Twitchin can regulate the ATPase cycle of actomyosin in a phosphorylation-dependent manner in skinned mammalian skeletal muscle fibres

Archives of Biochemistry and Biophysics 521 (2012) 1–9

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Twitchin can regulate the ATPase cycle of actomyosin in a phosphorylation-dependent manner in skinned mammalian skeletal muscle fibres Stanislava V. Avrova a, Nikita A. Rysev a, Oleg S. Matusovsky b, Nikolay S. Shelud’ko b, Yurii S. Borovikov a,⇑ a b

Laboratory of Mechanisms of Cell Motility, Institute of Cytology, Russian Academy of Sciences, 4 Tikhoretsky Avenue, St. Petersburg 194064, Russia Laboratory of Cell Biophysics, Institute of Marine Biology, Far East Branch of the Russian Academy of Sciences, Vladivostok 680068, Russia

a r t i c l e

i n f o

Article history: Received 9 February 2012 and in revised form 27 February 2012 Available online 10 March 2012 Keywords: Mollusc catch muscle Glycerol-skinned muscle fibres Twitchin ATPase cycle Polarized fluorescence

a b s t r a c t The effect of twitchin, a thick filament protein of molluscan muscles, on the actin–myosin interaction at several mimicked sequential steps of the ATPase cycle was investigated using the polarized fluorescence of 1.5-IAEDANS bound to myosin heads, FITC-phalloidin attached to actin and acrylodan bound to twitchin in the glycerol-skinned skeletal muscle fibres of mammalian. The phosphorylation-dependent multistep changes in mobility and spatial arrangement of myosin SH1 helix, actin subunit and twitchin during the ATPase cycle have been revealed. It was shown that nonphosphorylated twitchin inhibited the movements of SH1 helix of the myosin heads and actin subunits and decreased the affinity of myosin to actin by freezing the position and mobility of twitchin in the muscle fibres. The phosphorylation of twitchin reverses this effect by changing the spatial arrangement and mobility of the actin-binding portions of twitchin. In this case, enhanced movements of SH1 helix of the myosin heads and actin subunits are observed. The data imply a novel property of twitchin incorporated into organized contractile system: its ability to regulate the ATPase cycle in a phosphorylation-dependent fashion by changing the affinity and spatial arrangement of the actin-binding portions of twitchin. Ó 2012 Elsevier Inc. All rights reserved.

Introduction Muscle contraction results from the ATP-powered cyclic interaction between myosin heads on the thick filaments and actin subunits in the thin filaments, causing the thick and thin filaments to slide past one another [1]. Biochemical and mechanical studies have determined that ATP hydrolysis results in force production due to the changes in the molecular structure of the actomyosin motor components. It is widely accepted that actomyosin complex can alternate between two groups of states: the weakbinding non-force-generating states and the strong-binding forcegenerating states [2]. The weak-binding states of actomyosin include actomyosinATP (state AM⁄ATP) and actomyosinADPPi, (state AM⁄⁄ADPPi); these states are characterized by the fast kinetics of attachment/detachment of myosin to actin and a lack of cooperative activation of the regulated actin filaments [3]. The strong-binding states are found when myosin binds to actin in the absence of nucleotide (state AM) or in the presence of MgADP (state AM^ADP); these states are characterized by a high myosin affinity to actin, slow kinetics of myosin attachment/detachment from actin and the ability of myosin heads to activate regulated thin filaments cooperatively [4].

⇑ Corresponding author. Fax: +7 812 297 0341. E-mail address: [email protected] (Y.S. Borovikov). 0003-9861/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.abb.2012.03.004

Contraction is regulated by molecular switches on the thin filaments or on both the thin and thick filaments [5]. The actin-linked regulation in skeletal muscle occurs due to the movement of regulatory proteins of the tropomyosin–troponin (TM–TN)1 complex located on actin filaments in response to the change in Ca2+ concentration. It is suggested that TM strands block a specific myosin-binding site on the actin filament in low-Ca2+ resulting in the inhibition of the actin–myosin interaction [6,7]. The binding of Ca2+ to TN eliminates this inhibition causing an azimuthal movement of TM strands, thus allowing myosin to interact freely with actin [6]. Smooth muscles of vertebrates and molluscan muscles have a dual Ca2+-regulation. In these muscles, the primary myosin-based regulatory system involves the phosphorylation of myosin regulatory light chains in the presence of Ca2+ or direct Ca2+ binding to myosin neck [8]. It is believed that the secondary actin-based system of regulation involves caldesmon, TM and calmodulin [9,10]. Some molluscan muscles have the ability to maintain muscle tension for extended periods of time, even upon cessation of stimulation. This phenomenon is called ‘‘catch state’’ (for review, see [11–14]). Induction and release of the ‘‘catch state’’ involve 1 Abbreviations used: S1, myosin subfragment-1; TM, tropomyosin; TN, troponin complex; IAEDANS, N-iodoacetyl-N’-(5-sulfo-1-naphtylo)ethylenediamine; FITC-Ph, fluorescein phalloidin; DTT, dithiothreitol; PKA, cAMP-dependent protein kinase; EGTA, ethylene glycol-bis (2-aminoethylether)-N,N,N’,N’-tetraacetic acid.

