HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 280, Issue 15, 15071-15083, April 15, 2005

This Article Abstract Full Text (PDF) All Versions of this Article: 280/15/15071    most recent M500616200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kovács, M. Articles by Sellers, J. R. Search for Related Content PubMed PubMed Citation Articles by Kovács, M. Articles by Sellers, J. R. Social Bookmarking                      What's this?

Mechanism of Action of Myosin X, a Membrane-associated Molecular Motor^*

Mihály Kovács, Fei Wang, and James R. Sellers

From the Laboratory of Molecular Physiology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-1762

Received for publication, January 18, 2005 , and in revised form, February 9, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
We have performed a detailed biochemical kinetic and spectroscopic study on a recombinant myosin X head construct to establish a quantitative model of the enzymatic mechanism of this membrane-bound myosin. Our model shows that during steady-state ATP hydrolysis, myosin X exhibits a duty ratio (i.e. the fraction of the cycle time spent strongly bound to actin) of around 16%, but most of the remaining myosin heads are also actin-attached even at moderate actin concentrations in the so-called "weak" actin-binding states. Contrary to the high duty ratio motors myosin V and VI, the ADP release rate constant from actomyosin X is around five times greater than the maximal steady-state ATPase activity, and the kinetic partitioning between different weak actin-binding states is a major contributor to the rate limitation of the enzymatic cycle. Two different ADP states of myosin X are populated in the absence of actin, one of which shows very similar kinetic properties to actomyosin·ADP. The nucleotide-free complex of myosin X with actin shows unique spectral and biochemical characteristics, indicating a special mode of actomyosin interaction.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Myosin X is a recently described member of the myosin superfamily that is expressed in vertebrate tissues as a single isoform (1, 2). The heavy chain of myosin X consists of an N-terminal motor domain containing the actin- and ATP-binding sites, a neck region that binds three calmodulin (or possibly other) light chains, a putative coiled-coil region that may bring about heavy chain dimerization, and a tail region consisting of several domains of effector function (three pleckstrin homology (PH3)¹ domains, a MyTH4, and a FERM domain) (1). The presence of the PH3 domains in the myosin X tail is a unique feature among myosins, and it enables this myosin to directly bind to the plasma membrane via phosphatidylinositol phospholipids (1, 3). Myosin X has been shown to localize to regions of dynamic actin and to exhibit remarkable patterns of intrafilopodial motility (1, 4). Most interestingly, myosin X localizes to the tips of filopodia, which appears to be an active process requiring myosin X motor function (4). Myosin X induces elongation of filopodia by transporting the Mena-VASP complex (an inhibitor of actin filament capping) to filopodial tips (5). Myosin X activity is also necessary for phagocytosis (6) as well as the localization and function of integrins (7).

The above functional studies indicate a cellular role for myosin X as a plasma membrane-associated cargo transporter. This setting is unique within the myosin superfamily, which implies that this class of motors may have adapted to its role by acquiring distinctive molecular properties. All myosins exert their motile activity during a cyclic interaction with actin filaments and ATP. The enzymatic parameters are key determinants of the motile output of motor proteins, and it has been shown in numerous cases that the biochemistry of different myosins reflects precise and profound functional adaptations to their widely differing cellular roles (for a review see Ref. 8). Studies on a heavy meromyosin-like recombinant fragment of myosin X have shown that myosin X exhibits actin-activated ATPase activity and in vitro motility directed toward the barbed end of actin filaments (9). Its mechanism of action, however, has not been investigated in detail. To assess the biochemical and enzymatic properties of myosin X, we used the baculovirus-Sf9 expression system to produce a single-headed myosin X construct (named mX-S1) that retains the catalytically active portion of the molecule. We performed steady-state and transient kinetic, fluorescence spectroscopic, and other biochemical measurements to show that myosin X spends a relatively small fraction (16%) of its ATPase cycle time in the so-called strong actin-binding states. This behavior suggests that myosin X does not use a myosin V-like single molecule processive stepping mechanism. However, the total actin attachment ratio of myosin X during steady-state ATP hydrolysis is notably high even at moderate actin concentrations (>50% attachment at 20 µM actin). This feature is due to the kinetic partitioning between different "weak" actin-binding states, which may greatly aid in maintaining continuous actin attachment and therefore proper functioning of a small array of membrane-bound motors.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cloning, Expression, and Purification of mX-S1—A truncated (subfragment-1-like) fragment of the bovine myosin X heavy chain cDNA (gift of Dr. David Corey, Harvard Medical School) encoding the motor domain and all three light chain-binding motifs (first 810 amino acids of the heavy chain) was cloned into the pVL1392 expression vector. This construct (referred to as mX-S1 throughout this paper) was coexpressed with calmodulin in the baculovirus-Sf9-expression system. A C-terminal FLAG tag (sequence DYKDDDDK) was appended to the heavy chain that enabled high purity (>99%) preparation of recombinant mX-S1. Expression and purification procedures were as described earlier for non-muscle myosin IIA heavy meromyosin (10). Typically, 2–5 mg of mX-S1 could be purified from 4 x 10⁹ Sf9 cells. Purified mX-S1 was stored in a high ionic strength (I = 525 mM) buffer and used within 4 days of preparation or flash-frozen immediately. The construct was not soluble at low ionic strengths in the absence of actin and nucleotide. Therefore, ionic strengths of >30 mM had to be used in most experiments. A 2-fold molar excess of extra calmodulin was added to mX-S1 after purification in order to fully saturate the binding sites on the mX-S1 heavy chain, thereby preventing protein aggregation. Protein concentrations were determined using the Bio-Rad assay using smooth muscle myosin subfragment-1 as a standard. Stopped-flow-based active site titrations with mant-ATP indicated that the error of protein concentration determinations was below 10%.

