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.     

Aggregation of partially unfolded Myosin subfragment-1 into spherical oligomers with amyloid-like dye-binding properties

Proteolytic myosin subfragment 1 (S1) is known to be partially unfolded in its 50-kDa subdomain by mild heat treatment at 35 degrees C [Burke et al. (1987) Biochemistry 26, 1492-1496]. Here, we report that this partial unfolding is accompanied by aggregation of S1 protein. Characteristics of the aggregate thus formed were: (i) formation of transparent sediment under centrifugation at 183,000 x g; (ii) amyloid-like, dye-binding properties such as Congo red-binding and Thioflavin T fluorescence enhancement; (iii) a uniformly sized spherical appearance in electron micrographs; and (iv) sensitivity to tryptic digestion. Gel filtration analysis of the aggregation process indicates that the spheroid was formed through an intermediate oligomeric stage. The aggregate inhibited spontaneous aggregation of an isolated 50 kDa fragment into a large amorphous mass. The remaining native regions in the partially unfolded S1 were probably responsible for this effect. These results show that, unlike the 50-kDa fragment, the partially unfolded S1 molecules do not form amorphous aggregates but assemble into spherical particles. The native regions in partially unfolded S1 may be a determinant of aggregate morphology.     

Kinetic properties of binding of myosin subfragment-1 with F-actin in the absence of nucleotide

The rate constant for the binding of myosin subfragment-1 (S-1) with F-actin in the absence of nucleotide, k1, and that for dissociation of the F-actin-myosin subfragment-1 complex (acto-S-1), k-1, were measured independently. The rate of S-1 binding with F-actin was measured from the time course of the change in the light scattering intensity after mixing S-1 with various concentrations of F-actin and k1 was found to be 2.55 X 10(6) M-1 X S-1 at 20 degrees C. The dissociation rate of acto-S-1 was determined using F-actin labeled with pyrenyl iodoacetamide (Pyr-FA). Pyr-FA, with its fluorescence decreased by binding with S-1, was mixed with acto-S-1 complex and the rate of displacement of F-actin by Pyr-FA was measured from the decrease in the Pyr-FA fluorescence intensity. The k-1 value was calculated to be 8.5 X 10(-3) S-1 (or 0.51 min-1). The value of the dissociation constant of S-1 from acto-S-1 complex, Kd, was calculated from Kd = k-1/k1 to be 3.3 X 10(-9) M at 20 degrees C. Kd was also measured at various temperatures (0-30 degrees C), and the thermodynamic parameters, delta G degree, delta H degree, and delta S degree, were estimated from the temperature dependence of Kd to be -11.3 kcal/mol, +2.5 kcal/mol, and +47 cal/deg . mol, respectively. Thus, the binding of the myosin head with F-actin was shown to be endothermic and entropy-driven.     

Myosin subfragment-1 attachment to actin. Expected effect on equatorial reflections

The characteristic equatorial X-ray pattern from a relaxed vertebrate skeletal muscle changes when the muscle is activated. In particular, there is a simultaneous decrease in the intensity of the first reflection (I10) and increase in the intensity of the second (I11). This observed change is almost reciprocal. When compared with the predictions of computer modeling, it produces a strong argument that the intensity change is due to a redistribution of myosin heads (myosin subfragment-1 or S-1), which results from the formation and configuration changes of actin-myosin links. Computer modeling shows that different actin-S-1 configurations will give different numerical values for I10 and I11, assuming the same number of attachments. For a given configuration, the intensity changes are a nonlinear function of attachment number, so that direct scaling of force to reflection intensity may be difficult. Data from active muscle are consistent with the notion that in different states of active muscle, i.e. shortening or isometric, there are different average configurations of actin-myosin attachment and different numbers of actin-myosin links.     

The magnesium-ion-dependent adenosine triphosphatase of bovine cardiac Myosin and its subfragment-1.