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Ca2+-dependent phosphatase [15] and cAMP-dependent protein kinase [16], respectively. The target for cAMP-dependent protein kinase is twitchin [17], a thick filament protein belonging to the titin-like giant proteins family. The phosphorylation of twitchin increases the relaxation rate in the accessory radula closer muscle of the mollusc Aplysia californica [17] and releases the ‘‘catch state’’ in skinned fibres from smooth muscles of the bivalve mollusc Mytilus edulis [18,19]. On the other hand, twitchin has the ability to interact with F-actin and this interaction is regulated by twitchin phosphorylation in the same way as twitchin phosphorylation regulates the ‘‘catch’’ of the muscle [20]. Considering this, the existence of regulated passive twitchin bridges between the thin and thick filaments that maintain the catch tension was postulated [11,20–22]. According to this hypothesis, a transient increase in the intracellular Ca2+-level under cholinergic stimulation of molluscan catch muscle activates the thin and thick filaments and twitchin phosphatase. As a result, two processes must start in parallel: the active muscle contraction and the formation of twitchin links between the thick and thin filaments. However, at the initial stage of active contraction twitchin links do not prohibit filament sliding [22]. This observation was explained by the assumption that twitchin and the myosin heads compete for binding to actin, and it allowed the strong-binding myosin heads to displace twitchin from actin even though twitchin was nonphosphorylated [22–24]. Further assumption was that the mechanism of interdependent influence of the active and passive bridges on one another involves the regulating effect of thin filaments on the actin–myosin interaction [25]. Recently, we have obtained some evidence in support of the latter assumption [26,27]. We used a model system of reconstructed ghost muscle fibres to study the effect of twitchin on the actin–myosin interaction at various intermediate stages of the ATP hydrolysis cycle and showed that the addition of nonphosphorylated twitchin to the ghost fibres ‘‘freezed’’ actin and tropomyosin in the position typical of relaxation of muscle fibres, and the myosin heads in the weak-binding states. The addition of PKA catalytic subunit reverses the effect of twitchin. These data suggest that twitchin can control the actin–myosin interaction during the ATPase cycle in a phosphorylation-dependent manner by changing the conformational state of actomyosin, which results in the inhibition of the transformation of the weak-binding states into the strong-binding ones. In this work, we used a different model system: skinned skeletal muscle fibres contained incorporated molluscan twitchin. The use of skinned fibres from a skeletal muscle is based on the ability of mussel twitchin interacts specifically not only with molluscan contractile proteins but also with actin and myosin from rabbit skeletal muscle [24,25]. We have shown that nonphosphorylated acrylodan–twitchin incorporated in skinned skeletal muscle fibres has an ordered orientation in fibres and can inhibit the movements of myosin heads and actin subunits, and decreases the affinity of the myosin heads for actin as well as in the ghost fibres [27], where twitchin does not form linkages. The phosphorylation of twitchin reverses its effects on the actin–myosin interactions and changes in the position and mobility of twitchin.

Materials and methods Preparation and labeling of proteins and muscle fibres Single glycerol-skinned skeletal muscle fibres were isolated from the bundles dissected from rabbit psoas muscles and glycerinated using the method of Rome [29]. The single fibres were washed out using a solution containing 67 mM phosphate buffer (pH 7.0), 100 mM KCl, 3 mM MgCl2 to remove glycerol. Then myosin and actin in the fibres were labeled with fluorescent

probes. Modification of myosin with 1,5-IAEDANS was carried out as described by Borejdo and Putnam [30] and followed by preferential labeling with Cys707 (belonging to SH1 helix). The probe to protein molar ratio was about 0.8:1. The fibres are referred to as being AEDANS-labeled because the iodide atom of 1,5-IAEDANS is lost during the covalent modification of sulfhydryl side chains. FITC-phalloidin was covalently bound to F-actin of the fibres by their incubation in a solution containing 6.7 mM phosphate buffer (pH 7.0), 50 mM KCl, 3 mM MgCl2 (solution A) and 40 lM FITCphalloidin for 2,5 h at room temperature [31]. Cystein residues of twitchin were labeled with acrylodan (Molecular Probes); the probe to protein molar ratio was about 0.5:1. Mussel twitchin, skeletal actin and myosin were isolated from the posterior adductor of the mussel Crenomytilus grayanus and rabbit skeletal muscles, respectively, as described earlier [20,25]. The labeled twitchin retained the ability to inhibit the MgATPase activity of actomyosin. Twitchin was incorporated into the fibres by incubation in a solution containing 20 mM MOPS (pH 7.0), 30 mM KCl, 3 mM MgCl2, 2 mM DDT, 1 mM NaN3, and twitchin (2–3 mg/ml) for 2 h at room temperature [28]. The molar ratio of twitchin to actin in the glycerol-skinned fibres was 1:10 (±2), as shown by SDS-electrophoresis [32] with subsequent densitometry of the gels (UltroScan XL, Pharmacia LKB). In some experiments, we used myosin-free ghost fibres prepared as described earlier [33]. The incorporated twitchin was phosphorylated by incubating the fibres in a solution containing PKA catalytic subunit as previously described [28]. The MgATPase activity of synthetic actomyosin was determined in the medium containing (mM): 30 KCl, 2 MgCl2, 0.5 DTT, 10 imidazole–HCl (pH 6.8), and 0.1 CaCl2. The reaction was started by adding MgATP to 0.4 mM, and terminated after 5–10 min by adding trichloroacetic acid to 5 mM. Liberated inorganic phosphate was determined colorimetrically. Fluorescence polarization measurement Steady-state fluorescence polarization measurements on single glycerol-skinned muscle fibres were made using a flow-through chamber and photometer [34]. The polarized fluorescence from AEDANS-labeled myosin and acrylodan-labeled twitchin, FITC-phalloidin-labeled actin were excited at 407 ± 5 nm and 489 ± 5 nm, respectively, and recorded at 500–600 nm. Probes were excited by a 250 W DRSH-250 mercury lamp source of light which passed through a quartz lens and a double monochromator, and split into two polarized beams by a polarizing prism. The ordinary polarized beam was reflected by the dichroic mirror and condensed by a quartz objective (UV 58/0.80) on the fibre in the chamber at the microscope stage. The emitted light from the fibre was collected by the objective and led to a concave mirror with a small hole. After passing through the lens and barrier filter, the beam was split by a Wollaston prism into two polarized beams perpendicular and parallel to the fibre axis. The measurements were carried out in solution A containing 0.5 mM PMSF, 0.1 mM DDT, 0.1 mM NaN3, 4 mM EGTA in the absence or presence of 2.5 mM ADP, 15 mM AMP-PNP, 5 mM ATPcS or 5 mM ATP. The concentration of MgCl2 was 3 mM in the absence and presence of ADP. In the presence of ATPcS, ATP and AMP-PNP, the concentration of MgCl2 was 8 and 18 mM, respectively [35]. The solution containing ATP also contained 1 mg/ml creatine phosphokinase (100–200 units/mg activity) [36]. All the solutions were prepared at room temperature (20 °C). The absence of nucleotides modeled the AM state of the actomyosin complex. MgADP, MgAMP-PNP, MgATPcS, and MgATP were used for mimicking intermediate states of actomyosin, AM^ADP, AM’ADP, AM⁄⁄ADPPi and AM⁄ATP respectively [37,38], where A is actin and M, M^, M’, M⁄ and M⁄⁄ are various conformational states of the myosin head.

