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

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

Proteolysis and binding of myosin subfragment 1 to actin

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

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

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

The regulation of myosin binding to actin filaments by Lethocerus troponin

Lethocerus indirect flight muscle has two isoforms of troponin C, TnC-F1 and F2, which are unusual in having only a single C-terminal calcium binding site (site IV, isoform F1) or one C-terminal and one N-terminal site (sites IV and II, isoform F2). We show here that thin filaments assembled from rabbit actin and Lethocerus tropomyosin (Tm) and troponin (Tn) regulate the binding of rabbit myosin to rabbit actin in much the same way as the mammalian regulatory proteins. The removal of calcium reduces the rate constant for S1 binding to regulated actin about threefold, independent of which TmTn is used. This is consistent with calcium removal causing the TmTn to occupy the B or blocked state to about 70% of the total. The mid point pCa for the switch differed for TnC-F1 and F2 (pCa 6.9 and 6.0, respectively) consistent with the reported calcium affinities for the two TnCs. Equilibrium titration of S1 binding to regulated actin filaments confirms calcium regulated binding of S1 to actin and shows that in the absence of calcium the three actin filaments (TnC-F1, TnC-F2 and mammalian control) are almost indistinguishable in terms of occupancy of the B and C states of the filament. In the presence of calcium TnC-F2 is very similar to the control with approximately 80% of the filament in the C-state and 10-15% in the fully on M-State while TnC-F1 has almost 50% in each of the C and M states. This higher occupancy of the M-state for TnC-F1, which occurs above pCa 6.9, is consistent with this isoform being involved in the calcium activation of stretch activation. However, it leaves unanswered how a C-terminal calcium binding site of TnC can activate the thin filament.     

Two-step ligand binding and cooperativity. A model to describe the cooperative binding of myosin subfragment 1 to regulated actin.

The binding of actin to myosin subfragment 1 (S1) has been shown to occur as a two-step reaction. In the first step actin is weakly bound and then the complex isomerizes to the "rigor type" acto-S1 complex (Coates, J. H., A. H. Criddle, and M. A. Geeves, 1985 Biochem. J., 232:351-356). We propose here a model in which troponin/tropomyosin (Tn/Tm) controls the actin-S1 interaction by inhibiting the isomerization step. In this model the (actin)7 Tn/Tm unit is assumed to exist in two states: open and closed. S1 can bind to either of the two states but only the open form allows the isomerization reaction to take place. We demonstrate that this model can account for the cooperative binding of S1 and S1 nucleotide complexes to actin. The model provides a way of integrating both the effects of calcium and nucleotide on actin-S1 interactions.     

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

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

Binding of troponin I and the troponin I-troponin C complex to actin-tropomyosin. Dissociation by myosin subfragment 1

Troponin I (TnI) is the component of the troponin complex, TnI, TnC, TnT, that is responsible for inhibition of actomyosin ATPase activity. Using the fluorescence of pyrene-labeled tropomyosin (Tm), we probed the interaction of TnI and TnIC with Tm on the reconstituted muscle thin filament. The results indicate that TnI and TnIC(-Ca(2+)) bind specifically and strongly to actin-Tm with a stoichiometry of 1 TnI or 1 TnIC/1 Tm/7 actin, in agreement with previous results. The binding of myosin heads (S1) to actin-Tm at low levels of saturation caused TnI and TnIC to dissociate from actin-Tm. These results are interpreted in terms of the S1-binding state allosteric-cooperative model of the actin-Tm thin filament, closed/open. Thus, TnI and TnIC(-Ca(2+)) bind to the closed state of actin-Tm and their binding is greatly weakened in the S1-induced open state, indicating that they act as allosteric inhibitors. The fluorescence change and the stoichiometry indicate that the TnI-binding site is composed of regions from both actin and Tm probably in the vicinity of Cys 190.     

Ca(2+)-dependent, myosin subfragment 1-induced proximity changes between actin and the inhibitory region of troponin I.

