Addition of lysines to the 50/20 kDa junction of myosin strengthens weak binding to actin without affecting the maximum ATPase activity

Much interest has centered on two surface loops in the motor domain to explain the differences in enzymatic and mechanical properties of myosin isoforms. We showed that two invariant lysines at the C-terminal end of loop 2, which is part of the actin-binding interface, are required to obtain actin activation [Joel et al. (2001) J. Biol. Chem. 276, 2998-3003]. Here we investigate the effects of increasing positive charge in the variable portion of loop 2 of smooth muscle heavy meromyosin (smHMM). Increasing the net positive charge by +4 increased the affinity for actin in the presence and absence of ATP. The K(m) for actin-activated ATPase activity decreased 15-fold, but V(max) was unchanged, showing that "weak binding" of myosin for actin can be significantly strengthened without increasing the rate-limiting step for V(max). The mutant HMM had slower rates of in vitro motility and ADP release compared to WT HMM. ADP release and motility, which were both salt-dependent, correlated linearly with each other. Loop 2 thus plays a major role in setting the affinity for actin but also affects ADP release and motility. Because the actin- and nucleotide-binding regions communicate, mutations to one region can impact multiple facets of myosin's mechanical and enzymatic properties.     

Mechanism for the inhibition of acto-heavy meromyosin ATPase by the actin/calmodulin binding domain of caldesmon.

Caldesmon, an actin/calmodulin binding protein, inhibits acto-heavy meromyosin (HMM) ATPase, while it increases the binding of HMM to actin, presumably mediated through an interaction between the myosin subfragment 2 region of HMM and caldesmon, which is bound to actin. In order to study the mechanism for the inhibition of acto-HM ATPase, we utilized the chymotryptic fragment of caldesmon (38-kDa fragment), which possesses the actin/calmodulin binding region but lacks the myosin binding portion. The 38-kDa fragment inhibits the actin-activated HMM ATPase to the same extent as does the intact caldesmon molecule. In the absence of tropomyosin, the 38-kDa fragment decreased the KATPase and Kbinding without any effect on the Vmax. However, when the actin filament contained bound tropomyosin, the caldesmon fragment caused a 2-3-fold decrease in the Vmax, in addition to lowering the KATPase and the Kbinding. The 38-kDa fragment-induced inhibition is partially reversed by calmodulin at a 10:1 molar ratio to caldesmon fragment; the reversal was more remarkable in 100 mM ionic strength at 37 degrees C than in 20 or 50 mM at 25 degrees C. Results from these experiments demonstrate that the 38-kDa domain of caldesmon fragment of myosin head to actin; however, when the actin filament contains bound tropomyosin, caldesmon fragment affects not only the binding of HMM to/actin but also the catalytic step in the ATPase cycle. The interaction between the 38-kDa domain of caldesmon and tropomyosin-actin is likely to play a role in the regulation of actomyosin ATPase and contraction in smooth muscle.     

The binding of smooth muscle heavy meromyosin to actin in the presence of ATP. Effect of phosphorylation

Phosphorylation of the 20,000-dalton light chains of smooth muscle heavy meromyosin (HMM) from turkey gizzards results in a large increase in the actin-activated MgATPase activity over that observed with unphosphorylated HMM. In an attempt to define which step in the kinetic cycle is affected by phosphorylation, we have measured the binding of both unphosphorylated and phosphorylated HMM to actin in the presence of ATP using sedimentation. There was only a 4-fold difference in the actin binding constants of unphosphorylated HMM (5.35 x 10(3) M-1) and fully phosphorylated HMM (2.35 x 10(4) M-1). In contrast, the maximum rate of the actin-activated MgATPase activity (Vmax) of phosphorylated HMM was 25 times greater than that for unphosphorylated HMM. These data rule out a mechanism whereby the unphosphorylated light chain of myosin regulates actin-myosin interaction by directly or indirectly blocking the binding of HMM to actin. This implies that some step in the kinetic cycle other than the binding of HMM to actin must be regulated. We have also measured the rate constant for ATP hydrolysis (the initial phosphate burst) under the same conditions and found that this step was very fast compared to the steady state ATPase rate and was unaffected by phosphorylation. This suggests that the step which is regulated by phosphorylation is either phosphate release or a step preceding phosphate release but following ATP hydrolysis.     