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The intensities of the four components of polarized fluorescence and \Ik were measured. The subscripts k and \ designate the orientation of polarization plane parallel and perpendicular to the fibre axis, the former denoting the direction of polarization of the incident and the latter that of the emitted light. Fluorescence polarization ratios were defined as: Pk = (kIk  kI\)/ (kIk + kI\) and P\ = (\I\  \Ik)/(\I\ + \Ik). The fluorescence polarization ratios can be used qualitative assessment of the orientational distribution of the fluorescence probe with respect to the muscle fibre axis as follows: if P\ < Pk, then the average angle of the probes with respect to the muscle fibre axis is less than the ‘‘magic angle’’ (54.7°), typical of random orientation; if P\ > Pk, then the average angle of the probes with respect to the muscle fibre axis is greater than 54.7°; if Pk = P\, then the probes are either oriented at 54.7° with respect to the muscle fibre axis or isotropically disordered [36]. For a quantitatively assessment of the changes in the probe orientation, we used the ‘‘helix plus isotropic model’’ [39] that fits the data assuming two populations of probes: the population of helically ordered oscillators of absorption and emission probe at angles /A and /E to the axis of the muscle fibre, respectively, and the population (N) that is completely disordered (isotropic; the angle orientation probes was close to 54.7°). The angle between the axis of the oscillators of absorption and emission (c) was close to 20° (for AEDANS-S1 and FITC-phalloidin-actin) and 14° (for acrylodan–twitchin) [34]. The thin filament is flexible and can deviate from the fibre axis by the maximal angle h1/2 [40,41]. For this model, the changes in UA, UE, and N of 1,5-IAEDANS, UA, UE, and h1/2 of FITC-phalloidin are considered to reflect the alterations in orientation and mobility of the fluorophore-containing sites in the muscle fibres, showing the changes in the tilt angle of myosin SH1 helix (or the myosin head [30]) and actin subunits [42], respectively. h1/2 reflects the thin filament flexibility [40–42]. Since a probe upon its attachment to a protein molecule can become available for a solvent as well as affected by adjacent amino acid residues, the orientation and mobility of absorption and emission dipoles of the probe may be also sensitive to a change in its local environment. The information about such changes can be obtained by analyzing the fluorescent spectrum of the probes. In our work, we measured the position of the fluorescence spectrum maximum in all the experiments with an accuracy of 0.3 nm, and did not find any reliable shifts of the spectrum of the proteins modified by 1.5-IAEDANS, FITC-phalloidine and acrylodan. Based on these data was the suggestion that the changes in polarized fluorescence registered in our experiments reflected mainly the changes in orientation and mobility of the absorption and emission dipoles of the probes. In all experiments the pattern of /E changes was similar to that of /A changes, therefore only /E, h1/2 and N values were presented in the figures. The statistical reliability of the changes was evaluated using Student’s t-test. kIk, kI\, \I\

Results and discussion To study the effect of twitchin on conformational changes in F-actin and myosin motor at the sequential steps of the actomyosin ATPase cycle, we used glycerol-skinned skeletal muscle fibre of mammalian as a highly organized model system. Myosin and actin within such fibres were alternatively labeled with fluorescent dyes: 1,5-IAEDANS was cross-linked to Cys707 (SH1) of myosin [30], and FITC-phalloidin was bound to F-actin in the region of contact of three adjacent actin subunits [43]. Twitchin was incorporated into the fibres. In some experiments we used acrylodan–labeled twitchin. The polarized fluorescence measurements were carried out in the presence and absence of twitchin at various simulated intermediate stages of the ATPase cycle. The AM state of the actomyosin