Other Materials—Rabbit skeletal muscle myosin and subfragment-1 (S1) were prepared as described in Refs. 11 and 12, respectively. Recombinant non-muscle myosin IIB S1 was prepared as described earlier for non-muscle myosin IIA heavy meromyosin (10). Actin was prepared as described in Ref. 13 and pyrene labeled as described in Ref. 14. Actin filaments were stabilized by addition of a 1.5-fold molar excess of phalloidin (Calbiochem) in all experiments. Mant-ATP and pyreneiodoacetamide were purchased from Molecular Probes. MDCC-PBP (fluorescently labeled bacterial P_i-binding protein) (15) was generously provided by Dr. Howard D. White (Eastern Virginia Medical School). Other reagents were from Sigma.

Fluorescence Spectroscopy—Pyrene fluorescence excitation and emission spectra were recorded using a SPEX FluoroMax-3 spectrofluorometer at 25 °C in a buffer comprising 20 mM MOPS (pH 7.0), 5 mM MgCl₂, 100 mM KCl, and 0.1 mM EGTA (I = 125 mM). In acrylamide quenching, steady-state fluorescence anisotropy, and temperature dependence measurements, samples containing pyrene-actin were excited at 347 nm (1 nm bandwidth), and emission was monitored at 406 nm (5 nm bandwidth). Background signal measured with assay buffer alone was subtracted from all fluorescence records before analysis.

Acto-S1 Cosedimentation—Samples were set up at room temperature under the same buffer conditions as in the spectroscopic measurements (or using 20 mM instead of 100 mM KCl to yield a final I = 45 mM where indicated) and then ultracentrifuged at 100,000 rpm in a Beckman TLA-100 rotor for 15 min at 4 °C, and the supernatants and pellets were run on 4–20% SDS-polyacrylamide gels. In samples involving ATP but not ADP, contaminating amounts of ADP were removed by the addition of 200 units/ml pyruvate kinase and 1 mM phosphoenolpyruvate. Protein amounts in electrophoretic bands were quantified by densitometric analysis using the Kodak ID 3.5 software.

Steady-state Kinetics—Steady-state MgATPase activities were measured by a NADH-linked assay as described earlier (16, 17) in a buffer containing 4 mM MOPS (pH 7.0), 2 mM MgCl₂, 0.1 mM EGTA, 1 mM ATP, 40 units/ml lactate dehydrogenase, 200 units/ml pyruvate kinase, 1 mM phosphoenolpyruvate, 0.2 mM NADH plus various concentrations of KCl (I = 10 mM + [KCl]). Data were corrected for background ATPase activity of actin.

Transient Kinetics—Stopped-flow experiments were performed in a KinTek SF-2001 apparatus. Tryptophan fluorescence was excited at 295 nm (6 nm bandwidth), and emission was selected using a 347-nm band-pass filter with a 50 nm bandwidth. Pyrene fluorescence was excited at 365 nm (6 nm bandwidth), and emission monitored through a 400 nm long-pass filter. Light scattering was measured at 420 nm in samples containing pyrene-actin. Mant-ATP was excited using energy transfer from nearby tryptophan residues (excitation at 280 nm with 6 nm bandwidth; emission, 400 nm long-pass filter). MDCC-PBP fluorescence was excited at 436 nm (6 nm bandwidth), and emission was collected through a 450 nm long-pass filter. Quenched-flow experiments were performed in a KinTek RQF-3 apparatus using [-³²P]ATP as described earlier (17).

Post-mixing concentrations are indicated throughout the text unless stated otherwise. The volume ratio was 1:1 in all single mixing, and 5:2:5 in double-mixing experiments. Nucleotide-free mX-S1 and acto-mX-S1 were prepared by incubation with 0.01 units/ml apyrase for 15 min at room temperature. In phosphate release measurements, all solutions were preincubated with a "P_i mop" consisting of 0.02 units/ml purine nucleoside phosphorylase and 0.5 mM 7-methylguanosine.

Data Analysis and Kinetic Modeling—Reported means and S.D. are those of two to six rounds of experiment. Fitting of the data sets to mathematical functions was done using the KinTek SF-2001 software and OriginLab 7.0 (Microcal Corp.). Kinetic simulations and global fitting analyses were performed using the Gepasi version 3.21 software (Pedro Mendes, Virginia Bioinformatics Institute).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Basal ATPase Mechanism of mX-S1—In all experiments of the present study, we used a single-headed myosin X construct (mX-S1) that contains the catalytically active motor domain as well as the entire neck region comprising three light chain-binding IQ motifs and three calmodulin molecules as light chains.

The tryptophan fluorescence emission of mX-S1 showed a 6% increase and a small (3 nm) blue shift on the addition of ADP (Fig. 1A). The addition of ATP to mX-S1 caused a similar blue shift and a 20% fluorescence enhancement compared with the nucleotide-free state, indicating that at least one intermediate of higher fluorescence than the mX-S1·ADP complex is significantly populated during steady-state ATP hydrolysis (Fig. 1A). Mixing mX-S1 with ATP under pseudo first-order conditions in the stopped-flow apparatus yielded single exponential tryptophan fluorescence transients (traces not shown). The dependence of the fitted observed rate constants (k_obs) of these traces on ATP concentration showed signs of saturation above 250 s^-1 as shown in Fig. 1B. This behavior suggests that ATP binding to mX-S1 is a two-step process (K₁ and k₂ in Scheme 1), and the maximal k_obs value largely reflects a first-order isomerization step during the binding process (k₂). The ATP hydrolysis step (k₃ + k_-3) is associated with a further increase in tryptophan fluorescence (cf. Scheme 1), and it may therefore have contributions to the ATP-binding k_obs values of Fig. 1B. The quasi-linear part of the plot at low [ATP] indicated an apparent second-order ATP-binding rate constant (k₂/K₁) of 1.1 µM^-1 s^-1 (Fig. 1B and Table I). k_obs values for ATP binding obtained by monitoring the signal of mant-ATP, a fluorescently labeled substrate, showed a very similar profile to the tryptophan results (Fig. 1B and Table I).

