Báo cáo khoa học: Dynamic reorganization of the motor domain of myosin subfragment 1 in different nucleotide states docx

Eur J Biochem 270, 4835–4845 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03883.x Dynamic reorganization of the motor domain of myosin subfragment in different nucleotide states } ´ ´ ´ Emoke Bodis1, Krisztina Szarka2, Miklos Nyitrai2 and Bela Somogyi1,2 Department of Biophysics, Faculty of Medicine, University of Pe´cs, Hungary; 2Research Group for Fluorescence Spectroscopy, Office for Academy Research Groups Attached to Universities and Other Institutions, Department of Biophysics, Faculty of Medicine, University of Pe´cs, Hungary Atomic models of the myosin motor domain with different bound nucleotides have revealed the open and closed conformations of the switch element [Geeves, M.A & Holmes, K.C (1999) Annu Rev Biochem 68, 687–728] The two conformations are in dynamic equilibrium, which is controlled by the bound nucleotide In the present work we attempted to characterize the flexibility of the motor domain in the open and closed conformations in rabbit skeletal myosin subfragment Three residues (Ser181, Lys553 and Cys707) were labelled with fluorophores and the probes identified three fluorescence resonance energy transfer pairs – The effect of ADP, ADP.BeFx, ADP.AlF4 and ADP.Vi on the conformation of the motor domain was shown by applying temperature-dependent fluorescence resonance energy transfer methods The 50 kDa lower domain was found to maintain substantial rigidity in both the open and closed conformations to provide the structural basis of the interaction of myosin with actin The flexibility of the 50 kDa upper domain was high in the open conformation and further increased in the closed conformation The converter region of subfragment became more rigid during the open-to-closed transition, the conformational change of which can provide the mechanical basis of the energy transduction from the nucleotide-binding pocket to the lightchain-binding domain The mechanisms underlying the contraction of muscle involve the cyclic interaction of actin with myosin The binding and hydrolysis of ATP by the myosin induces a series of conformational changes within the motor domain of myosin, which lead to the sliding of the thick and thin filaments relative to each other Some of the intermediate states of ATP hydrolysis are short-lived and thus stable structural analogues are required to study these states [1–3] Recently, the structures of the recombinant truncated Dictyostelium discoideum myosin subfragment (S1) in the apo-state [4], or with ATP [4], ADP, ADP.BeFx [5], – ADP.AlF4 [5] or ADP.Vi [6], were shown to provide an excellent structural framework for using to understand the mechanism of muscle contraction According to these D discoideum structures, S1.ADP.BeFx resembles the – S1.ATP conformation, whereas S1.ADP.AlF4 and S1 ADP.Vi resemble the S1.ADP.Pi conformation On the other hand, the smooth muscle myosin S1 atomic structures – with ADP.BeFx and ADP.AlF4 were almost identical [7] Analysis of these atomic models revealed that a key structural part of the nucleotide induced conformational changes in the core of the motor domain is the switch (SWII) element, which consists of the SWII helix (residues 475–509) and the SWII loop (residues 511–520) The SWII element can be in an open or closed conformation in the individual states of the ATPase cycle [8] The two conformations are in a dynamic equilibrium, which is controlled by the bound nucleotide The open state is dominant in the pre- and postpower-stroke states, such as the apo-enzyme or S1 with bound ATP or ADP, or in the nucleotide states mimicked by b-c-imidoadenosine 5¢-triphosphate or ATPcS [8] The closed conformation was attributed to the transition state and was observed with bound ADP.Pi analogues, – ADP.Vi or ADP.AlF4 In the ADP.BeFx bound motor domain, both the open and closed conformation could be detected [5,7] During the open-to-closed transition, the SWII element moves towards the c-phosphate [8] This transition step can be followed by the hydrolysis of ATP and the closure of the active site through the relative rotation of the 50 kDa upper domain and the 50 kDa lower domain The helix consisting of residues 648–666 is in the fulcrum of this rotation In conjunction with this transition, the converter domain rotates by 60°, which induces the movement of the C–terminal end of S1 by 12 nm [9] Tryptophan fluorescence has proved to be a powerful experimental tool when used to characterize the different aspects of myosin interaction with nucleotides [10–13] Rapid kinetic experiments using tryptophan fluorescence indicated Correspondence to B Somogyi, Department of Biophysics, ´ ´ University of Pecs, Faculty of Medicine, Pecs, Szigeti Str 12, H-7624, Hungary Fax: + 36 72 536261, Tel.: + 36 72 536260, E-mail: somogyi.publish@aok.pte.hu Abbreviations: ANN, 9-anthroylnitrile; FHS, 6-(fluorescein-5-carboxamido)-hexanoic acid succinimidyl ester; FRET, fluorescence resonance energy transfer; IAEDANS, N-[[(iodoacetyl)amino]ethyl]5-naphthylamine-1-sulfonate; IAF, 5-(iodoacetamido)-fluorescein; S1, myosin subfragment (Received 13 August 2003, revised 10 October 2003, accepted 21 October 2003) Keywords: protein dynamics and conformation; myosin; muscle; nucleotides; fluorescence resonance energy transfer Ó FEBS 2003 ´ 4836 E Bodis et al (Eur J Biochem 270) that the delicately poised equilibrium between the closed and open conformations was influenced by temperature changes in a nucleotide dependent manner [14–16] The apo-form and ADP bound form of either a single tryptophan D discoideum myosin II motor domain construct [15] or skeletal muscle myosin S1 [16] were predominantly in the – open conformation, while the ADP.AlF4 bound forms were