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complex was simulated in the absence of nucleotides. MgADP, MgAMP-PNP, MgATPcS, and MgATP were used to mimic the AM^ADP, AM’ADP, AM⁄⁄ADPPi and AM⁄ATP states, respectively [37,38]. The nucleotides and twitchin altered the polarized fluorescence parameters of AEDANS–myosin and FITC-phalloidin–actin. The labeling of Cys707 with fluorescent probes can inhibit the ATPase activity of myosin (see example [36]). However, as it was shown in the experiments performed in the laboratory of Dr. F. Morales more than 30 years ago, the modification of Cys707 by 1.5-IAEDANS does not have any conspicuous effect on the ability of myosin to change their conformation. The muscle fibres containing myosin heads modified by dyes are able to generate force [44]. It is interesting to note that it was by using fluorescent probes that the cyclic work of the cross-bridges during muscle contraction was shown for the first time and the correlation between the conformational changes in the myosin heads and the force developed at muscle contraction revealed [45]. The effect of labeled myosin on the functional properties of this protein essentially depends on the probe species, the method of modification, the dye used and the firm where the dye has been synthesized. We used 1.5-IAEDANS, obtained from Molecular Probes and applied the method developed by Borejdo and Putnam [30]. These data suggest that the modification of Cys707 by 1.5-IAEDANS does not significantly change the functional property of myosin. Also, the modification by fluorescent probes seems to have no significant effect on the functional properties of actin in muscle fibres [46,47], therefore the observed changes in the conformational state of the labeled proteins were likely to reflect those occurring during muscle contraction (for a overview see [48,49]). It should be emphasized that in our steady-state experiments polarized fluorescence of the studied protein reflects the average structural state of the protein molecules population as a whole. The effect of twitchin on the conformational state of actin during the ATPase cycle It has been previously shown that the weak- and strong-binding of myosin heads to FITC-phalloidin labeled actin is accompanied by different changes in the orientation and mobility of excitation and emission dipoles that can be attributed to different modes of interaction between actin and myosin (for a review, see [48,49]). In agreement with earlier findings [46–50], the muscle fibres containing FITC-phalloidin-labeled actin filaments had a high positive Pk value and a low positive P\ value (Table 1), which indicated that FITC-phalloidin dipoles were predominantly oriented parallel to the fibre axis with an average angle smaller than the ‘‘magic angle’’ of 54.7°. When the helix plus isotropic model (see Materials and methods) was fitted to the fluorescence polarization of FITC-actin in the absence of nucleotides, the values of the angles of the emission dipole /E and the angles of h1/2 were 46.5° and 8.0°, respectively. The addition of nucleotides to the fibres devoid of twitchin resulted in statistically significant (p < 0.05) changes in these parameters. In the presence of MgADP (state AM^ADP), these values increased. In contrast, at mimicking the AM’ADP, AM⁄⁄ADPPi and AM⁄ATP states, the values of /E and h1/2 decreased (Fig. 1). Since FITC-phalloidin is located in the groove of the thin filament and is specifically bound to three adjacent actin subunits [43], it may be suggested that the changes in the values of /E and h1/2 of FITC-phalloidin reflect the conformational states of F-actin followed by the axial rotation of the actin subunits and the thin filament flexibility [49–51], respectively. While it is believed that at the strong-binding of myosin F-actin is in the ONconformational state and at the weak-binding in the OFF-state (for a review, see [52]), our results indicate that at transformation from AM⁄⁄ADPPi to AM states the switching on of actin subunits

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Table 1 The effect of the nucleotides, S1 or/and nonphosphorylated (PKA) or phosphorylated (+PKA) twitchin (TW) on polarization ratios of FITC-phalloidine bound to F-actin in the skinned muscle fibers. Nucleotide

TW

PKA

n

Pk ± SEM

P\ ± SEM

None

 + +  + +  + +  + +  + +

±  +   +   +   +   +

10 7 5 7 6 6 7 5 5 7 6 5 7 6 5

0.381 ± 0.002 0.400 ± 0.002 0.390 ± 0.002 0.365 ± 0.002 0.406 ± 0.002 0.323 ± 0.002 0.388 ± 0.002 0.421 ± 0.002 0.410 ± 0.002 0.399 ± 0.002 0.426 ± 0.002 0.414 ± 0.002 0.411 ± 0.002 0.419 ± 0.002 0.408 ± 0.002

0.123 ± 0.002 0.091 ± 0.002 0.126 ± 0.002 0.171 ± 0.002 0.038 ± 0.002 0.127 ± 0.002 0.022 ± 0.003 0.007 ± 0.002 0.085 ± 0.002 0.018 ± 0.003 0.015 ± 0.003 0.029 ± 0.003 0.061 ± 0.003 0.052 ± 0.003 0.051 ± 0.003

ADP

AMP-PNP

ATPcS

ATP

Pk and P\ were calculated as described in Materials and methods. n is the number of fibres used in the experiments. TW and the nucleotides have a pronounced effect on the values of Pk and P\, indicating the changes in the conformational state of F-actin in the skinned muscle fibres (p < 0.05).

leads to an axial rotation of the actin monomers to the muscle fibre periphery (Fig. 1A). It should be noted that each intermediate state of myosin induces a definite conformational state and a specific position of actin subunits in the thin filament [51]. Thus, in the presence of MgADP, the values of /E were remarkably higher than in the absence of nucleotides. This means that the ON-state of actin in the actin–myosin complex, is different from the ON-state in the actin–myosin–MgADP complex (Fig. 1). MgANP-PNP, MgATPS or MgATP decrease /E and h1/2 (Fig. 1), showing that actin subunits tilt to the thin filament center, which is indicative of the weak-binding of myosin to actin [34]. The rotation of actin subunits at transition from AM⁄⁄ADP-Pi to AM^ADP state was followed by the maximal axial tilting of actin towards the muscle fibre axis periphery (Fig. 1A), suggesting that at this transition force generation by the actomyosin motor seemed to occur [53]. The binding of nonphosphorylated twitchin to F-actin in the muscle fibres changed the parameters of polarized fluorescence of FITC-phalloidin in the absence or presence of nucleotides. In the absence or presence of nucleotides, such twitchin decreases /E (Fig. 1A), which is the evidence of rotation of the actin subunits

to the muscle fibre center. Interestingly, the values of /E at mimicking AM, AM^ADP and AM’ADP states in the presence of nonphoshorylated twitchin were close to those in the absence of twitchin at mimicking of AM’ADP, AM⁄⁄ADPPi and AM⁄ATP states, respectively, i.e. close to the weaker-binding states. It is known that skeletal and cardiac muscles contain a giant protein titin, which spans half of the sarcomere length in parallel with the actin and myosin filaments and anchors the myosin thick filament to the Z-line. The I-band region of titin contains a large PEVK domain. Since titin filaments are in close proximity to the thin filaments, it was hypothesized that titin PEVK domain may interact with actin filaments. Co-sedimentation assays revealed interaction between actin filaments and cardiac titin and its fragments, but detected no interaction between actin and the skeletal PEVK titin [54–57]. The absence of interaction was further confirmed by a number of mechanical experiments [58,59]. It was suggested that titin was responsible for the passive tension of muscle fibres that acted as an entropic spring [60]. Therefore, there is no reason to suppose that titin in the skeletal fibres might markedly bind to the actin filaments. This means that there is no binding of titin to actin in the glycerol-skinned fibres, or the effect of titin both on the actin–myosin interaction and the ability of twitchin to modulate this interaction is so insignificant that it cannot be revealed by the method used. It was previously found that the binding of nonphoshorylated twitchin to actin resulted in a decreased the number of unordered fluorescent dipoles (AEDANS), specifically bound to Cys-374 of actin subdomain-1. The thin filaments containing this twitchin practically lost their ability to respond with changes in subdomain-1 orientation; thus, they lost their ability to respond with conformational changes to the binding of the myosin heads [27]. The results were interpreted as the ‘‘freezing’’ of actin in the OFF-conformation. In another work such ‘‘freezing’’ of actin structure served as an explanation of the decrease in the angle between the thin filament axis and the fibre axis (h1/2) induced by troponin at lowCa2+ [40]. Therefore, the decrease in h1/2 in the presence of nonphoshorylated twitchin that was observed in our work (Fig. 1), can be interpreted in terms of the ‘‘freezing’’ of actin structure in the OFFstate. In our work, we also observed that nonphoshorylated twitchin decreased the amplitude of the axial rotation of the actin subunits by 34% (Fig. 1A). The decrease in the axial rotation of the actin subunits correlates with the inhibition of actin’s capability to activate

