Magnesium regulates ADP dissociation from myosin V

Processivity in myosin V is mediated through the mechanical strain that results when both heads bind strongly to an actin filament, and this strain regulates the timing of ADP release. However, what is not known is which steps that lead to ADP release are affected by this mechanical strain. Answering this question will require determining which of the several potential pathways myosin V takes in the process of ADP release and how actin influences the kinetics of these pathways. We have addressed this issue by examining how magnesium regulates the kinetics of ADP release from myosin V and actomyosin V. Our data support a model in which actin accelerates the release of ADP from myosin V by reducing the magnesium affinity of a myosin V-MgADP intermediate. This is likely a consequence of the structural changes that actin induces in myosin to release phosphate. This effect on magnesium affinity provides a plausible explanation for how mechanical strain can alter this actin-induced acceleration. For actomyosin V, magnesium release follows phosphate release and precedes ADP release. Increasing magnesium concentration to within the physiological range would thus slow both the ATPase activity and the velocity of movement of this motor.     

Kinetic and spectroscopic evidence for three actomyosin:ADP states in smooth muscle

Smooth muscle myosin II undergoes an additional movement of the regulatory domain with ADP release that is not seen with fast skeletal muscle myosin II. In this study, we have examined the interactions of smooth muscle myosin subfragment 1 with ADP to see if this additional movement corresponds to an identifiable state change. These studies indicate that for this myosin:ADP, both the catalytic site and the actin-binding site can each assume one of two conformations. Relatively loose coupling between these two binding sites leads to three discrete actin-associated ADP states. Following an initial, weakly bound state, binding of myosin:ADP to actin shifts the equilibrium toward a mixture of two states that each bind actin strongly but differ in the conformation of their catalytic sites. By contrast, fast myosins, including Dictyostelium myosin II, have reciprocal coupling between the actin- and ADP-binding sites, so that either actin or nucleotide, but not both, can be tightly bound. This uncoupling, which generates a second strongly bound actomyosin ADP state in smooth muscle, would prolong the fraction of the ATPase cycle time that this actomyosin spends in a force-generating conformation and may be central to explaining the physiologic differences between this and other myosins.     

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.     

AMP and IMP dissociate actomyosin into actin and myosin

We investigated to determine why heating of squid muscle at 60 degrees C induced the liberation of actin from myofibrils. When a mixture of a myofibrillar fraction and a low-molecular sarcoplasmic fraction prepared from squid muscle was heated at 60 degrees C, actin liberation occurred. When a myofibrillar fraction was heated with ATP, AMP, or IMP, actin liberation occurred. Hence, AMP is perhaps one of the factors causing actin liberation in postmortem squid muscle. It was found that AMP and IMP reversibly dissociated actomyosin of chicken, bovine, and porcine skeletal muscles into actin and myosin on incubation at 0 degrees C at pH 7.2 in 0.2 M KCl. These results led us to conclude that AMP and IMP were the most responsible factors causing actin liberation from myofibrils in the heated muscle and causing reversible dissociation of actomyosin on storage of skeletal muscle at a low temperature. Hence, AMP and IMP are possible factors causing the resolution of rigor mortis in muscles.     

Thermodynamics of nucleotide binding to actomyosin V and VI: a positive heat capacity change accompanies strong ADP binding

We have measured the energetics of ATP and ADP binding to single-headed actomyosin V and VI from the temperature dependence of the rate and equilibrium binding constants. Nucleotide binding to actomyosin V and VI can be modeled as two-step binding mechanisms involving the formation of collision complexes followed by isomerization to states with high nucleotide affinity. Formation of the actomyosin VI-ATP collision complex is much weaker and slower than for actomyosin V. A three-step binding mechanism where actomyosin VI isomerizes between two conformations, one competent to bind ATP and one not, followed by rapid ATP binding best accounts for the data. ADP binds to actomyosin V more tightly than actomyosin VI. At 25 degrees C, the strong ADP-binding equilibria are comparable for actomyosin V and VI, and the different overall ADP affinities arise from differences in the ADP collision complex affinity. The actomyosin-ADP isomerization leading to strong ADP binding is entropy driven at >15 degrees C and occurs with a large, positive change in heat capacity (DeltaC(P) degrees ) for both actomyosin V and VI. Sucrose slows ADP binding and dissociation from actomyosin V and VI but not the overall equilibrium constants for strong ADP binding, indicating that solvent viscosity dampens ADP-dependent kinetic transitions, presumably a tail swing that occurs with ADP binding and release. We favor a mechanism where strong ADP binding increases the dynamics and flexibility of the actomyosin complex. The heat capacity (DeltaC(P) degrees ) and entropy (DeltaS degrees ) changes are greater for actomyosin VI than actomyosin V, suggesting different extents of ADP-induced structural rearrangement.     