View larger version (16K): [in this window] [in a new window]   FIG. 1. ATP binding and hydrolysis by mX-S1 in the absence of actin. A, tryptophan fluorescence emission spectra of 5 µM mX-S1 in the absence of nucleotide (apo) and after the addition of 300 µM ADP or 300 µM ATP. Fluorescence was excited at 295 nm with 1 nm bandwidth, and emission was detected using a bandwidth of 5 nm. B, observed rate constants (kobs) of single exponential fits to tryptophan fluorescence transients recorded on mixing 0.5 µM mX-S1 with different concentrations of ATP in the stopped-flow apparatus (solid circles). A hyperbolic fit of the dependence of kobs on [ATP] yielded a maximal kobs of 430 ± 80 s-1 with half-saturation at 310 ± 90 µM ATP. The open triangle shows the kobs of the exponential burst phase of Pi production in a quenched-flow experiment, where 1.7 µM mX-S1 was mixed with 50 µM ATP (kobs = 67 ± 15 s-1, see text). The open squares indicate the kobs values of the single exponential increase in mant-ATP fluorescence on mixing 0.5 µM mX-S1 with various mant-ATP concentrations in the stopped flow. C, time course of ATP hydrolysis upon mixing 2.3 µM mX-S1 with 1 µM [-32P]ATP in the quenched-flow apparatus (single turnover conditions). A double exponential approximation to the reaction time profile indicated a burst phase (kobs = 2.1 s-1) with a fractional amplitude (= Afast/(Afast + Aslow)) of 0.28, whereas the slow phase of the reaction had a kobs of 0.03 s-1. Conditions are as follows: 25 °C, 20 mM MOPS (pH 7.0), 5 mM MgCl2, 100 mM KCl and 0.1 mM EGTA. Error bars indicate S.D. for n = 3.

View larger version (7K): [in this window] [in a new window]   SCHEME 1. Equilibrium constants are expressed as dissociation constants or for first-order transitions in a left-to-right direction in all schemes. Arrows for associating or dissociating components are omitted for clarity in all schemes except Scheme 4. Asterisks indicate states with elevated tryptophan fluorescence. Abbreviations used are as follows: A, actin; M, myosin; T, ATP; D, ADP; P, phosphate.

Phosphate release from the mX-S1·ADP·P_i complex was monitored using a fluorescently labeled P_i-binding protein (MDCC-PBP). The time course of P_i release on mixing 1 µM mX-S1 with 0.6 µM ATP (single turnover conditions) was a single exponential with a k_obs of 0.03 s^-1, a value identical to the steady-state ATPase activity of mX-S1 in the absence of actin, as measured by an NADH-linked coupled assay (traces not shown; Table I). This indicates that the P_i release is rate-limiting in the basal (i.e. in the absence of actin) ATPase cycle of mX-S1, with its k_obs being determined by the kinetic partitioning in the hydrolysis step (K₃) and the fundamental P_i release rate constant (k₄) as k_obs,Pi = k₄K₃/(1 + K₃) (Scheme 1 and Table I).

Although most of the above features of the basal mX-S1 ATPase cycle are similar to those of other myosins, the ADP interaction of mX-S1 showed some remarkably unusual properties. On mixing mX-S1 with ADP in the stopped-flow under pseudo first-order conditions, the single exponential k_obs values of the recorded tryptophan fluorescence transients slightly increased with increasing [ADP] and saturated around the low value of 4 s^-1 (Fig. 2A). At first glance, the data could seem to indicate a low affinity single step binding mechanism with a relatively high intercept (k_off 2 s^-1) and a small linear slope (k_on < 0.1 µM^-1 s^-1). However, the ADP binding affinity should then be low (K_d = k_off/k_on >20 µM), which is ruled out by the fact that the amplitudes of the transients showed saturation at much lower ADP concentrations (K_d = 1.0 ± 0.2 µM, Fig. 2A, inset). Another possible explanation for the obtained k_obs values comes from a two-step model (cf. the right part of Scheme 1 comprising K₅ and K₆, viewed right to left in the direction of ADP binding). In this model the ADP-binding process consists of an initial rapid equilibrium (K₆, no signal change on this step) followed by a slower reversible isomerization accompanied by a fluorescence increase (K₅). The intercept of Fig. 2A will thus be mostly determined by k₅ (1.5 s^-1, going left-to-right in Scheme 1); the maximal k_obs will indicate k₅ + k_-5 (4.5 s^-1), and half-saturation will occur at [ADP] = K₆ (5 µM). To test further the validity of this model, we performed ADP "chasing" stopped-flow experiments in which equilibrium mixtures of mX-S1 and ADP were rapidly mixed with mant-ATP in at least 5-fold excess over ADP to ensure that ADP dissociation after the rapid mix is essentially irreversible (Fig. 2B). The observed mant-ATP fluorescence transients were biphasic throughout the examined [ADP] range (Fig. 2B, inset). The fractional amplitude of the slow phase (A_slow/(A_fast + A_slow)) was saturated at 0.73 ± 0.02 and reached half-maximum at 1.7 ± 0.2 µM ADP. These data are consistent with the above two-step mechanism inasmuch as the fractional amplitude of the fast phase is determined by the combined fractional population of the M and MD species (cf. Scheme 1) that allows fast binding of mant-ATP, whereas the slow phase comes from M^*D first slowly isomerizing to MD, then losing ADP, and binding mant-ATP. At high [ADP], the A_fast/A_slow ratio will then equal [MD]/[M^*D] (because[M] = 0 in the preincubation mixture) and thus K₅ = 0.37 ± 0.03. Furthermore, at high [ADP], k_fast and k_slow will equal k₆ (= 15 s^-1, left-to-right in Scheme 1, cf. Fig. 2B, inset) and k₅ (= 1.3 s^-1), respectively. From the experimental data shown in Fig. 2, A and B, all rate and equilibrium constants of the two-step ADP-binding process can be calculated as shown in Table I.