Fig. 1. The effect of twitchin (TW) on the values of UE (A) and h1/2 (B) of the polarized fluorescence of FITC-phallodin–actin, revealed in glycerol-skinned fibres during the stimulation of the sequential steps of the ATPase cycle. Calculations of UE and h1/2 values, preparation of the fibres, their composition, and the conditions of the experiments are described in Materials and methods. The data represent the mean values of 5–10 fibres for each experimental condition (see Table 1). The UE and h1/2 values in the absence and in the presence of nucleotides are significantly altered by twitchin (p < 0.05). () Asterisks indicate unreliable differences in the values of N and /E in the absence and in the presence of PKA. Error bars indicate ±SEM. The values of N were close to 0.2.

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the ATPase of actomyosin reconstituted from the skeletal F-actin and myosin (Fig. 2) as well as in the case of actomyosin reconstituted from the mussel thin filaments and skeletal myosin [25]. It is possible that our data demonstrate the so-called negative allosteric effect of nonphosphorylated twitchin on actin-activated ATPase. A similar effect was observed earlier for troponin at lowCa2+ [61] and caldesmon [50,51]. Phosphorylation of incorporated twitchin by adding PKA catalytic subunit to the glycerol-skinned muscle fibres markedly changes the polarized parameters UE and h1/2 (Fig. 1). In contrast, no effect of PKA on these parameters in the absence of twitchin was observed. This means that there is no detectable effect of PKA catalytic subunits on other proteins in the glycerol-skinned muscle fibres, except for twitchin. According to Fig. 1, phosphorylated twitchin shifts the conformational states of actin subunits towards ON-states (the stronger-binding states) during the ATPase cycle, because the values of /E and h1/2 at mimicking AM^ADP, AM’ADP and AM⁄⁄ADPPi states in the presence of phosphorylated twitchin were close to those in the presence of nonphoshorylated twitchin at mimicking AM, AM^ADP and AM’ADP states, respectively, i.e. close to the stronger-binding states (Fig. 1). The maximal effect of phosphorylation of twitchin on the proportion of strong-binding sub-states in the actin population can be expected at mimicking AM and AM^ADP states, where the generation of force by the actomyosin motor has been postulated [53]. The amplitude of change in UE at transition from AM⁄⁄ADP-Pi to AM^ADP state was essentially higher for phosphorylated twitchin than for nonphosphorylated or for the experiments in the absence of twitchin (Fig. 1A). The increase in the axial rotation of actin subunits correlates with actin’s capability to activate ATPase of actomyosin reconstituted from skeletal F-actin and myosin (Fig. 2). It seems that phosphorylation of twitchin induces a positive allosteric effect on the ability of actin to activate the ATPase. A similar effect was observed earlier for troponin at high-Ca2+ [61]. These data are in a good agreement with the data obtained in the experiments employing AEDANS-labeled myosin (Fig. 3). The effect of nucleotides and twitchin on the conformational state of myosin during the ATPase cycle Polarized fluorescence measurements in the glycerol-skinned muscle fibres containing 1.5-IAEDANS-labeled myosin revealed

Fig. 2. The effect of phosphorylated, TW(+P), and nonphosphorylated, TW(P), twitchin on MgATPase activity of synthetic skeletal actomyosin. Conditions: 30 mM KCl, 0.5 mM MgCl2, 1 mM NaN3, 2 mM DTT,10 mM imidazole–HCl (pH 7.2). Rabbit myosin, 0.2 lmole; rabbit F-actin, 2.4 lmole; skeletal tropomyosin, 0.05 mg/ml; mussel twitchin, 0–0.4 lmole. The ATPase activity was measured in the presence of 0.3 mM MgATP at 25 °C for 10 min. Representative curves of three experiments with different protein preparations are shown.