Cryoenzymic studies on actomyosin ATPase: kinetic evidence for communication between the actin and ATP sites on myosin.

The post-ATP binding steps of myosin subfragment 1 (S1) and actomyosin subfragment 1 (actoS1) ATPases were studied at -15 degrees C with 40% ethylene glycol as antifreeze. The cleavage and release of Pi steps were studied by the rapid-flow quench method and the interaction of actin with S1 plus ATP by light scattering in a stopped-flow apparatus. At -15 degrees C, the interaction of actin with S1 remains tight, and the Km for the activation of S1 ATPase is very small (0.3 microM). The chemical data were interpreted by E + ATP----E*.ATP----E**.ADP.Pi----E*.ADP----products, where E is S1 or actoS1. In Pi burst experiments with S1, there was a large Pi burst of free Pi, but E**.ADP.Pi could not be detected. Here the predominant complex in the seconds time range is E*.ATP and in the steady-state E*.ADP. With actoS1, there was a small Pi burst of E**.ADP.Pi, evidence that the cleavage steps for S1 and actoS1 are different. From the stopped-flow experiments, the dissociation of actoS1 by ATP was complete, even at actin concentrations 60X its Km. Further, no interaction of actin with the key intermediate M*.ATP could be detected. Therefore, at -15 degrees C, actoS1 ATPase occurs by a dissociative pathway; in particular, the cleavage step appears to occur in the absence of actin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic characterization of the weak binding states of myosin V

Myosin V is a molecular motor shown to move processively along actin filaments. We investigated the properties of the weak binding states of monomeric myosin V containing a single IQ domain (MV 1IQ) to determine if the affinities of these states are increased as compared to conventional myosin. Further, using a combination of non-hydrolyzable nucleotide analogues and mutations that block ATP hydrolysis, we sought to probe the states that are populated during ATP-induced dissociation of actomyosin. MV 1IQ binds actin with a K(d) = 4 microM in the presence of ATP gamma S at 50 mM KCl, which is 10-20-fold tighter than that of nonprocessive class II myosins. Mutations within the switch II region trapped MV 1IQ in two distinct M.ATP states with very different actin binding affinities (K(d) = 0.2 and 2 microM). Actin binding may change the conformation of the switch II region, suggesting that elements of the nucleotide binding pocket will be in a different conformation when bound to actin than is seen in any of the myosin crystal structures to date.     

Switch I closure simultaneously promotes strong binding to actin and ADP in smooth muscle myosin

The motor protein myosin uses energy derived from ATP hydrolysis to produce force and motion. Important conserved components (P-loop, switch I, and switch II) help propagate small conformational changes at the active site into large scale conformational changes in distal regions of the protein. Structural and biochemical studies have indicated that switch I may be directly responsible for the reciprocal opening and closing of the actin and nucleotide-binding pockets during the ATPase cycle, thereby aiding in the coordination of these important substrate-binding sites. Smooth muscle myosin has displayed the ability to simultaneously bind tightly to both actin and ADP, although it is unclear how both substrate-binding clefts could be closed if they are rigidly coupled to switch I. Here we use single tryptophan mutants of smooth muscle myosin to determine how conformational changes in switch I are correlated with structural changes in the nucleotide and actin-binding clefts in the presence of actin and ADP. Our results suggest that a closed switch I conformation in the strongly bound actomyosin-ADP complex is responsible for maintaining tight nucleotide binding despite an open nucleotide-binding pocket. This unique state is likely to be crucial for prolonged tension maintenance in smooth muscle.     

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.     

Intrinsic tryptophan fluorescence identifies specific conformational changes at the actomyosin interface upon actin binding and ADP release.