The kinetics of the Mg2+-dependent ATPase (adenosine triphosphatase) activity of bovine cardiac myosin and its papain subfragment-1 were studied by using steady-state and pre-steady-state techniques, and results were compared with published values for the corresponding processes in the ATPase mechanism of rabbit skeletal-muscle myosin subfragment-1. The catalytic-centreactivity for cardiac subfragment-1 is 0.019s-1, which is less than one-third of that determined for the rabbit protein. The ATP-induced isomerization process, measured from enhancement of protein fluorescence on substrate binding, is similarly decreased in rate, as is also the isomerization process associated with ADP release. However, the equilibrium constant for ATP cleavage, measured by quenched-flow by using [gamma-32P]ATP, shows little difference in the two species. Other experiments were carried out to investigate the rate of association of actin with subfragment-1 by light-scattering changes and also the rate of dissociation of the complex by ATP. The dissociation rate increases with increasing substrate concentration, to a maximum at high ATP concentrations, with a rate constant of about 2000s-1. It appears that isomerization processes which may involve conformational changes have substantially lower rate constants for the cardiac proteins, whereas equilibrium constants for substrate binding and cleavage are not significantly different. These differences may be related to the functional properties of these myosins in their different muscle types. Kinetic heterogeneity has been detected in both steady-state and transient processes, and this is discussed in relation to the apparent chemical homogeneity of cardiac myosin.     

Interaction of a new fluorescent ATP analogue with skeletal muscle myosin subfragment-1

A new fluorescent ribose-modified ATP analogue, 2'(3')-O-[6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoic]-ATP (NBD-ATP), was synthesized and its interaction with skeletal muscle myosin subfragment-1 (S-1) was studied. NBD-ATP was hydrolysed by S-1 at a rate and with divalent cation-dependence similar to those in the case of regular ATP. Skeletal HMM supported actin translocation using NBD-ATP and the velocity was slightly higher than that in the case of regular ATP. The addition of S1 to NBD-ATP resulted in quenching of NBD fluorescence. Recovery of the fluorescence intensity was noted after complete hydrolysis of NBD-ATP to NBD-ADP. The quenching of NBD-ATP fluorescence was accompanied by enhancement of intrinsic tryptophan fluorescence. These results suggested that the quenching of NBD-ATP fluorescence reflected the formation of transient states of ATPase. The formation of S-1.NBD-ADP.BeF(n) and S-1.NBD-ADP.AlF(4)(-) complexes was monitored by following changes in NBD fluorescence. The time-course of the formation fitted an exponential profile yielding rate constants of 7.38 x 10(-2) s(-1) for BeF(n) and 1.1 x 10(-3) s(-1) for AlF(4)(-). These values were similar to those estimated from the intrinsic fluorescence enhancement of trp due to the formation of S-1.ADP.BeF(n) or AlF(4)(-) reported previously by our group. Our novel ATP analogue seems to be applicable to kinetic studies on myosin.     

Myosin 1E interacts with synaptojanin-1 and dynamin and is involved in endocytosis

Myosin 1E is one of two "long-tailed" human Class I myosins that contain an SH3 domain within the tail region. SH3 domains of yeast and amoeboid myosins I interact with activators of the Arp2/3 complex, an important regulator of actin polymerization. No binding partners for the SH3 domains of myosins I have been identified in higher eukaryotes. In the current study, we show that two proteins with prominent functions in endocytosis, synaptojanin-1 and dynamin, bind to the SH3 domain of human Myo1E. Myosin 1E co-localizes with clathrin- and dynamin-containing puncta at the plasma membrane and this co-localization requires an intact SH3 domain. Expression of Myo1E tail, which acts in a dominant-negative manner, inhibits endocytosis of transferrin. Our findings suggest that myosin 1E may contribute to receptor-mediated endocytosis.     

Transient kinetics of the interaction of actin with myosin subfragment-1 in the absence of nucleotide.