Inhibition of actin-tropomyosin activation of myosin MgATPase activity by the smooth muscle regulatory protein caldesmon.

Caldesmon inhibition of actin-tropomyosin activation of myosin MgATPase activity was investigated. greater than 90% inhibition of ATPase activation correlated with 0.035-0.1 caldesmon bound per actin monomer over a wide range of conditions. Caldesmon inhibited sheep aorta actin-tropomyosin activation of skeletal muscle heavy meromyosin (HMM) by 85%, but had no effect on the binding affinity of HMM.ADP.Pi to actin. At ratios of 2 and 0.12 subfragment 1 (S1):1 actin, addition of caldesmon inhibited the ATPase activation by up to 95%, but did not alter the fraction of S1.ADP.Pi associated with actin-tropomyosin. We concluded that caldesmon inhibited actomyosin ATPase by slowing the rate-limiting step of the activation pathway. At concentrations comparable to the ATPase measurements, S1 displaced caldesmon from native thin filaments both in the absence (rigor) and the presence of MgATP. We therefore concluded that caldesmon could displace S1.ADP.Pi from actin-tropomyosin only under exceptional circumstances. An expressed mutant of caldesmon comprising just the C-terminal 99 amino acids bound actin 10 times weaker than whole caldesmon but otherwise inhibited actin-tropomyosin activation with the same potency and same mechanism as intact caldesmon. Thus, the entire inhibitory function of caldesmon resides in its extreme C terminus.     

Calcium-sensitive binding of heavy meromyosin to regulated actin requires light chain 2 and the head-tail junction

Sedimentation in a preparative ultracentrifuge was used to determine the affinity of heavy meromyosin, HMM, for regulated actin, F-actin plus troponin-tropomyosin, in the presence of MgATP at pH 7.0, 20 degrees C, and mu = 18 mM. HMM was prepared from vertebrate striated muscle myosin by a mild chymotryptic digestion. This HMM contained 85-90% intact 19 000-dalton light chains, LC2. In the presence of calcium, 90% of the HMM bound to regulated actin with an association constant of (2-4) X 10(4) M-1. In the absence of calcium, while one-third of the HMM bound with an affinity similar to that observed in the presence of calcium, the rest bound much more weakly. It was not possible to accurately determine the association constant for this weakly binding HMM, but a 20-fold reduction in affinity is consistent with the binding data. The binding of single-headed heavy meromyosin to regulated actin was similarly sensitive to the calcium concentration. Since removal of calcium inhibits the regulated actin-activated ATPase of HMM greater than 20-fold, troponin-tropomyosin must be capable of inhibiting both the binding of HMM to regulated actin and a step which occurs after binding but prior to product release. Removal of LC2 increased the fraction of HMM with calcium-insensitive binding, and adding LC2 back to this depleted HMM restored most of the calcium sensitivity. Chymotryptic cleavage of LC2 to a 17 000-dalton fragment destroyed the calcium-sensitive binding of HMM to regulated actin. Phosphorylation of LC2, however, had no detectable effect on this binding. Thus, the calcium-sensitive binding of HMM to regulated actin requires that both the head-tail junction and the N-terminal part of LC2 be intact. Binding studies with cross-linked regulated actins and kinetic measurements of the rates of change in turbidity demonstrate that this calcium sensitivity is due to calcium binding to troponin and not to LC2.     