predominantly in the closed conformation over the 4–30 °C temperature range The open/closed equilibrium was shifted towards the closed conformation by increased temperature when the motor domain bound ADP.BeFx [15,16] In the work presented here we attempted to characterize the protein flexibility of the open and closed motor domain conformations By applying temperature-dependent fluorescence resonance energy transfer (FRET) methods [17,18], we investigated how the dynamic properties of the rabbit skeletal S1 motor domain adapted to the biological function in different nucleotide states We labelled three residues of S1 with suitable fluorophores, as follows: in the first case Ser181 was labelled with 9-anthroylnitrile (ANN) and Lys553 was labelled with 6-(fluorescein-5-carboxamido)hexanoic acid succinimidyl ester (FHS); in the second case Cys707 (SH1) was labelled with N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonate (IAEDANS) and Lys553 was labelled with FHS; and in the third case Ser181 was labelled with ANN and Cys707 (SH1) was labelled with 5-(iodoacetamido)-fluorescein (IAF) The – effects of ADP, ADP.BeFx, ADP.AlF4 and ADP.Vi on the flexibility of the motor domain were characterized The results suggest that the 50 kDa lower domain of S1 maintains substantial rigidity in both open and closed conformations, which may be important for the optimal interaction with actin The upper 50 kDa domain was flexible in all nucleotide states, which may be important for providing the permeability of the back door of the myosin for surrounding water or for the dissociating phosphate product The binding of ADP or ADP.BeFx to apo-S1, which is thought to be an open conformation, had little effect on the overall flexibility of the motor domain The flexibility of the motor domain was different in the – S1.ADP.AlF4 state from either apo-S1 or S1.ADP.Vi states The largest reorganization of the domains was observed in S1.ADP.Vi The observed changes suggest that in the closed conformation the flexibility of the 50 kDa upper domain is further increased The relative internal fluctuation of the 50 kDa upper domain and actin binding domain was suppressed, which reflected the stiffening of the converter region between the nucleotide-binding site and the lightchain-binding domain The transition to this rigid structure may be part of the mechanism by which the energy from ATP hydrolysis is transferred to the lever arm Materials and methods Reagents Tes, Mops, Tris, Na2HPO4, MgCl2, CaCl2, NaCl, KCl, NaOH, glycine-ethyl-esther, a-chymotrypsin, trypsin, phenylmethanesulfonyl fluoride, EDTA, EGTA, 2-mercaptoethanol, dimethylformamide, dithiothreitol, IAEDANS, NaF, AlCl3, Na3VO4, NADH, pyruvate kinase, lactate dehydrogenase and phosphoenol pyruvic acid were obtained from Sigma Chemical Co.; ADP and ATP were obtained from Merck; ANN, FHS and IAF were purchased from Molecular Probes; BeSO4 was purchased from Fluka; N,N,N¢,N¢-tetramethylethyliendiamine (TEMED) and the Coomassie Protein Micro-Assay were purchased from BioRad; and SDS was from US Biochemical Protein preparations and modifications Both myosin and actin were prepared from rabbit skeletal muscle according to the methods described by Margossian & Lowey [19] and Spudich & Watt [20], respectively S1 was prepared by a-chymotrypic digestion of myosin [21] The labelling of S1 with ANN [22], IAEDANS [23], FHS [24] or IAF [23] was performed according to previously published procedures The concentrations of S1 and G-actin were determined from absorption data using the extinction coefficient of A1%1cm ¼ 7.45 at 280 nm [25] and A1%1cm ¼ 6.30 at 290 nm [26], respectively The concentrations of ANN, IAEDANS, FHS and IAF were determined at pH 7.0 using the absorption coefficients of 8400 M)1Ỉcm)1 at 361 nm [22], 6100 M)1Ỉcm)1 at 336 nm [23], 68 000 M)1Ỉcm)1 at 495 nm [24] and 55 000 M)1Ỉcm)1 at 496 nm (determined for pH 7.0 based upon the work of Takashi [27]), respectively The labelling ratio was calculated as the ratio of the dye concentration to protein concentration When S1 was labelled with fluorophores, the absorbance measured for determining the protein concentration at 280 nm was corrected for the contribution of the labels using A280 ¼ A361 for the bound ANN; A280 ¼ 0.21A336 for the bound IAEDANS; A280 ¼ 0.3A495 for the bound FHS; and A280 ¼ 0.3A496 for the bound IAF Relying on the absorption data, the labelling ratios of different samples were found to be 0.4–1.0, 0.6–0.9, 0.8–1.0 and 0.7–1.0 mol probe per mol S1 for ANN, IAEDANS, FHS and IAF, respectively – The complexes of S1 and phosphate analogues, as AlF4 and BeFx, were formed by incubating S1 with 0.2 mM ADP, mM NaF and either 0.2 mM AlCl3 or BeSO4 [28] The complex of S1, ADP and the VO4 anion was formed by incubating S1 with 0.2 mM ADP and 0.2 mM VO4 [29], and is referred to hereafter as S1.ADP.Vi Previously, nucleotide analogues were used successfully to study S1 labelled on Ser181 [30], Lys553 [31] or Cys707 [23,32] In this work, in order to provide optimal conditions for the formation of S1–analogue complexes, the ADP and the analogues were not removed from the samples during the experiments Labelled S1 was routinely characterized by determining the K+/EDTA- and Ca2+ ATPase activities through measuring the release of phosphate [33] The assays were performed at room temperature in 50 mM Tris/HCl, pH 8.0, 0.6 M KCl, 2.5 mM ATP and either 10 mM EDTA or mM CaCl2 The ATPase activities measured simultaneously for unlabelled S1 served as a reference Labelling S1 with either ANN or IAEDANS, at a ratio of 0.4 (ANN–S1) or 0.6 (IAEDANS–S1), modified the Ca2+ ATPase activity to 47% or to 190%, compared with that of the unlabelled protein, and decreased the K+/EDTA ATPase activity to 53% and 44%, respectively These observations are in