The kinetics of the association of actin with myosin subfragment-1 (S1) has been studied by using S1 labeled at the sulfhydryl group SH1 with 5-(iodoacetamido)fluorescein (S1-AF). Upon rapid mixing in a stopped-flow apparatus, the fluorescence intensity of the fluorescein moiety increased by 50%, followed by a slower increase that was well resolved. This slow phase of the fluorescence change could not be fitted to either a monoexponential or a biexponential function, but it could be fitted to a sum of three exponential terms yielding three observed first-order rate constants (lambda i). The dissociation of acto.-(S1-AF) was studied by displacement of S1-AF from the complex with native S1. The dissociation kinetics was characterized by a single rate constant (approximately 0.012 s-1 at 20 degrees C), and this constant was independent of S1 concentration. Together with previous equilibrium data that were obtained under identified conditions for formation of acto-subfragment-1 (Lin, S.-H., and H. C. Cheung. 1991. Biochemistry. 30:4317-4323), a six-state two-pathway model is proposed as a minimum kinetic scheme for formation of rigor acto.S1. In this model, unbound subfragment-1 exists in two conformational states (S1' and S1) which are in equilibrium with each other, one corresponding to the previously established low-temperature state and the other to the high-temperature state. Each subfragment-1 state can interact with actin to form a collision complex, followed by two isomerizations to form two acto-subfragment-1 states (A.S1' and A.S1). Both isomerizations were visible in stopped-flow experiments. Two special cases of the model were considered: 1) a rapid pre-equilibration of the initial collision complex with actin and S1, and 2) trace accumulation of the collision complex. The first case required that the three combinations of the three observed rate constants be independent of actin concentration. The data were incompatible with this approximation. The other special case required that the sum of the lambda i vary linearly with actin concentration and the other two combinations of lambda i vary with actin concentration in a quadratic fashion. The present data were in agreement with the second case. At 20 degrees C and in 60 mM KCl, 2 mM MgCl2, 30 mM 2-([-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino)ethanesulfonic acid, and pH 7.5, the biomolecular association rate constants for the interaction of actin with S1' and S1 were 8.58 x 10(5) and 1.11 x 10(6) M-1 s-1, respectively.     

Possible role of structural changes in ATP hydrolysis by subfragment-1 and myosin

Dependence of the rate of ATP hydrolysis with subfragment-I and temperature of SF-I, denaturation on the concentration of heavy water in solution was studied. The value of kinetic isotope effect V/Vx linearly increases with the rise of the volume portion of heavy water in solution and at X-1 it equals 1.9. The temperature of protein denaturacticn increases with X rise, the pattern of this relationship looking as an arched curve. The results differ from those earlier obtained on myosin which points to the absence of essential contribution of structural dynamic changes to enzymic hydrolysis of ATP by subfragment-I.     

Interaction of plasma gelsolin with G-actin and F-actin in the presence and absence of calcium ions

Plasma gelsolin formed a very tight 1:2 complex with G-actin in the presence of Ca2+, but no interaction between gelsolin and G-actin was detected in the presence of excess EGTA. However, the 1:2 complex dissociated into a 1:1 gelsolin:actin complex and monomeric actin when excess EGTA was added. Plasma gelsolin bound tightly to the barbed ends of actin filaments and also severed filaments in the presence of Ca2+ and bound weakly to the filament barbed end in the presence of EGTA. The 1:2 gelsolin-actin complex bound to the barbed ends of filaments but did not sever them. By blocking the barbed end of filaments with plasma gelsolin, we determined the critical concentration at the pointed end in 1 mM MgCl2 and 0.2 mM ATP to be 4 microM. The dissociation rate constant for ADP-G-actin from the pointed end was estimated to be about 0.4 s-1 and the association rate constant to be about 5 X 10(4) M-1 s-1. Finally, we obtained evidence that plasma gelsolin accelerates but does not bypass the nucleation step and, therefore, that the concentration of gelsolin does not directly determine the concentration of filaments polymerized in its presence. Thus, gelsolin-capped filaments may not provide an absolutely reliable method for determining the rate constant for the association of ATP-G-actin at the pointed ends of filaments, but a reasonable estimate would be 1 X 10(5) M-1 s-1 in 1 mM MgCl2 and 0.2 mM ATP.     