Smooth muscle myosin: regulation and properties

The relationship of the biochemical states to the mechanical events in contraction of smooth muscle cross-bridges is reviewed. These studies use direct measurements of the kinetics of Pi and ADP release. The rate of release of Pi from thiophosphorylated cycling cross-bridges held isometric was biphasic with turnovers of 1.8 s-1 and 0.3 s-1, reflecting properties and forces directly acting on cross-bridges through mechanisms such as positive strain and inhibition by high-affinity MgADP binding. Fluorescent transients reporting release of an ADP analogue 3'-deac-edaADP were significantly faster in phasic than in tonic smooth muscles. Thiophosphorylation of myosin regulatory light chains (RLCs) increased and positive strain decreased the release rate around twofold. The rates of ADP release from rigor cross-bridges and the steady-state Pi release from cycling isometric cross-bridges are similar, indicating that the ADP-release step or an isomerization preceding it may limit the ATPase rate. Thus ADP release in phasic and tonic smooth muscles is a regulated step with strain- and dephosphorylation-dependence. High affinity of cross-bridges for ADP and slow ADP release prolong the fraction of the duty cycle occupied by strongly bound AM.ADP state(s) and contribute to the high economy of force that is characteristic of smooth muscle. RLC thiophosphorylation led to structural changes in smooth muscle cross-bridges consistent with our findings that thiophosphorylation and strain modulate product release.     

ADP binding induces an asymmetry between the heads of unphosphorylated myosin

Light chain phosphorylation is the key event that regulates smooth and non-muscle myosin II ATPase activity. Here we show that both heads of smooth muscle heavy meromyosin (HMM) bind tightly to actin in the absence of nucleotide, irrespective of the state of light chain phosphorylation. In striking contrast, only one of the two heads of unphosphorylated HMM binds to actin in the presence of ADP, and the heads have different affinities for ADP. This asymmetry suggests that phosphorylation alters the mechanical coupling between the heads of HMM. A model that incorporates strain between the two heads is proposed to explain the data, which have implications for how one head of a motor protein can gate the response of the other.     

Disturbed communication between actin- and nucleotide-binding sites in a myosin II with truncated 50/20-kDa junction.

The kinetic and functional consequences of deleting nine residues from an actin-binding surface loop (loop 2) were examined to investigate the role of this region in myosin function. The nucleotide binding properties of myosin were not altered by the deletion. However, the deletion affected actin binding and the communication between the actin- and nucleotide-binding sites. The affinity of M765NL for actin (644 nM) was approximately 100-fold lower than that of wild-type construct M765 (5.8 nM). Despite this reduction in affinity, actin binding weakened the affinity of ADP for the motor to a similar extent for both mutant and wild-type constructs. The addition of 0.5 microM actin decreased ADP affinity from 0.6 to 34 microM for M765NL and from 1.6 to 39 microM for M765. In contrast, communication between the actin- and nucleotide-binding sites appears disturbed in regard to phosphate release: thus, basal ATPase activity for M765NL (0.19 s-1) was 3-fold larger than for M765 (0.06 s-1), and the stimulation of ATPase activity by actin was 5-fold lower for M765NL. These results indicate different paths of communication between the actin- and nucleotide-binding sites, in regard to ADP and Pi release, and they confirm that loop 2 is involved in high affinity actin binding.     

Functional role of loop 2 in myosin V

Myosin V is molecular motor that is capable of moving processively along actin filaments. The kinetics of monomeric myosin V containing a single IQ domain (MV 1IQ) differ from nonprocessive myosin II in that actin affinity is higher, phosphate release is extremely rapid, and ADP release is rate-limiting. We generated two mutants of myosin V by altering loop 2, a surface loop in the actin-binding region thought to alter actin affinity and phosphate release in myosin II, to determine the role that this loop plays in the kinetic tuning of myosin V. The loop 2 mutants altered the apparent affinity for actin (K(ATPase)) without altering the maximum ATPase rate (V(MAX)). Transient kinetic analysis determined that the rate of binding to actin, as well as the affinity for actin, was dependent on the net positive charge of loop 2, while other steps in the ATPase cycle were unchanged. The maximum rate of phosphate release was unchanged, but the affinity for actin in the M.ADP.Pi-state was dramatically altered by the mutations in loop 2. Thus, loop 2 is important for allowing myosin V to bind to actin with a relatively high affinity in the weak binding states but does not play a direct role in the product release steps. The ability to maintain a high affinity for actin in the weak binding states may prevent diffusion away from the actin filament and increase the degree of processive motion of myosin V.     

Effect of F-actin upon the binding of ADP to myosin and its fragments.