agreement with previous results [22,23] Subsequent labelling of ANN–S1 with FHS or IAF modified the Ca2+ Ó FEBS 2003 Dynamic properties of the myosin motor domain (Eur J Biochem 270) 4837 ATPase activity to 64% and 20%, respectively, while the K+/EDTA ATPase activity of these samples decreased to 14% or 15% of that of the unlabelled protein, respectively The modification of IAEDANS–S1 with FHS increased the Ca2+ ATPase activity to 119% and decreased the K+/EDTA ATPase activity to 19% compared with that of the unlabelled protein, respectively To characterize the biological activity of the labelled S1 samples, the Mg2+ ATPase activities were also measured in the presence or absence of actin (17 lM or 30 lM) by using the coupled enzyme assay [34] The experiments were carried out in 20 mM Mops, pH 7.0, 100 mM KCl, mM MgCl2, 0.5 mM ATP, mM PEP, 0.5 mM EGTA, 0.15 mM NADH, 200 mL)1 pyruvate kinase and 400 mL)1 lactate dehydrogenase The conversion of NADH to NAD+ (molar equivalent to the hydrolysis of ATP) was monitored by measuring the absorbance at 340 nm in a Shimadzu UV-2100 spectrophotometer The S1 concentration was 0.5 lM in the assays The Mg2+ ATPase results are presented in Table and discussed below, in the Results In order to test whether the dyes bound specifically to the desired residues, limited tryptic cleavage of donor and donor–acceptor labelled S1 was performed Labelled S1 in 20 mM Tris (pH 8.0), 50 mM NaCl, was incubated with 0.02 mgỈmL)1 trypsin for 10 at 25 °C [22] The sample was added to solubilizing solution and 20 mgỈmL)1 dithiothreitol in boiling water for to prepare for gel electrophoresis The tryptic digested samples were analysed by SDS/PAGE [35] using 12% acrylamide gels To detect the fluorescent bands, gels were washed with methanol and acetic acid and photographed After photographs had been taken, the gels were stained with Coomassie Blue to allow sizing of the digested fragments by comparison with the molecular mass marker Analysis of SDS/PAGE gels for the products of tryptic digestion of donor or donor–acceptor labelled S1 samples showed that ANN fluorescence appeared only in the 23 kDa peptide, IAEDANS and IAF fluorescence appeared only in the 20 kDa peptide, and FHS fluorescence only in the 50 kDa peptide of S1, confirming that the labelling sites were, as designed, in either the single- or double labelled S1 samples Fluorescence measurements Fluorescence was measured using a Perkin Elmer LS50B luminescence spectrometer The measurements were carried out in buffer comprising 25 mM Tes, pH 7.0, 80 mM KCl, mM MgCl2, mM EGTA and mM 2-mercaptoethanol, and the protein concentration was mgỈmL)1 To calculate the FRET efficiency, the fluorescence intensities of the donor (ANN or IAEDANS) were recorded in the presence and absence of acceptors (FHS or IAF) The excitation monochromator was set to 350 nm, and both the excitation and emission slits were set to nm The corrected fluorescence intensity of ANN and IAEDANS were monitored at 400–470 nm with the optical slits adjusted to nm The contributions of fluorescence by either of the applied acceptor molecules to the measured fluorescence intensity can be excluded over this wavelength range The fluorescence intensities were corrected for inner filter effect The FRET efficiency (Eobs) was calculated as: Eobs ẳ FDA =FD ị 1ị where FDA and FD are the fluorescence integrated intensities (between 400 and 470 nm) of the donor molecule in the presence and in the absence of the acceptors, respectively As the acceptor labelling ratio was less than 1, the calculated FRET efficiency (Eobs) was corrected as: E ẳ Eobs =b 2ị where E and Eobs are the corrected and observed FRET efficiencies, respectively, and b is the actual acceptor/protein molar ratio The distance between the donor and the acceptor (R) was calculated from: E ẳ R6 =R6 ỵ R6 ị o o ð3Þ where Ro is Forster’s critical distance, defined as the donor ă acceptor distance at which the FRET efciency is 0.5 The value of Ro, and the overlap integral required to calculate the donor–acceptor distances, were determined as described previously [36] The normalized FRET efficiency, f¢, was defined as [18]: f ¼ E=FDA ffi hkt i=kf % hRÀ6 j2 i ð4Þ where kt and kf are the rate constants for the energy transfer and donor emission, j2 is the orientation factor, and Ỉ ỉ denotes the average of the given parameter This method [17,18], assumes that the equilibrium distance (ỈRỉ) between the donor and the acceptor does not change with the temperature, while the R distribution becomes wider with the increase in temperature It comes from the nature of the method [18], that the term ÔflexibilityÕ (owing to normalization of the f¢) is not directly related to the width of the donor–acceptor distance distribution Instead, this term is related to how easily the donor–acceptor distance Table The Mg2+ ATPase activity of unlabelled and labelled rabbit skeletal muscle myosin subfragment (S1) in the presence and absence of actin filaments (as given in the left column) The activities were measured using the coupled enzyme assay [34] The labelling ratios of the fluorophores in these samples were as follows: ANN, 0.96; IAEDANS, 0.86; IAF, double labelled IAF–ANN–S1, 1.00; double labelled FHS–ANN–S1, 0.95; and double labelled FHS–IAEDANS–S1, 1.00 All the ATPase data are given in s)1 Probe(s) and labelled residue(s) Actin Unlabelled ANN–Ser181 ANN–Ser181/ IAF–Cys707 FHS–Lys553/ ANN–Ser181 IAEDANS–Cys707 IAEDANS–Cys707/ FHS–Lys553 lM 17 lM 30 lM 0.05 0.25 0.45 0.08 0.20 0.40 0.05 0.15 0.27 0.04 0.09 0.17 0.19 0.36 0.61 0.19 0.27 0.33 Ó FEBS 2003 ´ 4838 E Bodis et al (Eur J Biochem 270) distribution widens as a response to the additional energy represented by the increase in temperature Therefore, the temperature profile of f¢ is characteristic of the