Interactions of myosin subfragment 1 isozymes with G-actin

The polymerization of G-actin by myosin subfragment 1 (S-1) isozymes, S-1(A1) and S-1(A2), and their proteolytically cleaved forms was studied by light-scattering, fluorescence, and analytical ultracentrifugation techniques. As reported previously, S-1(A1) polymerized G-actin rapidly while S-1(A2) could hardly promote the assembly reaction (Chaussepied & Kasprzak, 1989a; Chen and Reisler, 1990). This difference between the isozymes of S-1 was traced to the very poor, if any, ability of G-actin-S-1(A2) complexes to nucleate the assembly of actin filaments. The formation of G-actin-S-1(A2) complexes was verified in sedimentation velocity experiments and by fluorescence measurements using pyrene-labeled actin. The G-actin-S-1(A2) complexes supported the growth of actin filaments and accelerated the polymerization of actin in solutions seeded with MgCl2-, KCl-, and S-1(A1)-generated nuclei. The growth rates of actin-S-1(A2) filaments were markedly slower than those for actin-S-1(A1) filaments. Proteolytic cleavage of S-1 isozymes at the 50/20-kDa junction of the heavy chain greatly decreased their binding to G-actin and thus inhibited the polymerization of actin by S-1(A1). These results are discussed in the context of G-actin-S-1 interactions.     

Polymerization of G-actin by myosin subfragment 1

The polymerization of actin from rabbit skeletal muscle by myosin subfragment 1 (S-1) from the same source was studied in the depolymerizing G-actin buffer. The polymerization reactions were monitored in light-scattering experiments over a wide range of actin/S-1 molar rations. In contrast to the well resolved nucleation-elongation steps of actin assembly by KC1 and Mg2+, the association of actin in the presence of S-1 did not reveal any lag in the polymerization reaction. Light scattering titrations of actin with S-1 and vice versa showed saturation of the polymerization reaction at stoichiometric 1:1 ratios of actin to S-1. Ultracentrifugation experiments confirmed that only stoichiometric amounts of actin were incorporated into a 1:1 acto-S-1 polymer even at high actin/S-1 ratios. These polymers were indistinguishable from standard complexes of S-1 with F-actin as judged by electron microscopy, light scattering measurements, and fluorescence changes observed while using actin covalently labeled with N-(1-pyrenyl)iodoacetamide. F-actin obtained by polymerization of G-actin by S-1 could initiate rapid assembly of G-actin in the presence of 10 mM KC1 and 0.5 mM MgCl2 and showed normal activation of MgATPase hydrolysis by myosin.     

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.     

Role of actin DNase-I-binding loop in myosin subfragment 1-induced polymerization of G-actin: implications for the mechanism of polymerization

Proteolytic cleavage of actin between Gly(42) and Val(43) within its DNase-I-binding loop (D-loop) abolishes the ability of Ca-G-actin to spontaneously polymerize in the presence of KCl. Here we show that such modified actin is assembled into filaments, albeit at a lower rate than unmodified actin, by myosin subfragment 1 (S1) carrying the A1 essential light chain but not by S1(A2). S1 titration of pyrene-G-actin showed a diminished affinity of cleaved actin for S1, but this could be compensated for by using S1 in excess. The most significant effect of the cleavage, revealed by measuring the fluorescence of pyrene-actin and light-scattering intensities as a function of actin concentration at saturating concentrations of S1, is strong inhibition of association of G-actin-S1 complexes into oligomers. Measurements of the fluorescence of dansyl cadaverine attached to Gln(41) indicate substantial inhibition of the initial association of G-actin-S1 into longitudinal dimers. The data provide experimental evidence for the critical role of D-loop conformation in both longitudinal and lateral, cross-strand actin-actin contact formation in the nucleation reaction. Electron microscopic analysis of the changes in filament-length distribution during polymerization of actin by S1(A1) and S1(A2) suggests that the mechanism of S1-induced polymerization is not substantially different from the nucleation-elongation scheme of spontaneous actin polymerization.     