The effect of F-actin upon the binding of ADP to rabbit skeletal muscle myosin, heavy meromyosin, and subfragment 1 was studied by equilibrium dialysis, ultracentrifuge transport, and light scattering techniques. Both myosin and H-meromyosin (HMM) bind a maximum of approximately 1.6 mol of ADP/mol of protein, while S-1 binds approximately 0.9 mol of ADP/mol of protein. The affinity for ADP of all three preparations was similar at a given ionic strength (approximately 10(6) M-1 at 0.05 M KCl) and decreased with increasing ionic strength. Under conditions similar to those used for the measurement of ADP binding, the binding sites of myosin, HMM, and subfragment 1 (S-1) are saturated with actin at molar ratios of 2, 2, and 1 mol of actin monomer/mol of protein, respectively, as determined by light scattering, ultracentrifuge transport, and in the case of myosin by ATPase measurements. F-actin was found to inhibit ADP binding, but even at an actin concentration at least twice that required for saturation of myosin, HMM, or S-1, significant ADP binding remained. This ADP binding was inhibited by 10(-4) M pyrophosphate. The observations are consistent with the formation of an actomyosin-ADP complex in which actin and ADP are bound to myosin at distinct but interacting sites.     

Myosin regulatory light chain phosphorylation and strain modulate adenosine diphosphate release from smooth muscle Myosin

The effects of myosin regulatory light chain (RLC) phosphorylation and strain on adenosine diphosphate (ADP) release from cross-bridges in phasic (rabbit bladder (Rbl)) and tonic (femoral artery (Rfa)) smooth muscle were determined by monitoring fluorescence transients of the novel ADP analog, 3'-deac-eda-ADP (deac-edaADP). Fluorescence transients reporting release of 3'-deac-eda-ADP were significantly faster in phasic (0.57 +/- 0.06 s(-1)) than tonic (0.29 +/- 0.03 s(-1)) smooth muscles. Thiophosphorylation of regulatory light chains increased and strain decreased the release rate approximately twofold. The calculated (k-ADP/k+ADP) dissociation constant, Kd of unstrained, unphosphorylated cross-bridges for ADP was 0.6 microM for rabbit bladder and 0.3 microM for femoral artery. The rates of ADP release from rigor bridges and reported values of Pi release (corresponding to the steady-state adenosine triphosphatase (ATPase) rate of actomyosin (AM)) from cross-bridges during a maintained isometric contraction are similar, indicating that the ADP-release step or an isomerization preceding it may be limiting the adenosine triphosphatase rate. We conclude that the strain- and dephosphorylation-dependent high affinity for and slow ADP release from smooth muscle myosin prolongs the fraction of the duty cycle occupied by strongly bound actomyosin.ADP state(s) and contributes to the high economy of force.     

A kinetic model that explains the effect of inorganic phosphate on the mechanics and energetics of isometric contraction of fast skeletal muscle

A conventional five-step chemo-mechanical cycle of the myosin–actin ATPase reaction, which implies myosin detachment from actin upon release of hydrolysis products (ADP and phosphate, Pi) and binding of a new ATP molecule, is able to fit the [Pi] dependence of the force and number of myosin motors during isometric contraction of skeletal muscle. However, this scheme is not able to explain why the isometric ATPase rate of fast skeletal muscle is decreased by an increase in [Pi] much less than the number of motors. The question can be solved assuming the presence of a branch in the cycle: in isometric contraction, when the force generation process by the myosin motor is biased at the start of the working stroke, the motor can detach at an early stage of the ATPase cycle, with Pi still bound to its catalytic site, and then rapidly release the hydrolysis products and bind another ATP. In this way, the model predicts that in fast skeletal muscle the energetic cost of isometric contraction increases with [Pi]. The large dissociation constant of the product release in the branched pathway allows the isometric myosin–actin reaction to fit the equilibrium constant of the ATPase.     