flexibility of the protein matrix between the fluorophores, provided that the average orientation of the fluorophores (j2) remains unchanged with the variation of the temperature Note that owing to the )6 power dependence of f¢ on R, the temperature profile of f¢ is dominated by the change of the R distribution, even in the case of a slight variation of Ỉj2ỉ [18] Comparison of the temperature induced changes in different forms of the protein therefore provides information regarding the differences of protein flexibility between the forms Steady-state anisotropy measurements Steady-state anisotropy measurements were carried out in a Perkin Elmer LS50B spectrofluorimeter to characterize the volume within which the fluorophores could wobble The temperature dependence (6–26 °C) of the steady-state anisotropy in the absence of nucleotides was measured The results were analysed using the Perrin equation: 1=r ẳ 1=r0 ẵ1 þ ðkT=VgÞsŠ ð5Þ where r is the steady-state anisotropy, r0 is the limiting anisotropy, k is the Boltzman constant, T is the absolute temperature, V is the volume of the rotating unit, g is the viscosity and s is the lifetime of the fluorophore The apparent limiting anisotropy (r0app) was determined from the y-intercept of linear fits to the 1/r vs T/g plots [36], while the value of V was determined from the slopes Fluorescence lifetime experiments Fluorescence lifetime experiments were carried out using an ISS K2 multifrequency phase fluorimeter, as described previously [37] The excitation wavelength was 350 nm for ANN and IAEDANS, and 495 nm for FHS and IAF The fluorescence emission was monitored through a WG335 (ANN and IAEDANS) or 550FL07-25 (FHS and IAF) optical filter The average fluorescence lifetime was calculated as: X X sav ¼ s2 an = sn a n ð6Þ n where sn is the nth component of the lifetime and an is the amplitude of the nth lifetime Results The aim of this study was to characterize the change of protein flexibility during the nucleotide-induced reorganization of the motor domain of rabbit skeletal S1 Three amino acids in the motor domain were labelled with fluorescent dyes (Fig 1), as follows (a) Ser181, a conservative amino acid of the nucleotide-binding pocket [38,39], was labelled with ANN [22,40,41]; (b) Lys553, in the actinbinding region, was labelled with FHS [24]; and (c) Cys707 (SH1), the cysteine of S1 with the highest reactivity, was labelled with either IAEDANS or IAF [23] The labelled residues determined three FRET donor–acceptor pairs (ANN–FHS, ANN–IAF, and IAEDANS–FHS) along the sides of a triangle, which lay over the protein matrix of the motor domain (Fig 1) By using temperature dependent FRET experiments, we investigated how the flexibility of the protein matrix between these labels depended on the binding of nucleotides and nucleotide – analogues such as ADP, ADP.BeFx, ADP.AlF4 and ADP.Vi We attempted to test the biological activity of the labelled S1 samples by measuring the Mg2+ ATPase activities in the absence of actin and in the presence of 17 lM or 30 lM actin filaments The results are summarized in Table The Mg2+ ATPase activity of the unlabelled S1 was 0.05 s)1, Fig Schematic representation of the motor domain of Dictyostelium discoideum myosin in apo-form The 50 kDa upper domain is labelled in dark blue, the 50 kDa lower domain is labelled in green, and the 25 kDa domain and the truncated 20 kDa domain are labelled in grey The SWII element (residues 466–500) is labelled in red, and the converter domain (residues 693–759) is labelled in light blue In this work, the Ser181, Lys553 and Cys707 residues of rabbit skeletal myosin subfragment (S1) were labelled with fluorophores The corresponding residues (Ser181, Lys546 and Thr688 [1]) are shown in the D discoideum motor domain with yellow surfaces The yellow dashed lines highlight the applied FRET pairs Atomic coordinates were obtained from the Protein Data Bank (accession number 1FMV) Ó FEBS 2003 Dynamic properties of the myosin motor domain (Eur J Biochem 270) 4839 similar to that observed previously [42] For the labelled S1 samples, the basal Mg2+ ATPase activities were similar to or greater, and the actin activation lower, than for the unlabelled S1 The results obtained after the binding of IAEDANS to Cys707, or of FHS to Lys553, were in agreement with previously published observations [24,43,44] The data show that although the binding of fluorescence labels modified the physiological Mg2+ ATPase activity of S1, the fundamental behavior of S1 was preserved The ATPase cycle was similar in the labelled samples to that operating in the unlabelled S1 In view of the fact that, in this study, we stabilized different states of the ATPase cycle in the absence of nucleotides, or by adding ADP or nucleotide analogues, we concluded that the fluorescence experiments reported on the proper characteristics of the individual ATPase cycle states Fluorescence lifetime and anisotropy The temperature dependence of the steady-state anisotropy of the fluorophores in the absence of nucleotides was measured between and 26 °C, and analysed using the Perrin equation (Eqn 5) For the analyses, the fluorescence lifetimes were also measured The average fluorescence lifetime (Eqn 6) of ANN (Ser181), FHS (Lys553), IAEDANS (Cys707) and IAF (Cys707) were 12.0 ns (varied 1.0 ns between °C and 26 °C), 3.9 ns (varied 0.1 ns between °C and 26 °C), 17.8 ns (varied 0.3 ns between °C and 26 °C) and 3.6 ns (varied < 0.1 ns between °C and 26 °C), respectively The temperature dependent anisotropy data were fitted to Eqn (5) by using the above average lifetimes and r0 ¼ 0.4 (data not shown) to obtain estimates for the apparent limiting anisotropy r0app (the intercept of the straight line with the 1/r axis) and the app volume of the rotating unit (V) The values obtained for