View larger version (21K): [in this window] [in a new window]   FIG. 2. ADP interaction of mX-S1 in the absence of actin. A, observed rate constants (kobs) of single exponential fits to tryptophan fluorescence transients obtained on mixing 0.4 µM mX-S1 with different concentrations of ADP in the stopped-flow. The inset shows relative amplitudes of the same reactions (fluorescence level before mix was taken as unity). A hyperbolic fit to the amplitude data yielded a maximal relative amplitude of 0.085 ± 0.002 with half-saturation at 1.0 ± 0.2 µM ADP. Note the breaks in the x axes of both panels. B, fractional amplitude of the slow phase (= Aslow/(Afast + Aslow))of the double exponential mant-ATP fluorescence transients on rapid mixing of 0.5 µM mX-S1 plus the indicated ADP concentrations with mant-ATP (in at least 5-fold molar excess over ADP) in the stopped flow. The data were fitted to a hyperbola to yield a maximal fractional amplitude of 0.73 ± 0.02 for the slow phase with half-saturation at 1.7 ± 0.2 µM ADP. The inset shows traces for two of the data points. Trace 1 was obtained on mixing 0.5 µM mX-S1 and 2 µM ADP with 50 µM mant-ATP (kfast = 48 s-1 (59% amplitude); kslow = 2.8 s-1 (41%)). In trace 2, 0.5 µM mX-S1 and 60 µM ADP was mixed with 300 µM mant-ATP (kfast = 13 s-1 (29% amplitude); kslow = 1.3 s-1 (71%)). Pre-mixing concentrations are indicated in this panel. C, observed rate constants (kobs) of single exponential fits to tryptophan fluorescence transients recorded on rapidly mixing 0.7 µM mX-S1 with a pre-mixture of 50 µM ATP plus ADP at the indicated concentrations (solid symbols). The open symbols indicate the fitted rate constants of simulated time courses obtained using the kinetic model described in the text. Traces 1–3 of the inset are experimental tryptophan fluorescence transients of the data points at 0, 50, and 500 µM ADP, respectively (fitted rate constants are 70, 12, and 3.9 s-1). Conditions were as in Fig. 1. Error bars indicate fitting errors of kobs.

A chasing experiment in which the pre-mixture of 0.7 µM mX-S1 and 50 µM ADP was rapidly mixed with 500 µM ATP gave a tryptophan fluorescence increase transient with a fitted single exponential k_obs of around 16 s^-1 (trace not shown). This value is remarkably higher than the intercept of the ADP-binding transients in Fig. 2A. This behavior is consistent with the two-step model described in the previous paragraph, which predicts this experiment to yield a transient consisting of a major phase dominated by k₆ (15 s^-1) and a slower phase (k₅ 1.2 s^-1) with a small amplitude (the two phases did not appear separate because of the low experimental signal-to-noise ratio).

Actin Activation of the Steady-state ATPase Activity—The steady-state ATPase activity of mX-S1 was 100-fold activated by actin to a V_max around 4 s^-1 as measured by an NADH-linked coupled enzyme assay (Table II and Fig. 3). Although the V_max value was fairly independent of solution ionic strength, the actin concentration needed for half-maximal activation (K_ATPase) markedly increased with increasing ionic strength (Table II). This behavior is characteristic of myosins in which the so-called weak actin-binding (ATP or ADP·P_i-bound) myosin states are the predominant steady-state intermediates in the actomyosin ATPase cycle. The steady-state data are in line with those reported by Homma et al. (9) for a heavy meromyosin-like myosin X construct (V_max = 4.7 s^-1, K_ATPase = 28 µM at 25 °C, I = 40 mM in the absence of Ca²⁺).

View larger version (17K): [in this window] [in a new window]   FIG. 3. Actin activation of the mX-S1 ATPase. Actin-activated steady-state ATPase activity of mX-S1 (50 nM) at ionic strengths of 10 mM (solid symbols) and 110 mM (open symbols). Hyperbolic fits to the data sets yielded a Vmax of 4.0 ± 0.1 s-1 and a KATPase of 5.7 ± 0.4 µM (I = 10 mM), and a Vmax of 3.0 ± 0.3 s-1 and a KATPase of 33 ± 5 µM (I = 110 mM). Conditions are as follows: 25 °C, 4 mM MOPS (pH 7.0), 2 mM MgCl2, 0.1 mM EGTA, 1 mM ATP, 40 units/ml lactate dehydrogenase, 200 units/ml pyruvate kinase, 1 mM phosphoenolpyruvate, 0.2 mM NADH plus KCl to yield the stated ionic strengths. The basal ATPase activity of mX-S1 (0.03 s-1) was subtracted from all data points.