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that Pk values were notably higher than P\ values (Table 2). These findings indicate that the emission dipoles of this probe were predominantly oriented parallel to the fibre axis [30]. When the helix plus isotropic model (see Materials and methods) was fitted to the fluorescence polarization of AEDANS–myosin in the absence of nucleotides, the angles of /E as well as the fraction N of disordered probes indicated a well-ordered orientational distribution (Fig. 3). This condition mimics the strong-binding state of myosin to actin which is close to the AM state of actomyosin [37], and the emission dipole of 1.5-IAEDANS is oriented at an angle close to 48.9°, i.e. practically parallel to the bulk of the myosin heads. The nucleotides and twitchin had a distinct effect on the parameters (Pk, P\, /E and N) of the polarized fluorescence of AEDANS–myosin (Table 2 and Fig. 3), which can be interpreted in terms of a change in the affinity of myosin to actin and the tilt angle of the SH1 helix of the myosin motor domain (or the myosin head) relative to the fibre axis (for a review, see [48,49]). In this work, the polarized fluorescence of AEDANS–myosin was studied at several simulated stages of the ATP cycle. As seen in Fig. 3, both UE and N values are much higher at the stages when actin and myosin are bound weakly (AM⁄⁄ADPPi and AM⁄ATP), than when they are bound strongly (AM^ADP, and AM). As the probe is rigidly linked to the Cys707, an increase in the value of /E can be interpreted as a tilting of the myosin SH1 helix (or the myosin heads) towards the periphery of the thin filament. Similarly, a decrease in /E reflects the movement of the SH1 helix (or the myosin heads) in the opposite direction. A decrease in the values of N for AEDANS–myosin can be interpreted as an increase in the affinity of myosin to actin. Likewise, an increase in N can be seen as an indication of a decrease in myosin’s affinity for actin [61]. Basing on the above interpretation, it is possible to suggest that the conformational changes in myosin, occurring at transition from AM⁄ATP to AM state (Fig. 3), induce a multi-step tilting of the myosin SH1 helix (or the myosin heads) towards the thin filament and an increase in myosin’s affinity for actin. In the widely accepted model of molecular mechanism of actomyosin motility described by Geeves and Holmes [53], the authors suggested that the main structural change was the closing of a large cleft between the upper 50 kDa domain, spanning the ATPbinding site, and the lower 50 kDa domain, containing the actinbinding sites, leading to a distortion of the orientation of the three outer b-strands (b1, b2 and b3) of the upper 50 Da domain. In addition, the movement of these b-strands was tightly coupled to that of the SH1/SH2 helices and to subsequent transduction of these changes to the myosin ‘‘lever arm’’ [62–64] and to actin subunits [46,47,50]. Our data indicated that the closing of a large cleft between the upper and lower 50 kDa domains induced a multistage change in the conformation of myosin, which demonstrated a multi-step tilting of the myosin SH1 helix (or the myosin heads) towards the thin filament and an increase in the strong-binding of the myosin heads to actin. The incorporation of nonphosphorylated twitchin in the fibres induced concerted conformational changes, which decreased the proportion of strong-binding sub-states in the myosin population at mimicking the intermediate states of the ATPase cycle. In the presence of twitchin, the values of UE and N were increased in the strong-binding state (stage AM^ADP and AM) and faintly decreased in the weak-binding state (AM⁄ATP) (Fig. 3). The values of /E and N at mimicking AM and AM^ADP states in the presence of twitchin were close to those in the absence of this protein at mimicking AM’ADP state, indicating a shift in the conformational states of the myosin heads towards the weaker-binding states. As each mimicked state is most probably not uniform and consists of several different sub-states of myosin [65], the changes in the actomyosin conformational state may not necessarily be due to the formation of new sub-states but to the alterations in the

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Fig. 3. The effect of twitchin (TW) on the values of UE (A) and N (B) of the polarized fluorescence of AEDANS–myosin, revealed in glycerol-skinned fibres during the stimulation of the sequential steps of the ATPase cycle, where UE is the angle between the emission dipole of the probe and the thin filament axis; and N is the number of disorderly oriented fluorophores. Calculations of the UE and N values, preparation of the fibres, their composition, and the conditions of the experiments are described in Materials and methods. The data represent the mean values of 8–10 fibres for each experimental condition (see Table 2). The UE and N values in the absence or in the presence of nucleotides are significantly altered by twitchin (p < 0.05). () Asterisks indicate unreliable differences in the values of N and /E in the absence and in the presence of PKA. Error bars indicate ±SEM. The values of h1/2 were close to those shown in Fig. 1.

Table 2 The effect of the nucleotides and nonphosphorylated (PKA) or phosphorylated (+PKA) twitchin (TW) on polarization ratios of 1.5-IAEDANS bound to myosin in the skinned muscle fibers. Nucleotide

TW

PKA

n

Pk ± SEM

P\ ± SEM

None

 + +  + +  + +  + +  + +

±  +   +   +   +   +

11 7 5 7 6 5 7 5 5 7 6 5 6 5 5

0.350 ± 0.002 0.331 ± 0.002 0.355 ± 0.002 0.344 ± 0.002 0.342 ± 0.002 0.350 ± 0.002 0.325 ± 0.002 0.324 ± 0.002 0.327 ± 0.002 0.239 ± 0.002 0.237 ± 0.002 0.239 ± 0.002 0.263 ± 0.002 0.265 ± 0.002 0.261 ± 0.002

0.051 ± 0.002 0.058 ± 0.002 0.050 ± 0.002 0.033 ± 0.002 0.043 ± 0.002 0.033 ± 0.002 0.070 ± 0.003 0.080 ± 0.002 0.088 ± 0.002 0.211 ± 0.003 0.208 ± 0.003 0.211 ± 0.003 0.222 ± 0.003 0.214 ± 0.003 0.221 ± 0.003

ADP

AMP-PNP

ATPS

ATP

Pk and P\ were calculated as described in Materials and methods. n is the number of fibres used in the experiments. TW and the nucleotides have a pronounced effect on the values of Pk and P\, indicating the changes in the conformational state of myosin in the skinned muscle fibres (p < 0.05).

existing populations of sub-states [61]. Therefore it is possible that nonphosphorylated twitchin increases the proportion of ‘‘weakbinding’’ sub-states during the ATPase cycle, showing that the majority of the myosin heads lose their ability to form the strong-binding states. Similar enhancement of the proportion of ‘‘weak-binding’’ sub-states during the ATPase cycle was observed earlier for troponin at low Ca2+ [61] and caldesmon [51]. Nonphosphorylated twitchin also decreases the amplitude of the tilting of the myosin SH1 helix (Fig. 3a), showing that such twitchin inhibits the efficiency of the work of cross-bridges. These effects are in line with those observed in the complex of skeletal F-actin–myosin after adding twitchin (Fig. 2). As indicated in Fig. 2, nonphosphorylated twitchin heavily inhibited the MgATPase activity of this complex. The addition of PKA reverses the effect of nonphosphorylated twitchin-binding (Fig. 3). The value of UE is decreased at strongbinding (stages AM, AM^ADP, AM’ADP) and increased at mimicking AM⁄⁄ADPPi and AM’ATP weak-binding states; the value of N