r0 were 0.36, 0.37, 0.30 and 0.28 for ANN, FHS, IAEDANS ˚ and IAF, respectively The V-values were 2.9 · 104 A3, ˚ ˚ ˚ 1.08 · 104 A3, 5.8 · 104 A3 and 6.4 · 104 A3 for ANN, FHS, IAEDANS and IAF, respectively The donor–acceptor distances The shape of the emission spectra of donors (IAEDANS and ANN) was nucleotide and temperature independent except in the case of the S1.ADP.Vi complex, where the ANN spectrum was blue shifted compared with those measured in other nucleotide states The transfer efficiency (E), the quantum yield of the donors, the overlap integrals for each uorophore pairs and the Forster critical distances ă (R0) were determined from the experimental data in different nucleotide states at each temperature The calculated R0 values and the measured FRET efficiencies (E) are shown in Table The donor–acceptor distances (R) were determined using Eqn (3), and the results obtained at °C and 22 °C are shown in Table The distances did not show sharp temperature induced changes, and the data obtained at these two temperatures provided appropriate information regarding the overall effect of temperature The FRET ˚ ˚ ˚ distances were 32–36 A, 44–47 A and 3039 A for the ANNFHS, IAEDANSFHS and ANNIAF pairs, ă Table The nucleotide dependence of the Forster critical distance (R0) and the measured FRET efficiencies (E) for the three fluorophore pairs used in ˚ this study The data presented here were measured at 22 °C The standard deviations were 0.3–1.1 A for the R0 and 0.8–1.5% for the FRET efficiency data, as determined from the results of experiments on at least three independent preparations ANN–Ser181/ FHS–Lys553 IAEDANS–Cys707/ FHS–Lys553 ANN–Ser181/ IAF–Cys707 Nucleotide state ˚ R0 (A) E (%) ˚ R0 (A) E (%) ˚ R0 (A) E (%) Apo ADP.BeFx – ADP.AlF4 ADP.Vi ADP 38.9 36.6 40.7 37.7 36.3 68.6 69.6 68.9 66.2 66.9 46.3 44.5 46.2 45.6 45.5 55.5 49.8 53.0 45.2 52.3 39.7 37.3 41.5 38.4 37.1 63.1 74.8 72.4 84.7 76.3 Table The nucleotide dependence of the apparent donor–acceptor distances measured at °C and 22 °C in rabbit skeletal myosin subfragment ˚ (S1) The standard errors calculated from at least three independent experiments were smaller than A, in all cases Note that these errors provided the lower limit for the physically veritable errors For comparison, the distances from the chicken S1 structure [39], corresponding to the apo state, ˚ ˚ ˚ were determined: Ser181–Lys553, 33.8 A; Cys707–Lys553, 40.5 A; and Ser181–Cys707, 28.3 A The distances calculated from the Dictyostelium discoideum atomic models [4–6], between the corresponding residues (Ser181, Lys546 and Thr688) [8], are presented in columns labelled D.d ˚ All distances are given in A Ser181–Lys553 Cys707–Lys553 Ser181–Cys707 Nucleotide state °C 22 °C D.d °C 22 °C D.d °C 22 °C D.d Apo ADP.BeFx – ADP.AlF4 ADP.Vi ADP 35.9 33.9 37.2 34.2 33.9 34.2 31.8 35.7 33.7 32.3 36.5 36.1 32.6 33.9 36.2 44.9 45.2 46.5 46.6 45.2 44.6 44.5 45.3 47.1 44.8 44.6 43.4 46.3 45.8 43.2 38.9 33.1 37.0 31.7 32.9 36.3 31.1 35.3 28.8 30.5 29.8 29.7 28.3 28.7 29.4 Ó FEBS 2003 ´ 4840 E Bodis et al (Eur J Biochem 270) respectively The data indicated that the effect of nucleotides on these distances was small, with the greatest variation ˚ being 3–4 A (Table 3), in agreement with the observation that the position of the lever arm can be modulated with only minor changes in the motor domain conformation [45] The FRET distances were close to the distances obtained from the atomic model of chicken S1 [39] or the D discoideum myosin II motor domain [4–6] (Table 3), which will be discussed further below, in the Discussion One possible way to improve the reliability of FRET distances is to perform the experiments with different fluorophores In our experiments, the labelling of Ser181 and Lys553 has only been shown for the fluorophores used here and therefore these control experiments were not feasible Protein flexibility The temperature dependence of the f¢ (Figs and 3) was smooth and showed a monotonic increase with increasing temperature Major temperature induced conformational changes were not detected, except in the case of the ANN– – IAF pair in the ADP.AlF4 state This exceptional case will be discussed in more detail below, in the Discussion The absence of any major change in donor–acceptor distances (Table 3) indicates that there is no major conformational change over the temperature range studied Accordingly, the temperature dependence of the normalized transfer efficiency (f¢; Eqn 4) could be attributed solely to the flexibility of the protein matrix In general, the larger change of the f¢ results from greater flexibility of the protein matrix [17,18] Figure shows the results obtained in the absence of nucleotides or in the presence of ADP In the nucleotide-free S1, the temperature induced change in f¢ was substantially smaller for IAEDANS–FHS–S1 than for either the ANN– IAF–S1 or the ANN–FHS–S1 ADP binding had only minor effects on the temperature dependence of f¢ in the case of ANN–IAF or ANN–FHS pairs In the case of the IAEDANS–FHS pair, ADP increased the change of f¢ from less than 5%, measured in the apo-form, to 15% The f¢ data measured in ADP.BeFx, ADP.Vi and – ADP.AlF4 states are presented, for the individual donor– acceptor pairs, in Fig 3A (ANN–FHS), Fig 3B (IAEDANS–FHS) and Fig 3C (ANN–IAF) For comparison, the results obtained from ADP experiments (Fig 2) are shown in the figures as dotted lines In the ADP.BeFx state, the change of f¢ was only slightly smaller than that of the ADP states for all three fluorophore pairs Formation of the – ADP.AlF4–S1 complex did not change the temperature dependence of f¢ between ANN and FHS (Fig 3A) For the other two fluorophore pairs (ANN–IAF and IAEDANS– – FHS), the change in f¢ was smaller in ADP.AlF4 than in – ADP (Fig 3B,C) The largest effect of