View larger version (29K): [in this window] [in a new window]   FIG. 4. Strong binding actin interaction of mX-S1. A, excitation and emission spectra of 1 µM pyrene-actin alone (a) and in the presence of 2 µM mX-S1 (mX) or 2 µM skeletal muscle myosin II S1 (mII). Excitation wavelength was 344 nm for the emission spectra, whereas the excitation spectra were recorded at an emission wavelength of 406 nm. Bandwidths were 1 and 5 nm for the excitation and emission sides, respectively. Data were normalized to the 344 nm excitation and 406 nm emission peaks of pyrene-actin alone. B, pyrene fluorescence titration of 30 nM pyrene-actin with increasing concentrations of mX-S1. Excitation was at 344 nm (1 nm bandwidth), and emission was detected at 406 nm (5 nm bandwidth). Data were normalized to the fluorescence intensity of pyrene-actin alone. A hyperbolic fit to the data set yielded a KA of 0.19 ± 0.03 µM with a final fluorescence level of 0.84 ± 0.02. The inset shows the results of a stopped-flow titration where 50 nM pyrene-actin was preincubated with the indicated mX-S1 concentrations and then rapidly mixed with 10 µM ATP to fully dissociate acto-S1 (pre-mixing concentrations indicated). The [mX-S1] dependence of the relative amplitudes (expressed as F/Ffinal) of the recorded single exponential pyrene fluorescence transients (kobs = 4.3 ± 0.5 s-1) was fitted to a quadratic binding equation to yield a KA of 0.09 ± 0.04 µM and a maximal F/Ffinal value of 0.09 ± 0.005. C, observed rate constants (kobs) of pyrene fluorescence decrease transients recorded on mixing 0.1–0.3 µM mX-S1 with various concentrations of pyrene-actin in the absence (solid symbols) and presence (open symbols) of 1 mM ADP (in all syringes). Linear fits to the data sets yielded slopes of 2.2 ± 0.02 µM-1s-1 (k-A) and 0.26 ± 0.02 µM-1s-1 k-DA) for mX-S1 and mX-S1·ADP binding to pyrene-actin, respectively. The y intercepts indicating the dissociation rate constant from pyrene-actin were 0.41 ± 0.06 s-1 (kA) and 0.81 ± 0.04 s-1 (kDA) for mX-S1 and mX-S1·ADP, respectively. The inset shows traces of 0.3 µM mX-S1 binding to 5 µM pyrene-actin in the absence (trace 1, kobs = 11 s-1) and presence of 1 mM ADP (trace 2, kobs = 2.0 s-1). D, pyrene fluorescence transient recorded on rapid mixing of a pre-mixture of 0.2 µM pyrene-actin and 0.3 µM mX-S1 with 10 µM unlabeled actin in the absence of nucleotide. A single exponential fit of the transient gave a k of 0.36 s-1 for mX-S1 dissociation from pyrene-actin (kA) in the experiment shown. Conditions were as in Fig. 1. Data points shown in B and C are averages of three experiments.

View larger version (12K): [in this window] [in a new window]   SCHEME 2. Abbreviations used are as follows: A, actin; M, myosin; D, ADP.

ATP-induced Acto-mX-S1 Dissociation, ATP Hydrolysis, and the Weak Actin-binding Interaction—The ATP-induced dissociation of mX-S1 from pyrene-actin was followed by monitoring both pyrene fluorescence and light scattering. The changes in the two signals had opposite signs (pyrene fluorescence increased and light scattering decreased upon pyrene-acto-mX-S1 dissociation) and showed nearly identical dependence of the single exponential k_obs values on ATP concentration (Fig. 5A). This behavior shows that the dissociation of mX-S1 from pyrene-actin immediately follows the transition from a strongly to a weakly actin-bound state (k_T + k_-T > k₂' in Scheme 3). Similarly to other myosins, k_obs showed saturation at a high value (k₂' >600 s^-1), indicating that ATP binding to acto-mX-S1 is a two-step process (K₁' and k₂'; Scheme 3 and Table IV). The apparent second-order ATP-binding rate constant (k₂'/K₁') was markedly reduced with increasing ionic strength (0.55 ± 0.04 µM^-1 s^-1 at I = 125 mM as opposed to 1.8 ± 0.2 µM^-1 s^-1 at I = 35 mM; Fig. 5A and Table IV).

View larger version (20K): [in this window] [in a new window]   FIG. 5. ATP-induced dissociation of acto-mX-S1, ATP hydrolysis and weak actin binding. A, observed rate constants (kobs) of ATP-induced pyrene-acto-mX-S1 dissociation on rapid mixing of a pre-mixture of 0.25 µM mX-S1 and 0.15 µM pyrene-actin with the indicated ATP concentrations in the stopped-flow. The recorded transients were fitted to single exponentials. The solid circles show kobs values of pyrene fluorescence records (I = 35 mM), and the open circles indicate kobs values of light-scattering transients of the same reactions. Hyperbolic fits yielded maximal kobs values of 620 ± 50 and 630 ± 40 s-1 (k2' in Scheme 3) with half-saturation (K1') at 290 ± 50 and 240 ± 40 µM ATP for pyrene fluorescence and light scattering, respectively. The solid squares show pyrene fluorescence kobs values for the same reactions at I = 125 mM. The inset shows light scattering (trace 1, kobs = 60 s-1) and pyrene fluorescence (trace 2, kobs = 66 s-1) traces recorded at 40 µM ATP (I = 35 mM). B, time courses of ATP hydrolysis on mixing 1.4 µM mX-S1 and 9 µM actin with 50 µM (solid symbols) and 10 µM (open symbols) [-32P]ATP in the quenched-flow apparatus. Data were fitted to a single exponential burst with a linear steady-state phase. The kobs values of the burst phase were 98 s-1 (50 µM ATP) and 14 s-1 (10 µM ATP), whereas the burst amplitudes were 0.33 µM (corresponding to 0.24 mol of Pi/mol of mX-S1) in 50 µM ATP and 0.35 µM (0.25 mol Pi/mol mX-S1) in 10 µM ATP. The slope of the linear steady-state phase was fairly uncertain, but its value was in the range expected based on the steady-state ATPase measurements shown in Fig. 3 (0.2–0.5 s-1). Error bars in the graph indicate S.D. for n = 4. C, actin binding of mX-S1 in the presence of ATP and/or ADP as monitored by acto-mX-S1 cosedimentation. 2 µM mX-S1 was mixed with the indicated actin concentrations and 1 mM ATP (solid circles, I = 125 mM; open circles, I = 45 mM), a mixture of 1 mM ATP and 100 µM ADP (open squares, I = 125 mM), or 200 µM ADP alone (solid squares, I = 125 mM). After ultracentrifugation, the amount of mX-S1 heavy chain in the supernatants (S) and pellets (P) was analyzed by densitometry of SDS-polyacrylamide bands. Error bars represent S.D. for n = 2. The inset shows mX-S1 heavy chain (2 µM) SDS-polyacrylamide bands of representative samples at I = 125 mM (S = supernatant, P = pellet). Row 1, control in the absence of actin; row 2,20 µM actin and 1 mM ATP; row 3, 20 µM actin, 1 mM ATP, and 100 µM ADP; row 4, 20 µM actin and 1 mM ADP. To remove potential ADP contamination, 200 units/ml pyruvate kinase and 1 mM phosphoenolpyruvate were added to the samples in which only ATP and no ADP was included. Conditions were as in Fig. 1 except that in A and C ionic strengths were as indicated.