is decreased at strong-binding states (stage AM and AM^ADP) and shows an increase at weak-binding (stages AM’ADP, AM⁄⁄ADPPi and AM⁄ATP). The maximal effect of phosphorylated twitchin on the proportion of strong-binding sub-states in myosin population can be expected at mimicking AM^ADP state (Fig. 3), where the generation of force by the actomyosin motor has been postulated [53]. This means that phosphorylation of twitchin induces conformational changes in actomyosin, which can enhance the MgATPase activity (Figs. 1 and 2). The amplitude of change in UE at transition from AM⁄⁄ADP-Pi to AM^ADP state was higher for phosphorylated twitchin than for nonphosphorylated one by 71% (Fig. 3), indicating the enhancement of the efficiency of the work of cross-bridges in the presence of PKA. A similar enhancement of the amplitude of change in UE at transition from AM⁄⁄ADP-Pi to AM^ADP state was observed earlier for troponin at high-Ca2+ [61]. It is possible that twitchin in the same way as troponin can regulate the ATPase cycle by converting the weak-binding actomyosin states to the strong-binding ones. The modulation of the actin–myosin interaction seems to be realized by the changes in the conformation of twitchin. Such a suggestion is confirmed by the data obtained using the glycerolskinned fibres containing acrylodan-labeled twitchin (Fig. 4). The effect of nucleotides and PKA on the conformational state of twitchin during the ATPase cycle It is known that twitchin is composed of a single polypeptide of 530 kDa containing multiple repeats of immunoglobulin (Ig) and fibronectin type III motifs [11,24]. It is believed that the main body of twitchin is located on the thick filaments while its terminus, one [24,25] or both [23], promote a cross-links between the thick and thin filaments in a phosphorylation-dependent manner. Since both immunoglobulin and fibronectin motifs have cysteine residues [11], which can be labeled with a fluorescent dye. (Materials and methods), the polarized fluorescence from acrylodan–twitchin can be used to examine the conformational states of this protein in the glycerol-skinned muscle fibres during the ATPase cycle. The incorporation of acrylodan–twitchin to the glycerolskinned muscle fibres initiates polarized fluorescence, with Pk values being lower than P\ values (Table 3). In contrast, no anisotropy of polarized fluorescence (Pk = P\) was observed when acrylodan– twitchin was added to myosin-free ghost fibres. It means that only

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Fig. 4. The effect of PKA catalytic subunit on the values of UE and N of the polarized fluorescence of acrylodan–twitchin revealed in the glycerol-skinned fibers during the stimulation of the sequential steps of the ATPase cycle. The conditions of the measurements and designations are as in Fig. 3. The data represent the mean values of 5–6 fibers for each experimental condition (see Table 3). The UE and N values in the absence or in the presence of nucleotides are significantly altered by PKA catalytic subunit (p < 0.05). () Asterisks indicate unreliable differences in the values of N and /E in the absence and in the presence of PKA. Error bars indicate ±SEM. The values of h1/2 were close to those shown in Fig. 1.

Table 3 The effect of nucleotides and PKA on polarization ratios of acrylodan bound to twitchin (TW) in the glycerol-skinned muscle fibers. Nucleotide

PKA

n

Pk ± SEM

P\ ± SEM

None

 +  +  +  +  +

5 7 5 7 5 7 5 7 4 5

0.291 ± 0.002 0.284 ± 0.002 0.296 ± 0.002 0.302 ± 0.002 0.296 ± 0.002 0.300 ± 0.002 0.297 ± 0.002 0.295 ± 0.002 0.301 ± 0.002 0.318 ± 0.002

0.371 ± 0.002 0.432 ± 0.002 0.345 ± 0.002 0.371 ± 0.002 0.334 ± 0.003 0.345 ± 0.002 0.318 ± 0.003 0.320 ± 0.003 0.316 ± 0.003 0.282 ± 0.003

ADP AMP-PNP ATPS ATP

The nucleotides and PKA have a pronounced effect on the values of Pk and P\, indicating the changes in the conformational state of twitchin (p < 0.05). Designations are as in the legend to Table 1.

in the presence of the thick filaments the dipoles of acrylodan localized in twitchin were ordered primary perpendicular to the fibre axis. The angle of the emission dipole of acrylodan /E was found to be close to 55° (Fig. 4a), which also indicates an ordered orientation of acrylodan–twitchin in the glycerol-skinned fibres. This is in line with our finding that incorporated twitchin can induce a catch-like stiffness in skinned skeletal muscle fibres, which indicates the formation of the linkages between the thick and thin filaments [28]. The relative quantity of disorderly oriented fluorophores was something like 0.4–0.5 (Fig. 4b), that reflects high elasticity or flexibility of twitchin molecule. Nucleotides and PKA had a prominent effect on the parameters of polarized fluorescence (Pk, P\, /E b N) of acrylodan–twitchin incorporated in glycerol-skinned fibres (Table 3 and Fig. 4). According to Fig. 4, the transition from AM⁄ ATP to AM state was accompanied by a feebly marked multi-step increase in the values of UE (by 1.7 ± 0.1°) and a small decrease in the values of N (by 0.022 rel. units) for nonphosphorylated twitchin. This means that the transition was accompanied by changes in the conformation of this protein that are visualized by only a weak rotation of the oscillator emission towards the periphery of the muscle fibre and a small decrease in the mobility probe during the ATPase cycle. The addition of PKA dramatically changes this effect (Fig. 4). In this case, the value of UE markedly increases during the formation the strong-binding actomyosin states (AM and AM^ADP) and visibly