Dynamic interaction between actin and myosin subfragment 1 in the presence of ADP

The equilibrium and dynamics of the interaction between actin, myosin subfragment 1 (S1), and ADP have been investigated by using actin which has been covalently labeled at Cys-374 with a pyrene group. The results are consistent with actin binding to S1.ADP (M.D) in a two-step reaction, A + M.D K1 equilibrium A-M.D K2 equilibrium A.M.D, in which the pyrene fluorescence only monitors the second step. In this model, K1 = 2.3 X 10(4) M-1 (k+1 = 4.6 X 10(4) M-1 s-1) and K2 = 10 (k+2 less than or equal to 4 s-1); i.e., both steps are relatively slow compared to the maximum turnover of the ATPase reaction. ADP dissociates from both M.D and A-M.D at 2 s-1 and from A.M.D at greater than or equal to 500 s-1; therefore, actin only accelerates the release of product from the A.M.D state. This model is consistent with the actomyosin ATPase model proposed by Geeves et al. [(1984) J. Muscle Res. Cell Motil. 5, 351]. The results suggest that A-M.D cannot break down at a rate greater than 4 s-1 by dissociation of ADP, by dissociation of actin, or by isomerizing to A.M.D. It is therefore unlikely to be significantly occupied in a rapidly contracting muscle, but it may have a role in a muscle contracting against a load where the ATPase rate is markedly inhibited. Under these conditions, this complex may have a role in maintaining tension with a low ATP turnover rate.     

Subtilisin-cleaved actin: polymerization and interaction with myosin subfragment 1

Homogeneous preparations of actin cleaved into two fragments, the N-terminal 9- and C-terminal 36-kDa peptides, were achieved by proteolysis of G-actin with subtilisin at 23 degrees C at a 1:1000 (w/w) ratio of enzyme to actin. The subtilisin cleavage site was identified by sequence analysis to be between Met-47 and Gly-48. Although under nondenaturing conditions the two fragments remained associated to one another, the cleavage affected macromolecular interactions of actin. The rates of cleaved actin polymerization by MgCl2, KCl, and myosin subfragment 1 (S-1) were slower and the critical concentrations for this process were higher than in intact protein. Intact and cleaved actin formed morphologically indistinguishable filaments and copolymerized in the presence of MgCl2. The affinity of actin for S-1 was decreased by about 10-fold due to subtilisin cleavage, but the S-1 ATPase activity was activated to the same Vmax value by both intact and cleaved actins. DNase I inhibition measurements revealed lower affinity of cleaved actin for DNase I than that of intact protein. These results are discussed in terms of actin's structure.     

Nucleotide-induced changes in the interaction of myosin subfragment 1 with actin: detection by antibodies against the N-terminal segment of actin

The binding of myosin subfragment 1 (S-1) to actin in the presence and absence of nucleotides was determined under conditions of partial saturation of actin, up to 80%, by Fab(1-7), the antibodies against the first seven N-terminal residues on actin. In the absence of nucleotides, the binding constant of S-1 to actin (2 x 10(7) M-1) was decreased by 1 order of magnitude by Fab(1-7). The binding of S-1 to actin caused only limited displacement of Fab, and between 30 and 50% of actin appeared to bind both proteins. In the presence of MgAMP.PNP, MgADP, and MgPPi and at low S-1 concentrations, the same antibodies caused a large decrease in the binding of S-1 to actin. However, the binding of S-1.nucleotide to actin in the presence of Fab(1-7) increased cooperatively with the increase in S-1 concentration. Also, in contrast to rigor conditions, there was no indication for the binding of Fab(1-7) and S-1.nucleotide to the same actin molecules. These results show a nucleotide-induced transition in the actomyosin interface, most likely related to the different roles of the N-terminal segment of actin in the binding of S-1 and S-1.nucleotide. The possible implications of these findings to the regulation of actomyosin interactions are discussed.     

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.     

Immunochemical evidence for the binding of caldesmon to the NH2-terminal segment of actin

The binding of caldesmon and its actin-binding fragments to actin was studied by using peptide antibodies directed against two actin sites implicated in actomyosin interactions. Antibodies against residues 1-7 on skeletal alpha-actin strongly inhibited the binding of caldesmon to actin and perturbed to a smaller extent the interaction between actin and the actin binding fragments. Carbodiimide coupling of ethylenediamine to the NH2-terminal acidic residues on actin inhibited the binding of caldesmon and its fragments to actin to a similar extent as the (residues 1-7) antibodies. Antibodies against residues 18-28 showed only limited competition with caldesmon for the binding to actin. These results lead to the following conclusions. (i) The NH2-terminal residues on actin play an important role in the binding of caldesmon to actin, (ii) residues 18-28 on actin do not form a major caldesmon interaction site, and (iii) the actin-binding fragments do not contain the full actin-binding interface. These conclusions and other literature data suggest that caldesmon regulates the actomyosin ATPase by competing with myosin.ATP for the NH2-terminal segment on actin.     

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.     