View larger version (9K): [in this window] [in a new window]   SCHEME 3. Abbreviations used are as follows: A, actin; M, myosin; T, ATP; D, ADP; P, phosphate. Strong binding acto-mX-S1 states are underlined.

We assessed the fractional binding of mX-S1 to actin during steady-state ATP hydrolysis by SDS-PAGE analysis following sedimentation of actin-bound mX-S1 by ultracentrifugation (Fig. 5C). mX-S1 showed notably high steady-state actin attachment even at moderate actin concentrations and in the absence of any ADP "background" (50% attachment was attained at around 8 and 13 µM actin at ionic strengths of 45 and 125 mM, respectively, see Fig. 5C). Inclusion of ADP in the steady-state mixture (at an ATP:ADP ratio of 10:1) caused a further increase in actin attachment (Fig. 5C). As expected from the transient kinetics of mX-S1·ADP binding to actin (K_DA = 1–2 µM; see Fig. 4C and Table IV), practically all mX-S1 was bound to actin even at low actin concentrations in the presence of 200 µM ADP when ATP was not present (Fig. 5C).

Actin Activation of Phosphate Release—We investigated the kinetics of P_i release from acto-mX-S1 by double-mixing single turnover stopped-flow experiments in which mX-S1 was first mixed with substoichiometric amounts of ATP (typically, 2 µM mX-S1 and 1 µM ATP were present after the first mix), was incubated for 1–3 s for ATP binding and hydrolysis to occur, and was then mixed with a range of actin concentrations. P_i release was monitored using a fluorescently labeled phosphate-binding protein (MDCC-PBP) (15) present at a concentration of 4 µM in all syringes in all experiments. The ionic strength was reduced to 45 mM in these experiments to obtain measurable (i.e. relatively higher) affinities of the weak binding mX-S1 states to actin (K_T and K_DP in Scheme 3). The transients recorded after the second rapid mix were single exponential at low actin concentrations, but at higher [actin] two separate phases were observed (Fig. 6A). The k_obs of the slow phase saturated around 1 s^-1, whereas the fast phase k_obs increased with increasing actin concentration to >20 s^-1 at 50 µM actin, showing signs of saturation (Fig. 6A). Similarly to the work of White et al. (28) on skeletal muscle myosin II S1, we interpret this biphasic behavior as a result of the following processes. After the first mix, an equilibrium mixture of the pre- and post-hydrolysis MT and MDP states forms (Scheme 1). Upon mixing with actin, the fast phase of the MDCC-PBP fluorescence transients arises from actin binding of MDP (K_DP; see Scheme 3) followed by the release of P_i from the AMDP quaternary complex (k₄'). The k_obs of this phase will be defined as k₄' [actin]/(K_DP + [actin]). The slow phase splits off at higher actin concentrations as a result of the presence of an alternative pathway in which actin-attached ATP hydrolysis (k₃' + k_-3' 1 s^-1; see Scheme 3) limits the rate of the oncoming phosphate release and thus determines the k_obs of the slow phase of the transients. The fractional amplitudes of the fast and slow phases (30% fast phase, Fig. 6B, inset) remained more or less constant throughout the examined actin concentration range and are in line with the actin-detached ATP hydrolysis equilibrium constant (K₃ = 0.3 ± 0.1; see Table I). Lowering the ATP concentration to 0.5 µM after the first mix did not affect the k_obs values and fractional amplitudes (data not shown), demonstrating that the slow phase is not a result of binding of a second ATP molecule to mX-S1 because of an accidental molar excess of ATP over mX-S1 (e.g. because of a possible error in protein concentration).

View larger version (18K): [in this window] [in a new window]   FIG. 6. Phosphate release from acto-mX-S1. A, MDCC-PBP fluorescence transients recorded in double-mixing stopped-flow experiments at I = 45 mM. 2.3 µM mX-S1 was first mixed with 1.1 µM ATP (concentrations after the first mix), incubated for 1 s for ATP binding and hydrolysis to occur, and then rapidly mixed with increasing concentrations of actin (actin concentrations after the second mix are indicated; data acquisition started at the second mix). At 8 µM actin, a single exponential transient was observed with a kobs of 1.4 s-1 (trace 1). At 50 µM actin (trace 2), the transient was double exponential with kobs values of 25 s-1 (35% fractional amplitude) and 1.0 s-1 (65%). Traces were normalized to a total amplitude of 1. B, dependence of the kobs values of the fast (solid symbols) and slow (open symbols) phases on actin concentration in Pi release experiments described in A. A hyperbolic fit to the fast phase kobs values indicated a maximal kobs (k4' in Scheme 3) of 90 ± 30 s-1 with half-saturation at 130 ± 50 µM actin (KDP). A linear fit to the same data set (not shown) yielded an apparent second-order rate constant of actin binding to the mX-S1·ADP·Pi complex of 0.56 ± 0.03 µM-1s-1 (k4'/KDP). C, observed rate constants (kobs) of single exponential Pi release transients recorded in double-mixing stopped-flow experiments similar to those of A but at I = 125 mM. A linear fit to the dependence of kobs on actin concentration yielded 0.019 ± 0.002 µM-1s-1 for k4'/KDP. The y intercept of the fit was 0.03 ± 0.01 s-1, in line with the basal mX-S1 ATPase activity (Table I). The inset shows an MDCC-PBP fluorescence trace recorded at 5 µM actin that had a kobs of 0.13 s-1. 4 µM MDCC-PBP was present in all syringes. Conditions were as in Fig. 1, except that ionic strengths were as indicated. Error bars indicate S.D. for n = 3.