Myosin subfragment 1 and structural elements of G-actin: effects of S-1(A2) on sequences 39-52 and 61-69 in subdomain 2 of G-actin.

The effect of myosin on the structure of two sequences on G-actin, a loop between residues 39 and 52 and a segment between residues 61 and 69 from the NH2-terminus, was probed by limited proteolytic digestions of G-actin in the presence of the myosin subfragment 1 isozyme S-1(A2). Under the experimental conditions of this work, no polymerization of actin was induced by S-1(A2) [Chen & Reisler (1991) Biochemistry 30, 4546-4552]. S-1(A2) did not change the rates of subtilisin and chymotryptic digestion of G-actin at loop 39-52. In contrast to this, the second protease-sensitive region on G-actin, segment 61-69, was protected strongly by S-1(A2) from tryptic cleavage. The minor if any involvement of loop 39-52 in S-1 binding was confirmed by determining the binding constants of S-1(A2) for pyrene-labeled G-actin (1.2 x 10(6) M-1), subtilisin-cleaved pyrenyl G-actin (0.3 x 10(6) M-1), and DNase I-pyrenyl G-actin complexes (0.3 x 10(6) M-1). Consistent with this, the activity of DNase I, which binds to actin loop 39-52 [Kabsch et al. (1990) Nature 347, 37-44], was inhibited almost equally well by actin in the presence and absence of S-1(A2). These results confirm the observation that DNase I and S-1(A2) bind to distinct sites on actin [Bettache et al. (1990) Biochemistry 29, 9085-9091] and demonstrate myosin-induced changes in segment 61-69 of G-actin.     

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.     

Differences in G-actin containing bound ATP or ADP: the Mg2+-induced conformational change requires ATP

The role of adenosine 5'-triphosphate (ATP) in the Mg2+-induced conformational change of rabbit skeletal muscle G-actin has been investigated by comparing actin containing bound ADP with actin containing bound ATP. As previously described [Frieden, C. (1982) J. Biol. Chem. 257, 2882-2886], N-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine-labeled G-actin containing ATP undergoes a time-dependent Mg2+-induced fluorescence change that reflects a conformational change in the actin. Addition of Mg2+ to labeled G-actin containing ADP gives no fluorescence change, suggesting that the conformational change does not occur. The fluorescence change can be restored on the addition of ATP. Examination of the time courses of these experiments suggests that ATP must replace ADP prior to the Mg2+-induced change. The Mg2+-induced polymerization of actin containing ADP is extraordinarily slow compared to that of actin containing ATP. The lack of the Mg2+-induced conformational change, which is an essential step in the Mg2+-induced polymerization, is probably the cause for the very slow polymerization of actin containing ADP. On the other hand, at 20 degrees C, at pH 8, and in 2 mM Mg2+, the elongation rate from the slow growing end of an actin filament, measured by using the protein brevin to block growth at the fast growing end, is only 4 times slower for actin containing ADP than for actin containing ATP.     

The ubiquitin-like protein HUB1 forms SDS-resistant complexes with cellular proteins in the absence of ATP

Ubiquitin and ubiquitin-like modifiers (UBLs) form covalent complexes with other proteins by isopeptide formation between their carboxyl (C)-termini and -amino groups of lysine residues of acceptor proteins. A hallmark of UBLs is a protruding C-terminal tail with a terminal glycine residue, which is required for ATP-dependent conjugation. Recently, the highly conserved protein HUB1 (homologous to ubiquitin 1) has been reported to function as a UBL following C-terminal processing. HUB1 exhibits sequence similarity with ubiquitin but lacks a C-terminal tail bearing a glycine residue. Here we show that HUB1 can form SDS-resistant complexes with cellular proteins, but provide evidence that these adducts are not formed through covalent C-terminal conjugation of HUB1 to substrates. The adducts are still formed when the C-terminus of HUB1 was altered by epitope tagging, amino-acid exchange or deletion, or when cells were depleted of ATP. We propose that HUB1 may act as a novel protein modulator through the formation of tight, possibly noncovalent interactions with target proteins.     