Mechanism of inhibition of skeletal muscle actomyosin by N-benzyl-p-toluenesulfonamide

N-Benzyl-p-toluenesulfonamide (BTS) is a small organic molecule that specifically inhibits the contraction of fast skeletal muscle fibers. To determine the mechanism of inhibition by BTS, we performed a kinetic analysis of its effects on the elementary steps of the actomyosin subfragment-1 ATPase cycle. BTS decreases the steady-state acto-S1 ATPase rate approximately 10-fold and increases the actin concentration for half-maximal activation. BTS primarily affects three of the elementary steps of the reaction pathway. It decreases the rate of P(i) release >20-fold in the absence of actin and >100-fold in the presence of actin. It decreases the rate of S1.ADP dissociation from 3.9 to 0.8 s(-)(1) while decreasing the S1.ADP dissociation constant from 2.3 to 0.8 microM. BTS weakens the apparent affinity of S1.ADP for actin, increasing the K(d) from 7.0 to 29.5 microM. ATP binding to S1, hydrolysis, and the affinity of nucleotide-free S1 for actin are unaffected by BTS. Kinetic modeling indicates that the binding of BTS to myosin depends on actin association/dissociation and on nucleotide state. Our results suggest that the reduction of the acto-S1 ATPase rate is due to the inhibition of P(i) release, and the suppression of tension is due to inhibition of P(i) release in conjunction with the decreased apparent affinity of S1.ADP.P(i) and S1.ADP for actin.     

Intermediate oxygen exchange catalyzed by the actin-activated skeletal myosin adenosinetriphosphatase

Considerable effort has been devoted to understanding the mechanism of 18O exchange in skinned skeletal and insect muscle fibers. However, a full understanding of the mechanism of 18O exchange in muscle fibers requires an understanding of the mechanism of 18O exchange in the simpler in vitro systems employing myosin subfragment 1 (S-1) and heavy meromyosin (HMM). In the present study, using both S-1 and S-1 covalently cross-linked to actin, we show first that over a wide range of temperature, ionic strength, and actin concentration there is only one pathway of 18O exchange with S-1. This is also the case with HMM except at very low ionic strength and low actin concentration, and even here, the data can be explained if 20% of the HMM is denatured in such a way that it shows no 18O exchange. Our results also show that actin markedly decreases the rate of 18O exchange. If it is assumed that Pi release is rate limiting, the four-state kinetic model of the actomyosin ATPase cannot fit these 18O exchange data. However, if it is assumed that the ATP hydrolysis step is rate limiting and Pi release is very fast, the four-state kinetic model can qualitatively fit these data although the fit is not perfect. A better fit to the 18O exchange data can be obtained with the six-state kinetic model of the actomyosin ATPase, but this fit requires the assumption that, at saturating actin concentration, the rate of Pi rotation is about 9-fold slower than the rate of reversal of the ATP hydrolysis step.     

Caldesmon freezes the structure of actin filaments during the actomyosin ATPase cycle

Hybrid contractile apparatus was reconstituted in skeletal muscle ghost fibers by incorporation of skeletal muscle myosin subfragment 1 (S1), smooth muscle tropomyosin and caldesmon. The spatial orientation of FITC-phalloidin-labeled actin and IAEDANS-labeled S1 during sequential steps of the acto-S1 ATPase cycle was studied by measurement of polarized fluorescence in the absence or presence of nucleotides conditioning the binding affinity of both proteins. In the fibers devoid of caldesmon addition of nucleotides evoked unidirectional synchronous changes in the orientation of the fluorescent probes attached to F-actin or S1. The results support the suggestion on the multistep rotation of the cross-bridge (myosin head and actin monomers) during the ATPase cycle. The maximal cross-bridge rotation by 7 degrees relative to the fiber axis and the increase in its rigidity by 30% were observed at transition between A**.M**.ADP.Pi (weak binding) and A--.M--.ADP (strong binding) states. When caldesmon was present in the fibers (OFF-state of the thin filament) the unidirectional changes in the orientation of actin monomers and S1 were uncoupled. The tilting of the myosin head and of the actin monomer decreased by 29% and 90%, respectively. It is suggested that in the "closed" position caldesmon "freezes" the actin filament structure and induces the transition of the intermediate state of actomyosin towards the weak-binding states, thereby inhibiting the ATPase activity of the actomyosin.     

