding traces of myosin subfragment 1 (S1) binding to regulated actin filaments in the presence and absence of calcium, and both fit the experimental data reasonably well [Chen et al., Biophys. J., 80, 2338-2349]. Here, we compared the Hill model and the McK-G model for binding myosin S1 to regulated actin against three sets of experimental data: the titration of regulated actin with S1 and the kinetics of S1 binding of regulated actin with either excess S1 to actin or excess actin to S1. Each data set was collected for a wide range of specified calcium concentrations. Both models were able to generate reasonable fits to the time course data and to titration data. The McK-G model can fit all three data sets with the same calcium-concentration-sensitive parameters. Only K(B) and K(T) show significant calcium dependence, and the parameters have a classic pCa curve. A unique set of the Hill model parameters was extremely difficult to estimate from the best fits of multiple sets of data. In summary, the McK-G cooperativity model more uniquely resolves parameters estimated from kinetic and titration data than the Hill model, predicts a sigmoidal dependence of key parameters with calcium concentration, and is simpler and more suitable for practical use.     

Interaction of maleimidobenzoyl actin with myosin subfragment 1 and tropomyosin-troponin.

A chemical modification of G-actin with (m-maleimidobenzoyl)-N-hydroxysuccinimide ester (MBS) impairs actin polymerization [Bettache, N., Bertrand, R., & Kassab, R. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6028-6032]. MBS-actin recovers the ability to polymerize when a 2-fold molar excess of phalloidin is added in 30 mM KCl/2 mM MgCl2/20 mM Tris-HCl (pH 7.6). The resulting polymer (MBS-P-actin) is highly potentiated so that it activates the Mg(2+)-ATPase of S1 more strongly than native F-actin. The affinity of MBS-P-actin for S1 in the presence of ATP (KATPase) is about four times higher than that of native F-actin, although the maximum velocity at infinite actin concentration (Vmax) is almost the same. This high activation is not due to a cross-linking between MBS-P-actin and the S1 heavy chain, since no substantial amount of cross-linking was observed in SDS gel electrophoresis. Direct binding studies and ATPase measurements showed that the modification of actin with MBS impairs the binding of tropomyosin. Tropomyosin binding can be improved considerably by the addition of troponin. However, the regulation mechanism of the acto-S1 ATPase activity by troponin-tropomyosin is damaged. The addition of troponin-tropomyosin reduces the S1 ATPase activation by MBS-P-actin to the same level as that of native F-actin in 30 mM KCl/2.5 mM ATP/2 mM MgCl2, but there is no difference in the ATPase activation in the presence and absence of Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

The binding of myosin subfragment 1 to actin can be measured by proteolytic rates method

The initial rates of tryptic digestion at the 50/20-kDa junction in myosin subfragment 1 (S-1) were determined for free S-1, acto-S-1, and acto-S-1 in the presence of magnesium adenyl-5'-yl imidodiphosphate (Mg AMP-PNP) and MgATP under ionic strength conditions ranging from 30 to 124 mM. The percentage of S-1 bound to actin in the presence of Mg AMP-PNP and MgATP was calculated from these rates for each set of digestion experiments. Parallel experiments carried out in an Airfuge centrifuge on identical acto-S-1 solutions yielded independent information on the binding of S-1 to actin. The results of binding measurements by these two methods were in excellent agreement in all cases tested, covering the range from 15 to 95% binding of S-1 to actin. Tryptic digestions of synthetic mixtures of S-1 and p-phenylenedimaleimide S-1 in the presence of actin demonstrated that a two-component system of myosin heads with different affinities for actin can be resolved into its constituents by the proteolytic rates method. The results of this work justify applications of the proteolytic rates method to actomyosin binding studies in more complex systems.     

Effect of mild heat treatment on actin and nucleotide binding of myosin subfragment 1

Chymotryptic subfragment 1 (S-1) prepared from rabbit skeletal myosin has lost its ATPase activity upon incubation at 35 degrees C for 3 h. The loss in ATPase activity was accompanied by the perturbation of the structure of the 50K domain as indicated by a dramatic increase in the tryptic susceptibility of this domain without any change in the susceptibility of the other domains of S-1. The perturbation starts at the C-terminal region of the domain as suggested by the appearance of a 29K intermediate protein band in the tryptic peptide pattern of the heat-treated S-1. The heat-treated molecule essentially retained its actin and polyphosphate binding ability, and the actin binding was still sensitive to the presence of ATP or pyrophosphate. However, as opposed to native S-1, in heat-treated S-1 the addition of ATP does not induce an increase in tryptophan fluorescence, and, in the case of the treated species, the fluorescence of 1,N6-ethenoadenosine 5'-diphosphate added to the mixture is quenchable by acrylamide. This latter observation suggests that the binding of the adenine ring of the nucleotide has been altered following the heat treatment. The results indicate that the actin and polyphosphate binding sites of S-1 are distinct and that they are relatively independent of the adenine ring binding site.     