The helix-loop-helix (A-site) and myopathy loop (R-site) are located on opposite sides of the cleft that separates the proposed actin-binding interface of myosin. To investigate the structural features of the A- and R-sites, we engineered two mutants of the smooth muscle myosin motor domain with the essential light chain (MDE), containing a single tryptophan located either in the A-site (W546-MDE) or in the R-site (V413W MDE). W546- and V413W-MDE display actin-activated ATPase and actin-binding properties similar to those of wild-type MDE. The steady-state fluorescence properties of W546-MDE [emission peak (lambda(max)) = 344, quantum yield = 0.20, and acrylamide bimolecular quenching constant (k(q)) = 6.4 M(-)(1). ns(-)(1)] and V413W-MDE [lambda(max) = 338, quantum yield = 0.27, and k(q) = 3.6 M(-)(1).ns(-)(1)] demonstrate that Trp-546 and Trp-413 are nearly fully exposed to solvent, in agreement with the crystallographic data on these residues. In the presence of actin, Trp-546 shifts to a more buried environment in both the ADP-bound and nucleotide-free (rigor) actomyosin complexes, as indicated by an average lambda(max) of 337 or 336 nm, respectively, and protection from dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide (DHNBS) oxidation. In contrast, Trp-413 has a single conformation with an average lambda(max) of 338 nm in the ADP-bound complex, but in the rigor complex it is 50% more accessible to DHNBS oxidation and can adopt a range of possible conformations (lambda(max) = 341-347 nm). Our results suggest a structural model in which the A-site remains tightly bound to actin and the R-site adopts a more flexible and solvent-exposed conformation upon ADP release.     

Interactions of muscle beta-connectin with myosin, actin, and actomyosin at low ionic strengths

The interaction of the muscle elastic protein connectin with myosin and actin filaments was investigated by turbidimetry, viscosity, flow birefringence measurements, and electron microscopic observations. In KCl concentrations lower than 0.15 M at pH 7.0 at 25 degrees C, both myosin and actin filaments were aggregated by connectin. Myosin filaments were entangled with each other in the presence of connectin. Actin filaments were assembled into bundles under the influence of connectin just as under that of alpha-actinin. The physiological significance of the interactions of connectin with myosin and actin filaments is discussed in relation to the localization of connectin in myofibrils. The Mg2+-activated ATPase activity of actomyosin was appreciably enhanced by connectin in the presence of KCl concentrations lower than 0.1 M. The extent of activation by connectin was smaller than by alpha-actinin. The enhancement of the ATPase activity may be due to acceleration of the onset of superprecipitation of actomyosin.     

Regulation of the interaction between actin and myosin subfragment 1: evidence for three states of the thin filament.

Equilibrium titrations and kinetic experiments were used to define the cooperative binding of myosin subfragment 1 (S1) to actin-troponin-tropomyosin. Both types of experiment require an equilibrium between two states of the thin filament in which one state (the off state) binds S1 less readily than the other. Equilibrium titrations are compatible with > 95% of the actin7.Tn.Tm units being in the off state in the absence of calcium and 80% in the off state in the presence of calcium. Kinetic binding data suggest that the presence of calcium switches the thin filament from 70% in the off state to < 5%. The two experiments, therefore, define quite different populations of the off states. We propose a three-state model of the thin filament. A "blocked state" which is unable to bind S1, a "closed state" which can only bind S1 relatively weakly and an "open state" in which the S1 can both bind and undergo an isomerization to a more strongly bound rigor-like conformation. The equilibrium between the three states is calcium-dependent; KB = [closed]/[blocked] = 0.3 and > or = 16 and KT = [open]/[closed] = 0.09 and 0.25 in the absence and presence of calcium, respectively. This model can account for both types of experimental data.     

Two-state equilibria of myosin subfragment 1 and its complexes with ADP and actin