Myosin Vb interacts with Rab8a on a tubular network containing EHD1 and EHD3

Cells use multiple pathways to internalize and recycle cell surface components. Although Rab11a and Myosin Vb are involved in the recycling of proteins internalized by clathrin-mediated endocytosis, Rab8a has been implicated in nonclathrin-dependent endocytosis and recycling. By yeast two-hybrid assays, we have now demonstrated that Myosin Vb can interact with Rab8a, but not Rab8b. We have confirmed the interaction of Myosin Vb with Rab11a and Rab8a in vivo by using fluorescent resonant energy transfer techniques. Rab8a and Myosin Vb colocalize to a tubular network containing EHD1 and EHD3, which does not contain Rab11a. Myosin Vb tail can cause the accumulation of both Rab11a and Rab8a in collapsed membrane cisternae, whereas dominant-negative Rab11-FIP2(129-512) selectively accumulates Rab11a but not Rab8a. Additionally, dynamic live cell imaging demonstrates distinct pathways for Rab11a and Rab8a vesicle trafficking. These findings indicate that Rab8a and Rab11a define different recycling pathways that both use Myosin Vb.     

Slow Myosin ATP Turnover in the Super-Relaxed State in Tarantula Muscle

We measured the nucleotide turnover rate of myosin in tarantula leg muscle fibers by observing single turnovers of the fluorescent nucleotide analog 2′-/3′-O-(N′-methylanthraniloyl)adenosine-5′-O-triphosphate, as monitored by the decrease in fluorescence when 2′-/3′-O-(N′-methylanthraniloyl)adenosine-5′-O-triphosphate (mantATP) is replaced by ATP in a chase experiment. We find a multiexponential process with approximately two-thirds of the myosin showing a very slow nucleotide turnover time constant (∼ 30 min). This slow-turnover state is termed the super-relaxed state (SRX). If fibers are incubated in 2′-/3′-O-(N′-methylanthraniloyl)adenosine-5′-O-diphosphate and chased with ADP, the SRX is not seen, indicating that trinucleotide-relaxed myosins are responsible for the SRX. Phosphorylation of the myosin regulatory light chain eliminates the fraction of myosin with a very long lifetime. The data imply that the very long-lived SRX in tarantula fibers is a highly novel adaptation for energy conservation in an animal that spends extremely long periods of time in a quiescent state employing a lie-in-wait hunting strategy. The presence of the SRX measured here correlates well with the binding of myosin heads to the core of the thick filament in a structure known as the “interacting-heads motif,” observed previously by electron microscopy. Both the structural array and the long-lived SRX require relaxed filaments or relaxed fibers, both are lost upon myosin phosphorylation, and both appear to be more stable in tarantula than in vertebrate skeletal or vertebrate cardiac preparations.     

Effects of ATP and Actin-Filament Binding on the Dynamics of the Myosin II S1 Domain

Actin and myosin interact with one another to perform a variety of cellular functions. Central to understanding the processive motion of myosin on actin is the characterization of the individual states along the mechanochemical cycle. We present an all-atom molecular dynamics simulation of the myosin II S1 domain in the rigor state interacting with an actin filament. We also study actin-free myosin in both rigor and post-rigor conformations. Using all-atom level and coarse-grained analysis methods, we investigate the effects of myosin binding on actin, and of actin binding on myosin. In particular, we determine the domains of actin and myosin that interact strongly with one another at the actomyosin interface using a highly coarse-grained level of resolution, and we identify a number of salt bridges and hydrogen bonds at the interface of myosin and actin. Applying coarse-grained analysis, we identify differences in myosin states dependent on actin-binding, or ATP binding. Our simulations also indicate that the actin propeller twist-angle and nucleotide cleft-angles are influenced by myosin at the actomyosin interface. The torsional rigidity of the myosin-bound filament is also calculated, and is found to be increased compared to previous simulations of the free filament.     

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.