ADP.AlF4 was observed between the residues labelled with ANN and IAF (Fig 3C) In this case, the overall change of f¢ was only % 10%, much less than in other nucleotide states (60–80%) The temperature profile of f¢ showed a saturation tendency, reaching a maximum value between 14 and 18 °C The binding of ADP.Vi to the S1 provided the greatest effects amongst the nucleotide analogues on the protein flexibility of the motor domain The temperature induced change of f¢ was less than in any other nucleotide states (Fig 3), for either the IAEDANS–FHS (< 5%) or the ANN–FHS (% 15%) pairs (Fig 3A,B) Between the residues labelled by ANN and IAF in ADP.Vi, the overall change of f¢ was % 70% at a temperature range of 6–26 °C (Fig 3C) Discussion Fig Temperature dependence of the normalized FRET efficiency in the absence of nucleotides (black symbols) and in the presence of ADP (white symbols) Data are presented for ANN–Ser181 and FHS– Lys553 (circles), ANN–Ser181 and IAF–Cys707 (triangles), and IAEDANS–Cys707 and FHS–Lys553 (squares) fluorophore pairs The donors ANN or IAEDANS were excited at 350 nm and the emission was monitored between 400 and 470 nm in buffer comprising 25 mM Tes (pH 7.0), 80 mM KCl, mM MgCl2, mM EGTA and mM 2-mercaptoethanol In this study, the distances determined by the three donor– acceptor pairs highlighted three structural aspects of the motor domain of skeletal muscle myosin (Fig 1) The protein matrix between Cys707 (IAEDANS) and Lys553 (FHS) is located in the 50 kDa lower domain and is built up of a-helixes, which are quasi parallel to the direction of this side of the imaginary triangle (Fig 1) The data obtained by measuring the energy transfer between Ser181 (ANN) and Cys707 (IAF) characterize the part of the 50 kDa upper domain that is located more closely to the light-chain binding domain The third side of the triangle, Ser181 (ANN) and Lys553 (FHS), cross over the nucleotidebinding pocket The FRET experiments between ANN and FHS reported on the relative motion of the 50 kDa upper and 50 kDa lower domains Based upon the FRET results, the effects of nucleotides and nucleotide analogues follow each other in the order of apo-, ADP, ADP.BeFx, – ADP.AlF4 and ADP.Vi, in agreement with previous observations [46] Although the FRET distances were in good agreement with those obtained from either chicken or D discoideum atomic coordinates (Table 3), the results of analysis of the temperature dependence of steady-state anisotropy data Ó FEBS 2003 Dynamic properties of the myosin motor domain (Eur J Biochem 270) 4841 suggested that the agreement was coincidental The distances from FRET experiments were calculated using j2 ¼ 2/3, which assumes free rapid probe motion on a nanosecond timescale The high values (‡ 0.28) obtained for the r0app indicated that the dyes were rigidly attached to the protein segments, thus preventing the free rotation of the probes Therefore, the j2 ¼ 2/3 assumption is probably not valid and the calculated donor–acceptor distances can be taken as apparent distances The calculated values for the rotating volumes are approximately two orders of magnitude greater than the volumes of the spheres with a radius of the length ˚ of the fluorophores (< 103 A3), indicating that the motion of the labels reflects the motion of the protein segment to which they are attached The results suggested that the temperature profile of the f¢ is not sensitive to local probe motions, similarly to the case of actin monomers, where the IAEDANS on the Cys374 was sensitive to the cation exchange [37], but the temperature dependent FRET experiments between IAEDANS and FITC on Lys61 showed no changes in the dynamics of the smaller domain of actin [47] The apparent donor–acceptor distances showed no major change with the temperature (Table 3), i.e the equilibrium distances between the donor–acceptor pairs not change with the variation of the temperature in this range, in accordance with the basic assumption of the method [18] [The fact that the apparent donor–acceptor distances not change with the temperature let us conclude that the actual distances also remain unchanged Otherwise, one would have to use the very unlikely assumption that any change in the equilibrium donor– acceptor distance is compensated for by the appropriate change of j2 to leave the apparent distance unchanged.] We concluded that the changes in the f¢ were related to the increased width of donor–acceptor distance distribution, and the greater slope of the temperature dependence of f¢ indicated the more flexible protein matrix between the labels The FRET data will be interpreted based upon the structural model, which assumes that the motor domain can exist in two conformations – open and closed – defined by the conformation of the SWII element [8] The equilibrium between these conformations is controlled by the bound nucleotide and was characterized previously for unlabelled myosins by using temperature and pressure jump experiments [15,16] In the present study we applied external labels, which probably modified the open–closed equilibrium The tryptophan fluorescence measured for these labelled S1 samples would be informative regarding these undesired effects [15,16] However, the absorption and emission spectra of tryptophan overlap with those of the fluorophores used, which did not allow us to carry out these control experiments The results will be discussed therefore using the equilibrium constants determined previously for unlabelled myosins Fig The temperature dependence of the normalized FRET efficiency – in S1.ADP.BeFx (h), S1.ADP.AlF4 (d) and S1.ADP.Vi (m) Data are presented for the ANN–Ser181 and FHS–Lys553 pair (A), the IAEDANS–Cys707 and FHS–Lys553 pair (B), and the ANN–Ser181 and IAF–Cys707 pair (C) For comparison, the data obtained in