The effect of troponin-tropomyosin on the binding of heavy meromyosin to actin in the presence of ATP

In the presence of ATP and the absence of Ca2+, the binding of myosin subfragment-1 to actin is only slightly inhibited by troponin-tropomyosin, while the actin-activated subfragment-1 ATPase rate is 95% inhibited (Chalovich, J. M., Chock, P. B., and Eisenberg, E. (1981) J. Biol. Chem. 256, 575-578). On the other hand, it has been reported the troponin-tropomyosin markedly inhibits the binding of heavy meromyosin (HMM) to actin in the presence of ATP and the absence of Ca2+, providing that the HMM has intact light chain 2 (Wagner, P. D., and Stone, D. (1982) Biochemistry 22, 1334-1342). In the present study, we reinvestigated the binding of HMM with 85% intact light chain 2, to regulated actin. If we assume that only a single population of HMM is present, the binding constant of HMM to regulated actin at 19 mM ionic strength is only about 3 times larger in the presence of Ca2+ than in the absence of Ca2+ (2.4 X 10(4) M-1 compared to 8.8 X 10(3) M-1). On the other hand, if we correct for the population of HMM with degraded light chain 2, the difference in the binding constants in the presence and absence of Ca2+ may be as great as 5-fold. A double binding experiment also suggested that HMM with intact light chain 2 binds at most 5 times more strongly to regulated actin in the presence of Ca2+ than in its absence. We conclude that, just as with subfragment-1, the primary effect of troponin-tropomyosin in regulating the acto HMM ATPase activity is to inhibit a kinetic step in the ATPase cycle. However, our data with HMM also suggest that, in addition to this primary effect, troponin-tropomyosin may modulate the binding of the cross-bridge to actin in relaxed muscle to a small extent.     

Cooperative turning on of myosin subfragment 1 adenosinetriphosphatase activity by the troponin-tropomyosin-actin complex

In the field of muscle regulation, there is still controversy as to whether Ca2+, alone, is able to shift muscle from the relaxed to the fully active state or whether cross-bridge binding also contributes to turning on muscle contraction. Our previous studies on the binding of myosin subfragment 1 (S-1) to the troponin-tropomyosin-actin complex (regulated actin) in the absence of ATP suggested that, even in Ca2+, the binding of rigor cross-bridges is necessary to turn on regulated actin fully. In the present study, we demonstrate that this is also the case for the turning on of the acto.S-1 ATPase activity. By itself, Ca2+ does not fully turn on the acto.S-1 ATPase activity; at low actin concentration, there is almost a 10-fold increase in ATPase activity when the regulated actin is fully turned on by the binding of rigor cross-bridges in the presence of Ca2+. This large increase in ATPase activity does not occur because the binding of S-1.ATP to actin is increased; the binding of S-1.ATP is almost the same to maximally turned-off and maximally turned-on regulated actin. The increase in ATPase activity occurs because of a marked increase in the rate of Pi release so that when the regulated actin is fully turned on, Pi release becomes so rapid that the rate-limiting step precedes the Pi release step. These results suggest that, while Ca2+, alone, does not fully turn on the regulated actin filament in solution, the binding of rigor cross-bridges can turn it on fully. If force-producing cross-bridges play the same role in vivo as rigor cross-bridges in vitro, there may be a synergistic effect of Ca2+ and cross-bridge binding in turning on muscle contraction which could greatly sharpen the response of the muscle fiber to Ca2+.     

Mouse Myosin-19 Is a Plus-end-directed,High-duty Ratio Molecular Motor

Class XIX myosin (Myo19) is a vertebrate-specific unconventional myosin, responsible for the transport of mitochondria. To characterize biochemical properties of Myo19, we prepared recombinant mouse Myo19-truncated constructs containing the motor domain and the IQ motifs using the baculovirus/Sf9 expression system. We identified regulatory light chain (RLC) of smooth muscle/non-muscle myosin-2 as the light chain of Myo19. The actin-activated ATPase activity and the actin-gliding velocity of Myo19-truncated constructs were about one-third and one-sixth as those of myosin-5a, respectively. The apparent affinity of Myo19 to actin was about the same as that of myosin-5a. The RLCs bound to Myo19 could be phosphorylated by myosin light chain kinase, but this phosphorylation had little effect on the actin-activated ATPase activity and the actin-gliding activity of Myo19-truncated constructs. Using dual fluorescence-labeled actin filaments, we determined that Myo19 is a plus-end-directed molecular motor. We found that, similar to that of the high-duty ratio myosin, such as myosin-5a, ADP release rate was comparable with the maximal actin-activated ATPase activity of Myo19, indicating that ADP release is a rate-limiting step for the ATPase cycle of acto-Myo19. ADP strongly inhibited the actin-activated ATPase activity and actin-gliding activity of Myo19-truncated constructs. Based on the above results, we concluded that Myo19 is a high-duty ratio molecular motor moving to the plus-end of the actin filament.     