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)     

Cross-linking of actin to myosin subfragment 1 in the presence of nucleotides

Chemical cross-linking of actin to the 20K and 50K fragments of tryptically cleaved myosin subfragment 1 (S-1) by the zero-length cross-linking reagent 1-ethyl-3-[3-dimethylamino)propyl]carbodiimide (EDC) was used as a probe of the acto-S-1 interface in the presence of nucleotides. The course of the two reactions was monitored by measuring on sodium dodecyl sulfate (SDS)-polyacrylamide gels the time-dependent formation of the 20K-actin and 50K-actin cross-linked products. Both reactions were inhibited somewhat in the presence of MgADP, were slowed 3-4-fold in the presence of magnesium 5'-adenylyl imidodiphosphate (MgAMPPNP), and proceeded at least 7-fold slower with N,N'-p-phenylenedimaleimide (pPDM) modified S-1, as compared to the respective rates in the absence of nucleotides. However, neither the binding of the nucleotides MgADP and MgAMPPNP to S-1 nor the modification of S-1 by pPDM significantly changed the ratio of the cross-linking rates of actin to the 20K and 50K fragments. Similar to what was previously observed in the absence of nucleotides [Chen, T., Applegate, D., & Reisler, E. (1985) Biochemistry 24, 137-144], actin was cross-linked at an approximately 3-fold faster rate to the 20K fragment than to the 50K fragment under all reaction conditions tested. Thus, irrespective of the extent of acto-S-1 dissociation or the binding of nucleotides to acto-S-1, the 20K fragment remains the preferred cross-linking site for actin. These results show that the interaction of actin with each of the cross-linking sites on S-1 is not under selective or preferential control by nucleotides.     

Preparation and characterization of frog muscle myosin subfragment 1 and actin.

The preparation, structural and steady-state kinetic characteristics of contractile proteins from the leg muscle of frogs Rana temporaria and Rana pipiens are described. Actin and myosin from the two frog species are indistinguishable. The proteins have structural and steady-state kinetic properties similar to those from rabbit fast-twitch skeletal muscle. Chymotrypsin digestion of frog myosin or myofibrils in the presence of EDTA yields subfragment 1, which is separated by chromatography into two components that are distinguished by their alkali light-chain content.     

Interaction of caldesmon and myosin subfragment 1 with the C-terminus of actin.

The interactions of caldesmon and S1 with the C-terminus of actin were examined in co-sedimentation experiments using proteolytically truncated actin. It is shown that removal of 6 residues from the C-terminus of actin reduces the binding of caldesmon by about 50% while improving the binding of S1 to actin. We also show that S1 protects actin's C-terminus from enzymatic cleavage. Both S1 and caldesmon binding to actin are decreased in the presence of an actin C-terminal peptide. These results emphasize the importance of the C-terminus of actin in binding to S1 and caldesmon.     

Cross-linking myosin subfragment 1 Cys-697 and Cys-707 modifies ATP and actin binding site interactions.

Skeletal muscle myosin is an enzyme that interacts allosterically with MgATP and actin to transduce the chemical energy from ATP hydrolysis into work. By modifying myosin structure, one can change this allosteric interaction and gain insight into its mechanism. Chemical cross-linking with N,N'-p-phenylenedimaleimide (pPDM) of Cys-697 to Cys-707 of the myosin-ADP complex eliminates activity and produces a species that resembles myosin with ATP bound (Burke et al., 1976). Nucleotide-free pPDM-modified myosin subfragment 1 (S1) was prepared, and its structural and allosteric properties were investigated by comparing the nucleotide and actin interactions of S1 to those of pPDM-S1. The structural properties of the nucleotide-free pPDM-S1 are different from those of S1 in several respects. pPDM-S1 intrinsic tryptophan fluorescence intensity is reduced 28%, indicating a large increase of an internal quenching reaction (the fluorescence intensity of the related vanadate complex of S1, S1-MgADP-Vi, is reduced by a similar degree). Tryptophan fluorescence anisotropy increases from 0.168 for S1 to 0.192 for pPDM-S1, indicating that the unquenched tryptophan population in pPDM-S1 has reduced local freedom of motion. The actin affinity of pPDM-S1 is over 6,000-fold lower than that of S1, and the absolute value of the product of the net effective electric charges at the acto-S1 interface is reduced from 8.1 esu2 for S1 to 1.6 esu2 for pPDM-S1. In spite of these changes, the structural response of pPDM-S1 to nucleotide and the allosteric communication between its ATP and actin sites remain intact.(ABSTRACT TRUNCATED AT 250 WORDS)