In a multiple turnover, single-mixing stopped-flow experiment in which 20 µM actin and 1 µM mX-S1 was mixed with 50 µM ATP at I = 125 mM, a linear P_i release time profile was observed with no exponential burst phase (data not shown). This behavior indicates that the net rate of P_i release (influenced by all of the parameters k₃' + k_-3', K₃, K_T, K_DP, and k₄', see Scheme 3) is rate-limiting in the steady-state ATPase cycle in these conditions, in line with the results of the double-mixing experiments discussed above.

ADP Interaction of Acto-mX-S1—ADP binding to and release from pyrene-acto-mX-S1 did not result in a change in pyrene fluorescence, similarly to other myosins (data not shown). Therefore, we monitored the ADP interaction kinetics of acto-mX-S1 by utilizing the inhibitory effect of ADP on the ATP-induced dissociation of the pyrene-acto-mX-S1 complex. In a set of experiments shown in the main panel of Fig. 7A, pre-mixtures of 0.2 µM pyrene-actin, 0.25 µM mX-S1, and various ADP concentrations were rapidly mixed with 300 µM ATP in the stopped-flow apparatus (pre-mixing concentrations stated), and the resulting dissociation of mX-S1 from pyrene-actin was monitored by recording pyrene fluorescence increase transients. As a function of [ADP] in the pre-mixture, the fitted single exponential k_obs values of the transients rapidly decreased from 96 s^-1 in the absence of ADP to about 20 s^-1 at 1 µM ADP, and k_obs further converged to zero with increasing ADP concentration (Fig. 7A). The reduction of k_obs with increasing [ADP] can be deduced from two main causes. First, the rate constant of ADP dissociation from acto-mX-S1·ADP may be lower than that of the ATP-induced dissociation of pyrene-acto-mX-S1 at the ATP concentration used (k_AD <96 s^-1), and thus limit the rate of the reaction as the acto-mX-S1·ADP ternary complex becomes predominant at high [ADP] in the pre-mixture (Scheme 4 depicts the kinetic steps involved in this process). Second, with increasing [ADP], the transient rebinding of ADP instead of ATP to pyrene-acto-mX-S1 may inhibit the ATP-induced acto-S1 dissociation reaction (even if k_AD >96 s^-1). Theoretically, if the ADP release from acto-S1 is slower than ATP binding, then double exponential transients are to be expected with the fast phase arising from acto-S1 dissociation caused by ATP binding to ADP-free acto-S1 ([ATP]k₂'/K₁' = 96 s^-1), and the slow phase representing acto-S1 dissociation through the AMD AM AMT pathway where the first ADP dissociation step (k_AD) is rate-limiting (Scheme 4). The two phases, however, may not be resolvable because of the relatively low experimental signal-to-noise ratio due to the small pyrene fluorescence change (see Fig. 4 and the trace shown in Fig. 7B, inset). Therefore, further experiments were necessary to obtain the ADP dissociation and association rate constants (k_AD and k_-AD). The inset of Fig. 7A shows the k_obs values of single exponential fits to the pyrene fluorescence transients recorded on rapidly mixing 0.15 µM pyrene-actin plus 0.2 µM mX-S1 with a nucleotide mixture consisting of 200 µM ATP plus the indicated ADP concentrations (pre-mixing concentrations indicated). As discussed in a recent detailed investigation of the ADP interaction of pyrene-actomyosin VI (29), double exponential transients would be expected in this reaction, but the two phases were not resolved in our experiments. Nevertheless, the initial (zero [ADP]) k_obs decreased to half at 30 µM ADP (Fig. 7A, inset).

View larger version (21K): [in this window] [in a new window]   FIG. 7. ADP kinetics of acto-mX-S1. A, observed rate constants (kobs) of pyrene fluorescence increase transients recorded on mixing a pre-mixture of 0.25 µM mX-S1, 0.2 µM pyrene-actin, and the indicated ADP concentrations with 300 µM ATP in the stopped-flow. The inset shows kobs values of pyrene fluorescence transients recorded on rapid mixing of 0.2 µM mX-S1 plus 0.15 µM pyrene-actin with a mixture of 200 µM ATP and the indicated ADP concentrations. Note the breaks in the x axes of both panels. A and B, solid symbols show the fitted rate constants of the experimental traces, and open symbols indicate the rate constants resulting from kinetic simulations based on the model described in the text. B, observed rate constants (kobs) of pyrene fluorescence transients on rapid mixing of a pre-mixture of 0.2 µM mX-S1, 0.15 µM pyrene-actin, and 50 µM ADP with the indicated ATP concentrations in the stopped-flow. A hyperbolic fit to the data set yielded a maximal kobs (= kAD, Schemes 3 and 4) of 17.6 s-1. The inset shows a trace obtained at 200 µM ATP that had a kobs of 9.8 s-1. C, dependence of the observed rate constants (kobs) of mant-ATP fluorescence transients recorded on rapidly mixing a pre-mixture of 1 µM mX-S1, 12 µM actin, and 30 µM ADP with increasing mant-ATP concentrations in the stopped-flow. Data were fitted to a hyperbola to obtain a maximal kobs (= kAD) of 18.8 s-1. The inset shows a transient recorded at 100 µM mant-ATP (kobs = 15 s-1). Conditions were as in Fig. 1. Pre-mixing concentrations are stated throughout this figure. All traces were fitted to single exponentials (see text).