In order to help understand the spatial rearrangements of thin filament proteins during the regulation of muscle contraction, we used fluorescence resonance energy transfer (FRET) to measure Ca(2+)-dependent, myosin-induced changes in distances and fluorescence energy transfer efficiencies between actin and the inhibitory region of troponin I (TnI). We labeled the single Cys-117 of a mutant TnI with N-(iodoacetyl)-N'-(1-sulfo-5-naphthyl)ethylenediamine (IAEDANS) and Cys-374 of actin with 4-dimethylaminophenylazophenyl-4'-maleimide (DABmal). These fluorescent probes were used as donor and acceptor, respectively, for the FRET measurements. We reconstituted a troponin-tropomyosin (Tn-Tm) complex which contained the AEDANS-labeled mutant TnI, together with natural troponin T (TnT), troponin C (TnC) and tropomyosin (Tm) from rabbit fast skeletal muscle. Fluorescence titration of the AEDANS-labeled Tn-Tm complex with DABmal-labeled actin, in the presence and absence of Ca(2+), resulted in proportional, linear increases in energy transfer efficiency up to a 7:1 molar excess of actin over Tn-Tm. The distance between AEDANS on TnI Cys-117 and DABmal on actin Cys-374 increased from 37.9 A to 44.1 A when Ca(2+) bound to the regulatory sites of TnC. Titration of reconstituted thin filaments, containing AEDANS-labeled Tn-Tm and DABmal-labeled actin, with myosin subfragment 1 (S1) decreased the energy transfer efficiency, in both the presence and absence of Ca(2+). The maximum decrease occurred at well below stoichiometric levels of S1 binding to actin, showing a cooperative effect of S1 on the state of the thin filaments. S1:actin molar ratios of approximately 0.1 in the presence of Ca(2+), and approximately 0.3 in the absence of Ca(2+), were sufficient to cause a 50% reduction in normalized transfer efficiency. The distance between AEDANS on TnI Cys-117 and DABmal on actin Cys-374 increased by approximately 7 A in the presence of Ca(2+) and by approximately 2 A in the absence of Ca(2+) when S1 bound to actin. Our results suggest that TnI's interaction with actin inhibits actomyosin ATPase activity by modulating the equilibria among active and inactive states of the thin filament. Structural rearrangements caused by myosin S1 binding to the thin filament, as detected by FRET measurements, are consistent with the cooperative behavior of the thin filament proteins.     

Cooperative binding to the Ca2+-specific sites of troponin C in regulated actin and actomyosin

The Ca2+-binding component of troponin (TnC) and its proteolytic fragments containing Ca2+-binding sites I-III (TH1) or sites III and IV (TR2C) have been labeled with the fluorescent probes dansylaziridine (DANZ) at methionine 25 or 5-(iodoacetamidoethyl)amino-naphthalene-1-sulfonic acid (AEDANS) at cysteine-98. These probes report binding of Ca2+ to the low and high affinity sites, respectively. Fluorescence changes as a function of [Ca2+] were measured for the free peptides, their complexes with troponin I + troponin T, and these complexes bound to actin-tropomyosin in the presence of Mg2+ and ATP with and without myosin. An apparent Hill coefficient of 1.0-1.1 has been obtained for the Ca2+-induced fluorescence changes in TnC, its fragments, and their ternary complexes regardless of the label used. When a ternary complex containing appropriately labeled TnC or its fragment is bound to the actin-tropomyosin complex, the Hill coefficient for the titration of the low affinity sites increases to 1.5-1.6 and further increases to greater than 2 in the presence of myosin. To interpret the apparent Hill coefficients, we used a model containing two binding sites and a single reporter of the conformational change. Hill coefficients between 1.0 and 1.2 can be obtained for the fluorescence change without true cooperativity in metal binding, depending on the mechanism of the fluorescence change; i.e. the contribution of the singly or doubly occupied species to the fluorescence change. A Hill coefficient between 1.2 and 2, however, always indicates cooperativity in binding independently of the mechanism. Thus, our finding that fluorescence titrations of Ca2+ binding to TnCDANZ bound to actin-tropomyosin exhibit a Hill coefficient of 1.5 in the absence of myosin and 2.4 in its presence indicates the existence of true positive cooperativity in metal binding to sites I and II. No cooperativity was observed for AEDANS-labeled complexes that reflect Ca2+-binding to the high affinity sites. Plots of the Ca2+ dependence of myosin ATPase activity activated by actin-tropomyosin in the presence of any of the troponin complexes used had apparent Hill coefficients of approximately 4. The higher value suggests cooperative interactions in the activation of ATPase beyond those involved in Ca2+-binding to the Ca2+-specific sites.     