Interaction of myosin subfragment 1 with forms of monomeric actin

The ability of myosin subfragment 1 to interact with monomeric actin complexed to sequestering proteins was tested by a number of different techniques such as affinity absorption, chemical cross-linking, fluorescence titration, and competition procedures. For affinity absorption, actin was attached to agarose immobilized DNase I. Both chymotryptic subfragment 1 isoforms (S1A1 and S1A2) were retained by this affinity matrix. Fluorescence titration employing pyrenyl-actin in complex with deoxyribonuclease I (DNase I) or thymosin beta4 demonstrated S1 binding to these actin complexes. A K(D) of 5 x 10(-8) M for S1A1 binding to the actin-DNase I complex was determined. Fluorescence titration did not indicate binding of S1 to actin in complex with gelsolin segment 1 (G1) or vitamin D-binding protein (DBP). However, fluorescence competition experiments and analysis of tryptic cleavage patterns of S1 indicated its interaction with actin in complex with DBP or G1. Formation of the ternary DNase I-acto-S1 complex was directly demonstrated by sucrose density sedimentation. S1 binding to G-actin was found to be sensitive to ATP and an increase in ionic strength. Actin fixed in its monomeric state by DNase I was unable to significantly stimulate the Mg2+-dependent S1-ATPase activity. Both wild-type and a mutant of Dictyostelium discoideum myosin II subfragment 1 containing 12 additional lysine residues within an insertion of 20 residues into loop 2 (K12/20-Q532E) were found to also interact with actin-DNase I complex. Binding of the K12/20-Q532E mutant to the actin-DNase I complex occurred with higher affinity than wild-type S1 and was less sensitive to mono- and divalent cations.     

Specific cross-linking of the SH1 thiol of skeletal myosin subfragment 1 to F-actin and G-actin.

Recently, we reported that (maleimidobenzoyl)-G-actin (MBS-G-actin), which was resistant to the salt and myosin subfragment 1 (S-1) induced polymerizations, reacts reversibly and covalently in solution with the S-1 heavy chain at or near the strong F-actin binding region [Bettache, N., Bertrand, R., & Kassab, R. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6028-6032]. Here, we have readily converted the MBS-G-actin into MBS-F-actin in the presence of phalloidin and salts. The binding of S-1 to the two actin derivatives carrying on their surface free reactive maleimidobenzoyl groups was investigated comparatively in cross-linking experiments performed under various conditions to probe further the molecular structure of the actin-heavy chain complex before and after the polymerization process. Like MBS-G-actin, the isolated MBS-F-actin, which did not undergo any intersubunit cross-linking, bound stoichiometrically to S-1, generating two kinds of actin-heavy chain covalent complexes migrating on electrophoretic gels at 180 and 140 kDa. The relative extent of their production was essentially dependent on pH for both G-and F-actins. At pH 8.0, the 180-kDa species was predominant, and at pH 7.0, the amount of the 140-kDa adduct increased at the expense of the 180-kDa entity. The cross-linking of MBS-F-actin to S-1 led to the superactivation of the MgATPase substantiating the ability of this derivative to stimulate the S-1 ATPase as the native protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Potentiated state of the tropomyosin actin filament and nucleotide-containing myosin subfragment 1

The main purpose of this study was to determine whether potentiation of acto-S-1 ATPase activity (activity higher than that obtained with tropomyosin-free actin) could be caused by nucleotide-containing acto-S-1 complexes. In addition, we wanted to know whether these complexes also have a positive cooperative effect on their own apparent binding constant under conditions where nucleotide-free acto-S-1 complexes cause potentiation of ATPase activity. Using calcium-saturated troponin-tropomyosin actin filaments, we observed potentiation of ATPase activity in the presence of 5.0 mM magnesium 5'-adenylyl imidodiphosphate (MgAMPPNP) and calculated that the ability of acto-S-1-AMPPNP complexes to cause potentiation must have been very similar to that of nucleotide-free acto-S-1 complexes. In extension of earlier studies, potentiated acto-S-1 ATPase activity was characterized by an increase in Vmax and, as observed before, a lowering of the apparent Km for subfragment 1 (S-1). Under conditions similar to those that produce the potentiation of acto-S-1 ATPase activity, the apparent actin binding constant of nucleotide-free S-1 was increased about 3-5 fold while the apparent binding constant of AMPPNP to actin-bound S-1 was reduced to (2.5-10) x 10(2) M-1 compared to that of about (1-5) x 10(3) M-1 for S-1 bound to tropomyosin-free actin. Under the same conditions, the apparent binding constant of S-1-AMPPNP to actin was not increased. We suggest that a potentiated state of the tropomyosin actin filament is produced by the cooperative action of acto-S-1 or acto-S-1-AMPPNP complexes. The potentiated state is characterized by an increase in the Vmax of the acto-S-1 ATPase activity, increased binding constants for S-1 and S-1-ADP, and increased binding of tropomyosin to actin.     