View larger version (13K): [in this window] [in a new window]   SCHEME 4. Abbreviations used are as follows: A, actin; M, myosin; T, ATP; D, ADP.

We also applied another method for the determination of the k_DA rate constant. In these experiments a pre-mixture of 1 µM mX-S1, 12 µM actin, and 30 µM ADP was rapidly mixed with increasing concentrations of mant-ATP in the stopped flow, and the binding of mant-ATP to acto-mX-S1 was monitored by the increase in mant-ATP fluorescence (Fig. 7C). Similarly to the experiments of Fig. 7B, the ADP dissociation rate constant from acto-mX-S1 (k_AD) will limit the k_obs of the process at high mant-ATP concentrations (cf. Scheme 4). The fitted maximal k_obs in these experiments was 18.8 s^-1 (Fig. 7C), in agreement with the results of Fig. 7B.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Steady-state Distribution of Acto-mX-S1 ATPase Intermediates—We used the determined biochemical parameters to establish a kinetic model of the entire acto-mX-S1 ATPase cycle that can serve as a basis for calculations of functionally important steady-state parameters and predictions of the motile behavior of this myosin. A chief component of the actin activation of the mX-S1 ATPase is a robust (700-fold) acceleration of phosphate release from the mX-S1·ADP·P_i products complex by actin (k₄ = 0.13 s^-1, k₄' 90 s^-1; see Schemes 1 and 3 and Tables I and IV). However, both the rate constant of P_i release from acto-mX-S1 (k₄') and the ADP release rate constant (k_AD = 18 ± 1 s^-1) are markedly greater than the maximal steady-state ATPase activity of acto-mX-S1 (4–5 s^-1; see Table II). Other steps such as ATP binding to acto-mX-S1, dissociation of acto-mX-S1, or actin-detached ATP hydrolysis are also too fast to act as a single rate-limiting step of the enzymatic cycle. The contributions of multiple processes to steady-state rate limitation can therefore be determined only by global numerical analysis of the enzymatic cycle.

A large majority of the elementary rate constants used in the numerical analysis could be directly extracted from the experimental data. Large uncertainties remain in only two parameters as follows: the equilibrium constant of the actin-attached ATP hydrolysis step (K₃' in Scheme 3), and the actin affinity of the mX-S1·ATP complex (K_T). We could determine both K₃ and K_DP with reasonable accuracy, and therefore the values for K₃' and K_T must be interdependent to maintain thermodynamic consistency (K₃'/K_T = K₃/K_DP). The actin concentration dependences of the steady-state acto-mX-S1 ATPase activity (Fig. 3) and actin attachment ratio (Fig. 5C) were used as experimental steady-state data for parameter optimization of K₃' and K_T. (Note that the total actin attachment ratio is the sum of the fractional abundance of all actin-attached steady-state intermediates (AM, AMT, AMDP, and AMD in Scheme 3) and thus it is different from the duty ratio, which is the sum of the fractional abundance of only the strongly actin-bound states (AM and AMD).) Data related to an ionic strength of 45 mm were considered for the analysis because K_DP was determined at this ionic strength, and the actin affinity of the weak actin-binding myosin states (K_T and K_DP) are generally the parameters having a marked dependence on solution ionic strength. Simulations in which K_T was set to the same value as K_DP (=160 µM) and K₃' was thus defined as 0.3 (= K₃ K_T/K_DP) yielded a steady-state ATPase activity having a plateau (V_max) around 4.5 s^-1 with a half-saturation at 30 µM actin (K_ATPase). These values correspond reasonably well with the experimental data (a V_max of 3–5 s^-1 and a K_ATPase of 15–30 µM is expected (Table II)). However, the simulated steady-state actin attachment ratio was much lower than the experimental one (50% attachment was reached at 65 µM actin in the simulation as opposed to the experimental 10–15 µM (Fig. 5C)). Therefore, we next lowered K_T 10-fold (to 13 µM), accompanied by a 10-fold reduction in K₃' (to 0.03). These parameters resulted in a steady-state ATPase that rapidly saturated with increasing actin concentration (K_ATPase = 7 µM), but its maximal value (V_max = 1.3 s^-1) was well below the experimental one. A model with intermediate values, in which K_T was set to 43 µM and K₃' to 0.1, showed reasonably good agreement with the experimental data with regard to all three parameters (the model yielded a V_max of 3.5 s^-1 and a K_ATPase of 15 µM, with half-saturation of actin attachment occurring at 25 µM actin, see Fig. 8).

View larger version (21K): [in this window] [in a new window]   FIG. 8. Simulated duty ratio, actin attachment, and steady-state acto-mX-S1 ATPase activity. Results of computational kinetic simulations of the dependence of steady-state properties of mX-S1 on actin concentration are shown. The experimentally determined kinetic constants (Tables I and IV) were fed into a kinetic model of the entire mX-S1/acto-mX-S1 ATPase cycle according to Schemes 1 and 3. Additional parameters used are as follows: KT = 43 µM (rapid equilibrium); KDP = 130 µM (rapid equilibrium); k3 = 30 s-1; k-3 = 100 s-1; k3' = 0.1 s-1; k-3' = 1 s-1; k4' = 90 s-1 (irreversible step); [ATP] = 1 mM; and [ADP] = 0. The duty ratio (i.e. the sum of the fractional abundance of the strongly actin-bound states (AM and AMD in Scheme 3), shown as triangles, left y axis) acquired a maximal value of 0.16 at around 100 µM actin. The fractional actin attachment (i.e. the sum of the fractional abundance of all (weak and strong) actin-attached states, circles, left y axis) converged to 1 and showed half-saturation around 25 µM actin. The steady-state ATPase activity (squares, right y axis) exhibited a maximal value around 3 s-1 at 100 µM actin (with half-saturation around 15 µM actin) and then declined to 1 s-1 at high actin concentrations.