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.     

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.     

Conformational changes induced in troponin I by interaction with troponin T and actin/tropomyosin.

Troponin I (TnI) is the inhibitory component of the striated muscle Ca2+ regulatory protein troponin (Tn). The other two components of Tn are troponin C (TnC), the Ca2+-binding component, and troponin T (TnT), the tropomyosin-binding component. We have used limited chymotryptic digestion to probe the local conformation of TnI in the free state, the binary TnC*TnI complex, the ternary TnC*. TnI*TnT (Tn) complex, and in the reconstituted Tn*tropomyosin*F-actin filament. The digestion of TnI alone or in the TnC*TnI complex produced initially two major fragments via a cleavage of the peptide bond between Phe100 and Asp101 in the so-called inhibitory region. In the ternary Tn complex cleavage occurred at a new site between Leu140 and Lys141. In the absence of Ca2+ this was followed by digestion of the 1-140 fragment at Leu122 and Met116. In the reconstituted thin filament the same fragments as in the case of the ternary complex were produced, but the rate of digestion was slower in the absence than in the presence of Ca2+. These results indicate firstly that in both free TnI and TnI complexed with TnC there is an exposed and flexible site in the inhibitory region. Secondly, TnT affects the conformation of TnI in the inhibitory region and also in the region that contains the 140-141 bond. Thirdly, the 140-141 region of TnI is likely to interact with actin in the reconstituted thin filament when Ca2+ is absent. These findings are discussed in terms of the role of TnI in the mechanism of thin filament regulation, and in light of our previous results [Y. Luo, J.-L. Wu, J. Gergely, T. Tao, Biochemistry 36 (1997) 13449-13454] on the global conformation of TnI.     

The tail domain of myosin Va modulates actin binding to one head

Calcium activates full-length myosin Va steady-state enzymatic activity and favors the transition from a compact, folded "off" state to an extended "on" state. However, little is known of how a head-tail interaction alters the individual actin and nucleotide binding rate and equilibrium constants of the ATPase cycle. We measured the effect of calcium on nucleotide and actin filament binding to full-length myosin Va purified from chick brains. Both heads of nucleotide-free myosin Va bind actin strongly, independent of calcium. In the absence of calcium, bound ADP weakens the affinity of one head for actin filaments at equilibrium and upon initial encounter. The addition of calcium allows both heads of myosin Va.ADP to bind actin strongly. Calcium accelerates ADP binding to actomyosin independent of the tail but minimally affects ATP binding. Although 18O exchange and product release measurements favor a mechanism in which actin-activated Pi release from myosin Va is very rapid, independent of calcium and the tail domain, both heads do not bind actin strongly during steady-state cycling, as assayed by pyrene actin fluorescence. In the absence of calcium, inclusion of ADP favors formation of a long lived myosin Va.ADP state that releases ADP slowly, even after mixing with actin. Our results suggest that calcium activates myosin Va by allowing both heads to interact with actin and exchange bound nucleotide and indicate that regulation of actin binding by the tail is a nucleotide-dependent process favored by linked conformational changes of the motor domain.     

Differential binding of rabbit fast muscle myosin light chain isoenzymes to regulated actin

The direct binding of S1(A1) and S1(A2) to regulated actin has been investigated by centrifugation. Binding was measured in the presence of either Mg·AdoPP[NH]P or Mg·ADP at 24°C at various ionic strengths. At low ionic strength, in either the presence or absence of Ca²⁺, the binding of S1(A1) to regulated actin was always stronger than for S1(A2). As the ionic strength was increased the differential binding between S1(A1) and S1(A2) was still maintained in the presence of Ca²⁺ but not in its absence. These data are discussed in terms of a modifying role for the N-terminal region of the A1 light chain in regulation of the contractile process.     