The mechanism of smooth muscle caldesmon-tropomyosin inhibition of the elementary steps of the actomyosin ATPase

Caldesmon is a component of smooth muscle thin filaments that inhibits the actomyosin ATPase via its interaction with actin-tropomyosin. We have performed a comprehensive transient kinetic characterization of the actomyosin ATPase in the presence of smooth muscle caldesmon and tropomyosin. At physiological ratios of caldesmon to actin (1 caldesmon/7 actin monomers) actomyosin ATPase is inhibited by about 75%. Inhibitory caldesmon concentrations had little effect upon the rate of S1 binding to actin, actin-S1 dissociation by ATP, and dissociation of ADP from actin-S1 x ADP; however the rate of phosphate release from the actin-S1 x ADP x P(i) complex was decreased by more than 80%. In addition the transient of phosphate release displayed a lag of up to 200 ms. The presence of a lag phase indicates that a step on the pathway prior to phosphate release has become rate-limiting. Premixing the actin-tropomyosin filaments with myosin heads resulted in the disappearance of the lag phase. We conclude that caldesmon inhibition of the rate of phosphate release is caused by the thin filament being switched by caldesmon to an inactive state. The active and inactive states correspond to the open and closed states observed in skeletal muscle thin filaments with no evidence for the existence of a third, blocked state. Taken together these data suggest that at physiological concentrations, caldesmon controls the isomerization of the weak binding complex to the strong binding complex, and this causes the inhibition of the rate of phosphate release. This inhibition is sufficient to account for the inhibition of the steady state actomyosin ATPase by caldesmon and tropomyosin.     

Effect of caldesmon on the ATPase activity and the binding of smooth and skeletal myosin subfragments to actin

We have previously shown that inhibition of the ATPase activity of skeletal muscle myosin subfragment 1 (S1) by caldesmon is correlated with the inhibition of S1 binding in the presence of ATP or pyrophosphate (Chalovich, J., Cornelius, P., and Benson, C. (1987) J. Biol Chem. 262, 5711-5716). In contrast, Lash et al. (Lash, J., Sellers, J., and Hathaway, D. (1986) J. Biol. Chem. 261, 16155-16160) have shown that the inhibition of ATPase activity of smooth muscle heavy meromyosin (HMM) by caldesmon is correlated with an increase in the binding of HMM to actin in the presence of ATP. We now show, in agreement, that caldesmon does increase the binding of smooth muscle HMM to actin-tropomyosin while decreasing the ATPase activity. The effect of caldesmon on the binding of smooth HMM is reversed by Ca2+-calmodulin. Caldesmon strengthens the binding of smooth S1.ATP and skeletal HMM.ATP to actin-tropomyosin but to a lesser extent than smooth HMM.ATP. Furthermore, this increase in binding of smooth S1.ATP and skeletal HMM.ATP does not parallel the inhibition of ATPase activity. In contrast, in the absence of ATP, all smooth and skeletal myosin subfragments compete with caldesmon for binding to actin. Thus, the effect that caldesmon has on the binding of myosin subfragments to actin-tropomyosin depends on the source of myosin, the type of subfragment, and the nucleotide present. The inhibition of actin-activated ATP hydrolysis by caldesmon, however, is not greatly different for different smooth and skeletal myosin subfragments. Evidence is presented that caldesmon inhibits actin-activated ATP hydrolysis by attenuating the productive interaction between myosin and actin that normally accelerates ATP hydrolysis. The increased binding seen by some myosin subfragments, in the presence of ATP, may be due to binding of these subfragments to a nonproductive site on actin-caldesmon. The subfragments which show an increase in binding in the presence of ATP and caldesmon appear to bind directly to caldesmon as demonstrated by affinity chromatography.