We previously reported that the nucleotide complex of myosin subfragment 1, S1.epsilon ADP, exists in two states on the basis of the temperature dependence of the fluorescence decay of bound 1,N6-ethenoadenosine diphosphate (epsilon ADP) [Aguirre, R., Lin. S.-H., Gonsoulin, F., Wang, C.-K., & Cheung, H.C. (1989) Biochemistry 28, 799-809]. We have extended the previous study of the equilibrium between the two states, S1L.ADP in equilibrium S1H.ADP, by using a fluorescently labeled myosin S1 (S1-AF). In S1 alkylated with IAF [5-(iodoacetamido)fluorescein], the decay of the label emission was biexponential both in the presence and absence of ADP and/or actin. In the presence of ADP, the two decay times were 4.30 (alpha 1 = 0.55) and 0.80 ns (alpha 2 = 0.45) at 12.4 degrees C, in a medium containing 60 mM KCl, 30 mM TES (pH 7.5), and 2 mM MgCl2. The steady-state fluorescence intensities of S1-AF, (S1-AF).ADP, acto.(S1-AF), and acto.(S1-AF).ADP were dependent on temperature over the range of 5-30 degrees C. By combining lifetime and steady-state intensity data, we obtained for the two-state transition (S1-AF)L.ADP in equilibrium (S1-AF)H.ADP the following parameters: delta H degrees = 16.1 kcal/mol (67.3 kJ/mol) and delta S degrees = 55.8 cal/(deg.mol) [233.5 J/(deg.mol)], in agreement with previous results obtained with epsilon ADP. The delta H degrees values for the two-state transition of S1-AF, acto.(S1-AF), and acto.(S1-AF).ADP are 13.0, 21.6, and 5.2 kcal/mol, respectively. The corresponding delta S degrees values are 46.9, 79.5, and 17.4 cal/(deg.mol).(ABSTRACT TRUNCATED AT 250 WORDS)     

The binding of Mn2+ and ADP to myosin

The metal ion requirement of myosin-ADP binding was investigated by use of Mn²⁺. Mn²⁺ binds to two sets of noninteracting sites on myosin which are characterized by affinity constants of 10⁶ and 10³, M⁻¹ at 0.016 M KCl concentration. The maximum number of sites is 2 for the high affinity and 20–25 for the low affinity set. Binding of Mn²⁺ to the high affinity sites increases the affinity of ADP binding to myosin. F-actin inhibits ADP binding (Kiely, B., and Martonosi, A., Biochim. Biophys. Acta 172: 158–170 [1969]), but even at F-actin concentrations much higher than that required to saturate the actin binding sites of myosin or its proteolytic fragments, significant ADP binding remained. The actin insensitive portion of ADP binding was inhibited by 10⁻⁴ M inorganic pyrophosphate or ATP. The results are discussed on the basis of a model in which actin and ADP bind to myosin at distinct but interacting sites.     

"Twitchin-actin linkage hypothesis" for the catch mechanism in molluscan muscles: evidence that twitchin interacts with myosin, myorod, and paramyosin core and affects properties of actomyosin

"Twitchin-actin linkage hypothesis" for the catch mechanism in molluscan smooth muscles postulates in vivo existence of twitchin links between thin and thick filaments that arise in a phosphorylation-dependent manner [N.S. Shelud'ko, G.G. Matusovskaya, T.V. Permyakova, O.S. Matusovsky, Arch. Biochem. Biophys. 432 (2004) 269-277]. In this paper, we proposed a scheme for a possible catch mechanism involving twitchin links and regulated thin filaments. The experimental evidence in support of the scheme is provided. It was found that twitchin can interact not only with mussel myosin and rabbit F-actin but also with the paramyosin core of thick filaments, myorod, mussel thin filaments, "natural" F-actin from mussel, and skeletal myosin from rabbit. No difference was revealed in binding of twitchin with mussel and rabbit myosin. The capability of twitchin to interact with all thick filament proteins suggests that putative twitchin links can be attached to any site of thick filaments. Addition of twitchin to a mixture of actin and paramyosin filaments, or to a mixture of Ca(2+)-regulated actin and myosin filaments under relaxing conditions caused in both cases similar changes in the optical properties of suspensions, indicating an interaction and aggregation of the filaments. The interaction of actin and myosin filaments in the presence of twitchin under relaxing conditions was not accompanied by an appreciable increase in the MgATPase activity. We suggest that in both cases aggregation of filaments was caused by formation of twitchin links between the filaments. We also demonstrate that native thin filaments from the catch muscle of the mussel Crenomytilus grayanus are Ca(2+)-regulated. Twitchin inhibits the ability of thin filaments to activate myosin MgATPase in the presence of Ca(2+). We suggest that twitchin inhibition of the actin-myosin interaction is due to twitchin-induced switching of the thin filaments to the inactive state.