Caldesmon binding to actin is regulated by calmodulin and phosphorylation via different mechanisms

Smooth muscle caldesmon (CaD) binds F-actin and inhibits actomyosin ATPase activity. The inhibition is reversed by Ca2+/calmodulin (CaM). CaD is also phosphorylated upon stimulation at sites specific for mitogen-activated protein kinases (MAPKs). Because of these properties, CaD is thought to be involved in the regulation of smooth muscle contraction. The molecular mechanism of the reversal of inhibition is not well understood. We have expressed His6-tagged fragments containing the sequence of the C-terminal region of human (from M563 to V793) and chicken (from M563 to P771) CaD as well as a variant of the chicken isoform with a Q766C point mutation. By cleavages with proteases, followed by high-speed cosedimentation with F-actin and mass spectrometry, we found that within the C-terminal region of CaD there are multiple actin contact points forming two clusters. Intramolecular fluorescence resonance energy transfer between probes attached to cysteine residues (the endogenous C595 and the engineered C766) located in these two clusters revealed that the C-terminal region of CaD is elongated, but it becomes more compact when bound to actin. Binding of CaM restores the elongated conformation and facilitates dissociation of the C-terminal CaD fragment from F-actin. When the CaD fragment was phosphorylated with a MAPK, only one of the two actin-binding clusters dissociated from F-actin, whereas the other remained bound. Taken together, these results demonstrate that while both Ca2+/CaM and MAPK phosphorylation govern CaD's function via a conformational change, the regulatory mechanisms are different.     

Effect of ethylene glycol on the interaction of different myosin subfragment 1.nucleotide complexes with actin

To elucidate the structure of the cross-bridge intermediates in the actomyosin ATPase cycle, several laboratories have added both ethylene glycol and AMP-PNP to muscle fibers. These studies suggested that ethylene glycol shifts the structure of myosin.AMP-PNP toward the weak-binding conformation, i.e., toward the structure of myosin.ATP. Since only the weak-binding conformation of myosin subfragment 1 (S-1) binds with no apparent cooperativity to the troponin-tropomyosin-actin complex (regulated actin), we used this as a probe to examine the conformation of various S-1.nucleotide complexes in ethylene glycol. Our results show that ethylene glycol markedly weakens the binding strength of S-1, S-1.ADP, and S-1.AMP-PNP to actin but has almost no effect on the binding strength of S-1.ATP. As in muscle fibers, at 40% ethylene glycol, the binding strength of S-1.AMP-PNP to actin becomes very similar to the binding strength of S-1.ATP. In the presence of troponin-tropomyosin, the binding of S-1.AMP-PNP to actin shows no apparent cooperativity in 40% ethylene glycol. Therefore, our results confirm that ethylene glycol shifts the structure of the myosin.AMP-PNP toward the weak-binding conformation. However, our results also suggest that ethylene glycol has a direct effect on the regulated actin complex. This is shown by the fact that ethylene glycol markedly increases the cooperative binding of S-1.ADP to regulated actin both in the presence and in the absence of Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium-dependent movement of troponin I between troponin C and actin as revealed by spin-labeling EPR

We measured EPR spectra from a spin label on the Cys133 residue of troponin I (TnI) to identify Ca(2+)-induced structural states, based on sensitivity of spin-label mobility to flexibility and tertiary contact of a polypeptide. Spectrum from Tn complexes in the -Ca(2+) state showed that Cys133 was located at a flexible polypeptide segment (rotational correlation time tau=1.9ns) that was free from TnC. Spectra of both Tn complexes alone and those reconstituted into the thin filaments in the +Ca(2+) state showed that Cys133 existed on a stable segment (tau=4.8ns) held by TnC. Spectra of reconstituted thin filaments (-Ca(2+) state) revealed that slow mobility (tau=45ns) was due to tertiary contact of Cys133 with actin, because the same slow mobility was found for TnI-actin and TnI-tropomyosin-actin filaments lacking TnC, T or tropomyosin. We propose that the Cys133 region dissociates from TnC and attaches to the actin surface on the thin filaments, causing muscle relaxation at low Ca(2+) concentrations.