the presence of ADP (Fig 2) are also presented in the figures as dotted lines The experimental conditions were as described for Fig Comparison of the 50 kDa upper domain with the 50 kDa lower domain The temperature induced increase of f¢, along the Cys707– Lys553 direction, was much smaller than along the other two sides (Ser181–Lys553 and Ser181–Cys707) (Figs and 3), which raises the possibility that the motor domain Ó FEBS 2003 ´ 4842 E Bodis et al (Eur J Biochem 270) is heterogeneous from the dynamic point of view The sensitivity of the normalized energy transfer (f¢) depends on the r/R ratio (where r is the amplitude of the donor– acceptor fluctuation and R is the equilibrium distance), which is characteristic for the studied protein The temperature dependence of f¢ can also depend on the value of the Forster critical distance, which describes the sensitivity ă of the uorophore system applied In our study, the spectral properties of the individual donor–acceptor pairs were similar, giving R0 data in a relatively narrow range between ˚ ˚ 36 A and 48 A (Table 2) The measured distances were ˚ and 44 A The effect of these spectral and ˚ between % 30 A geometric parameters cannot account for the large deviations of f¢ found between the three sides of the triangle Accordingly, the direct juxtaposition of the flexibility data obtained along the three directions within the motor domain is reliable The smaller temperature induced change of f¢ along the Cys707–Lys553 direction (as compared to the other two directions) can only be attributed to the smaller relative amplitude of the donor–acceptor fluctuations The structure of the 50 kDa lower domain in the apo-enzyme is more rigid than that of the 50 kDa upper domain The rigidity of the 50 kDa lower domain could be provided by the set of a-helixes that run quasi parallel to the Cys707–Lys553 direction The binding of either ADP or ADP.Pi or ATP analogues had little effect on the flexibility of the protein matrix between Cys707 and Lys553, which implies that the 50 kDa lower domain behaves as a rigid body during the nucleotide induced reorganizations of the S1 The rigidity of this protein region can provide the structural stability for the proper interactions with actin This conclusion agrees with the observation that the protein matrix between Cys707 in S1 and the actin (labelled on Cys374) is rigid [48], and the width of the positional distribution of Cys707 is narrow in the absence of nucleotides [49], which suggests that the rigidity of the actin binding region is maintained during the interaction of S1 with actin In the apo-enzyme, the flexibility of the protein matrix along the Ser181–Cys707 direction was the greatest of the three directions This large flexibility was maintained in the ADP and ADP.BeFx states, although to differing extents, – and further increased in ADP.Vi In ADP.AlF4, the temperature dependence of the f¢ is more complex and will be discussed below The large flexibility along Ser181– Cys707, i.e in the 50 kDa upper domain, may be important in providing the structural frame for the motion and reorientation of the phosphate group and for its interaction with surrounding water molecules Oxygen exchange studies have shown that the cleavage of the myosin bound ATP is reversible, the equilibrium between myosin bound ATP and myosin-products complexes is rapid and the bound nucleotide is able to undergo a fast and reversible reaction with water to exchange all three oxygens [50,51] Such interactions require the rapid rotation and reorientation of the phosphate group Based on crystal structures it is assumed that the phosphate is coordinated by three strong bonds, in addition to the covalent bond in the strong conformation, with no indication of how would it rotate rapidly after hydrolysis [8] We assume that the amplitude and frequency of local protein fluctuations in this region should be sufficiently large to provide the motional freedom for the phosphate The flexibility of the 50 kDa upper domain is important in permitting such large-scale fluctuations In the back door enzyme model [52], it is believed that the dissociation of the phosphate product occurs through the back door of the motor domain on the opposite side of the head to the one where the ATP enters The atomic structures suggest that access to the back door, however, is partially blocked in either the open or closed conformations [4–6] In the absence of actin, the phosphate product is trapped in the nucleotide binding pocket and its dissociation from the motor domain is slow (% 0.05 s)1) The binding of actin to myosin can accelerate the phosphate release With the lack of data in the presence of actin we can only speculate that the large-scale breathing motion of the flexible upper 50 kDa domain may become important in the actin–myosin complex for the dissociation of the phosphate product The effect of nucleotides on the flexibility of the motor domain The binding of ADP to the apo-S1 influenced the protein dynamics only marginally The flexibility slightly increased between the Cys707 and Lys553 The atomic structures [4,5], and the results of rapid kinetic experiments [15,16], indicated that the motor domain is predominantly in the open conformation in either the apo-S1 or when ADP is bound, which suggests that the small ADP-induced change in the flexibility between Cys707 and Lys553 may not be directly related to the open-to-close transition It has been shown previously, by EPR [53,54], FRET [23,55,56] and covalent cross-linking [57] assays, that the binding of nucleotides loosens the structure of the essential SH/hinge region (involving Cys707) where the donor IAEDANS was located It is probable that melting of the SH helix was reflected by the slightly more flexible structure detected in our FRET