decreases in the weak-binding state (AM⁄ATP). At the same time, the value of N distinctly increases at transition from AM⁄ ATP to AM state (by 0.148 rel. units). As acrylodan molecules specifically and rigidly bind to cysteine residues of this protein, a change in the values of /E and N can be caused by a change in spatial organization and mobility of the twitchin molecule or a part of it. Since the main body of twitchin is located on the thick filaments and is immovable while the terminal portion(s) of molecule can attach and detach to actin, it is possible that the actin-binding part(s) of twitchin alter their position and mobility in the muscle fibres in a phosphorylation-dependent manner during the ATPase cycle. An increase in /E and N can be interpreted as a detachment or deflection of the actin-binding parts of twitchin from the thin filament body and an enhancement in the mobility or a decrease in the affinity of twitchin-binding to actin, respectively. In the same way, a decrease in the values of /E and N can be seen as a movement of the actin-binding parts of twitchin towards the thin filament body (as attachment) and a decrease in the mobility or an increase in the affinity of these parts for actin, respectively. According to this interpretation, the transition from AM⁄ ATP to AM state (Fig. 4) induces in nonphosphorylated twitchin very small changes in attachment of the actin-binding parts to the thin filament body and a small increase in the affinity of twitchin for actin (Fig. 4). In contrast, in phosphorylated twitchin, the ATPase cycle is accompanied by very marked changes in attachment of the actin-binding parts from the thin filament body and in the affinity of these terminal portions for actin. It is possible that the changes in the values of /E and N during the ATPase cycle can result from myosin-induced detachment or deflection of the actin-binding part(s) of twitchin from the thin filaments body and a decrease in its affinity for actin, respectively. As the value of N was low (close to 0.5) and the changes in the parameters of /E and N were insignificant for nonphosphorylated twitchin (Fig. 4) it is most likely that the actin-binding part(s) is strongly bound to actin in all intermediate states of the ATPase cycle. The influence of myosin and nucleotides on the position and affinity of the actin-binding part(s) markedly increased when twitchin was phosphorylated by PKA (Fig. 4). In this case, the actinbinding part(s) of twitchin bound weakly to actin or detached from the thin filaments at the strong-binding of the myosin heads to actin. On the contrary, at the weak-binding of myosin to actin, the actin-binding part(s) reattached to the thin filament or bound strongly to actin.

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It is known that the modulation of the actin–myosin interaction during the ATP hydrolysis cycle can be realized by the shifting or ‘‘rolling’’ [66] of the tropomyosin strands on the thin filament surface [67]. Recently, we have found that nonphosphorylated twitchin shifts tropomyosin towards the blocked position and restricts tropomyosin movements during the ATPase cycle. On the contrary, phosphorylated twitchin shifts TM strands to the center of the filament (to the open position) and enhances tropomyosin movements during the ATPase cycle [26]. Since each intermediate state of actomyosin corresponds to a definite conformational state and position of TM on the thin filament [61], it is possible to suggest that the motion of TM strands towards the blocked position can be a reason for the observed inhibition of the formation of strong-binding states, whereas the movement towards the open position can activate this formation. It is possible that the changes in the position and affinity of the actin-binding portions of twitchin can control the actin–myosin interaction during ATPase cycle by shifting or ‘‘rolling’’ tropomyosin strands, which can account for the transformation of the strong-binding actomyosin states into the weak-binding ones. To sum up, the application of the glycerol-skinned skeletal muscle fibres makes it possible to study the effect of twitchin on the conformational changes in actin subunits and myosin SH1 helix at different stages of the ATPase cycle. This technique allows the identification of the distinct equilibrium of the structural states presented at different intermediate stages of the ATPase cycle. Both the data presented here (Figs. 1, 3 and 4) and the data obtained by us earlier [26,27] provide an evidence that twitchin by changing its affinity for actin and its positioning on the thin filament can modify the structural state of the actomyosin motor during the ATPase cycle. Such a modification including the binding of nucleotides to the myosin head and the alteration of the structural state of actin due to the binding of twitchin to actin disturbs the equilibrium state of the ensemble of these proteins in a phosphorylationdependent manner thereby inducing a transition to another equilibrium state. The data obtained indicate that in the course of the ATP hydrolysis the intermediate states of the protein ensemble differ in mobility and spatial arrangement of myosin SH1 helix, actin subunits and the actin-binding portions of twitchin. Because twitchin interacts specifically not only with molluscan contractile proteins but also with actin and myosin from mammalian skeletal muscle [24,25], and this protein incorporated in skinned skeletal muscle fibres can induce a catch-like stiffness [28] we suggested that the ability of twitchin to regulate the ATPase cycle through its conformational changes in a phosphorylation-dependent manner can be also realized in molluscan contractile system. Acknowledgments We are grateful to Dr. Olga E. Karpicheva for the technical assistance provided in the course of a series of experiments. This work was supported by grants 11-04-00244a (to Y.B.) and 10-04-00550a (to O.M.) from the Russian Foundation for Basic Research, the Program 7 of the Presidium of the Russian Academy of Sciences (to Y.B.), and by a grant from the MCB program of the RAS (to N.S.). References [1] H.E. Huxley, Cold Spring Harbor Symp. Quant. Biol. 37 (1972) 361–376. [2] L.A. Stein, P.B. Chock, E. Eisenberg, Proc. Natl. Acad. Sci. U. S. A. 78 (1981) 1346–1350. [3] J.M. Chalovich, L.E. Greene, E. Eisenberg, Proc. Natl. Acad. Sci. U. S. A. 80 (1983) 4909–4913. [4] L.E. Greene, E. Eisenberg, Proc. Natl. Acad. Sci. U. S. A. 77 (1980) 2616–2620. [5] W. Lehman, A.G. Szent-Gyorgyi, J. Gen. Physiol. 66 (1975) 1–30. [6] W. Lehman, R. Craig, P. Vibert, Nature 368 (1994) 65–67. [7] W. Lehman, V. Hatch, V. Korman, M. Rosol, L. Thomas, R. Maytum, M.A. Geeves, J.E. Van Eyk, L.S. Tobacman, R. Craig, J. Mol. Biol. 302 (2000) 593–606.

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