Kinetic studies on the association and dissociation of myosin subfragment 1 and actin.

The reactions of pyrene-labeled actin with myosin subfragment 1 (S1) and S1-ligand complexes at low ionic strength are described by the schemes [formula: see text] where M refers to a myosin head; A is actin; L is ligand; the asterisk refers to a high fluorescence state of actin; and K1 and K3 are association constants. K1 is reduced approximately 10-fold for M.ADP or M.pyrophosphate versus M alone. The rate constant of the isomerization step (k2) is 150-200 s-1 for A*M, A*M.ADP, and A*M-pyrophosphate (20 degrees C). The interaction between the ligand the actin binding sites reduces K2 from 2,000 for A*M to 50-100 for A*M.ADP and to approximately unity for A*M-pyrophosphate. The A*M.ADP state is equated with the AM'.ADP state of Sleep and Hutton (Sleep, J., A., and Hutton, R. L. (1980) Biochemistry 19, 1276-1283).     

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)     

Interaction of fluorescently labeled myosin subfragment 1 with nucleotides and actin

Isolated myosin heads (subfragment 1) were modified by covalent attachment of 5-(iodoacetamido)fluorescein or 5-(iodoacetamido)salicylic acid to the essential sulfhydryl group SH1. The extrinsic fluorescence of the modified proteins was sensitive to binding of nucleotides and F-actin. With the fluorescein derivative [subfragment 1 (S1) modified with 5-(iodoacetamido)fluorescein (IAF) at SH1 (S1-AF)], association with MgADP decreased the probe fluorescence by 30%, whereas binding to actin increased the emission by a factor of 2. In the ternary complex acto-S1-AF X MgADP, the effect of nucleotide on the intensity of the attached fluorescein canceled the effect of actin. The fluorescence state of this ternary complex was similar to that of S1-AF X MgADP. The emission of S1-AF was resolved into two components with lifetimes of 4.3 and 0.6 ns and relative contributions of 33% and 67%, respectively. Interaction of S1-AF with nucleotides and actin did not alter the lifetimes but significantly shifted their fractional contributions. Quenching studies showed that the short lifetime likely arose from the fluorescein moiety statically quenched by internal groups. Binding of MgADP to the salicylate derivative [S1 modified with 5-(iodoacetamido)salicylic acid at SH1 (S1-SAL)] induced a 25% enhancement of the probe fluorescence, whereas formation of acto-S1-SAL decreased the emission by 10% regardless of whether MgADP was bound to the protein. Both labeled S1 species bound MgADP with a similar affinity, comparable to that of unmodified S1 previously reported by other investigators.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cross-linking of the skeletal myosin subfragment 1 heavy chain to the N-terminal actin segment of residues 40-113

Glutaraldehyde (GA) and N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ), a hydrophobic, carboxyl group directed, zero-length protein cross-linker, were employed for the chemical cross-linking of the rigor complex between F-actin and the skeletal myosin S-1. The enzymatic properties and structure of the new covalent complexes obtained with both reagents were determined and compared to those known for the EDC-acto-S-1 complex. The GA- or EEDQ-catalyzed covalent attachment of F-actin to the S-1 heavy chain induced an elevated Mg2+-ATPase activity. The turnover rates of the isolated cross-linked complexes were similar to those for EDC-acto-S-1 (30 s-1). The solution stability of the new complexes is also comparable to that exhibited by EDC-acto-S-1. The proteolytic digestion of the isolated AEDANS-labeled covalent complexes and direct cross-linking experiments between actin and various preformed proteolytic S-1 derivatives indicated that, as observed with EDC, the COOH-terminal 20K and the central 50K heavy chain fragments are involved in the cross-linking reactions of GA and EEDQ. KI-depolymerized acto-S-1 complexes cross-linked by EDC, GA, or EEDQ were digested by thrombin which cuts only actin, releasing S-1 heavy chain-actin peptide cross-linked complexes migrating on acrylamide gels with Mr 100K (EDC), 110K and 105K (GA), and 102K (EEDQ); these were fluorescent only when fluorescent S-1 was used. They were identified by immunostaining with specific antibodies directed against selected parts of he NH2-terminal actin segment of residues 1-113.(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.