experiments along the Cys707–Lys553 direction Accordingly, the small effect of ADP on the flexibility of the motor domain is attributed to local conformational changes around the Cys707 residue, and the binding of ADP did not change the overall structure and dynamics of the motor domain Recent results from electron microscopy experiments showed that the release of ADP from the acto–S1 ˚ complex is accompanied with a 35 A swing of the lever arm in the case of smooth muscle myosin [58] In accordance with these results, it was shown recently, by pressure-jump experiments, that the increase in molar volume for skeletal muscle S1 binding to ADP was half of that observed for smooth muscle S1 [59] ADP-induced movement of the light-chain binding domain was also found in brush border myosin-I [60], but was not detected in myosins from skeletal muscle The lack of ADP-induced swinging of the lever arm in skeletal muscle S1 agrees with our observation that the binding of ADP did not alter the dynamic properties of the motor domain The binding of BeFx to ADP–S1 slightly decreased the change in the normalized transfer efficiency measured for the three the donor–acceptor pairs between °C and 26 °C The interpretation of the temperature dependent FRET data, however, is complex in the case of ADP.BeFx The small decrease of the change in the normalized energy transfer efficiency could be a local conformational effect Ó FEBS 2003 Dynamic properties of the myosin motor domain (Eur J Biochem 270) 4843 induced by the binding of BeFx, or could reflect the temperature-induced shift of the open/closed equilibrium In skeletal S1 [16], or in the D discoideum myosin II motor domain [15], an increase in temperature shifted the equilibrium towards the closed conformation in the ADP.BeFx state The FRET data indicate that the motor domain adapts a more rigid conformation in the closed conformation than in the open state However, the observed changes of the FRET parameters were small and the overall structure of the motor domain was similar in the S1.ADP.BeFx to that observed in apo-S1 or S1.ADP As, in these latter two states, the open conformation is dominant, the FRET results suggest that the open–closed equilibrium was shifted towards the open conformation in S1.ADP.BeFx – ADP.AlF4 is thought to mimic the ADP.Pi state of S1 In – S1.ADP.AlF4, the temperature profile of f¢ showed a saturation curve between Ser181 and Cys707 (Fig 3C) The intramolecular events behind this observation can involve either temperature-induced changes in the protein structure, which alters the distance or average orientation between the donor and the acceptor, or steric constraints which limit the fluctuations of the protein segments where the donor or acceptor is located The presence of such an – effect in S1.ADP.AlF4, and the lack of it in the other nucleotide states (Fig 3), implies that the conformation of – the motor domain is different in ADP.AlF4 than in the apo-, ADP or ADP.BeFx conformations Accordingly, after the – binding of AlF4, the open conformation of S1 no longer dominated On the other hand, the binding of AlF4 could only partially reproduce the Vi effects (Fig 3) In the atomic models, the SWII element was in the closed – conformation in both S1.ADP.Vi and S1.ADP.AlF4 [5,6] However, according to the FRET results, the conforma– tions observed for S1 with bound ADP.Vi and ADP.AlF4 were different The interpretation of the FRET data, – measured between Ser181 and Cys707 in S1.ADP.AlF4, is not clear In the other two directions (Ser181–Lys553 and – Cys707–Lys553), the results for the ADP.AlF4 state were intermediate between the ADP.Vi and apo states, which – suggests that in S1.ADP.AlF4, the contribution of the open conformation of the motor domain was substantial This conclusion is in conflict with the temperature and pressure – jump results showing that in S1.ADP.AlF4, the closed conformation dominated between and 30 °C [15,16] It is possible that the tryptophan fluorescence, which was monitored in the cited studies and the FRET pairs, applied here, reported on different structural aspects of the S1 motor domain, which could account for the different – conclusions reached regarding the ADP.AlF4 state Alternatively, the shift towards the open conformation may have appeared in the present work owing to the application of external labels Our conclusion, that the S1 population is different with bound ADP.Vi from that with – bound ADP.AlF4, agrees with the observation that the – nucleotide-binding cleft is only half closed in the ADP.AlF4 X-ray structure [5,9] as compared to the ADP.Vi structure In this work, ADP.Vi was used to mimic the transition state as an alternative ADP.Pi analogue The atomic models suggested that S1 was predominantly in the closed conformation when ADP.Vi-bound [6] The effect of binding of ADP.Vi on the dynamic properties of S1 was the largest amongst the nucleotides investigated, and we interpret these observations as characteristic for the closed conformation The steady-state fluorescence experiments showed that the binding of Vi shifted the emission spectra of ANN to the blue by nm, indicating that the solvent accessibility of the ANN on Ser181 was reduced These observations suggest that the 50 kDa domain became more compact in the closed conformation of the motor domain The FRET results suggest that in the closed conformation the protein matrix between Ser181 and Cys707 became more flexible than in the open conformation, which could further accommodate the breathing motion of the 50 kDa upper domain In contrast, in the Ser181–Lys553 direction, the temperature induced increase of f¢ was substantially smaller in the closed conformation than in the open one, which suggests that the amplitude of the relative fluctuation of the 50 kDa upper and 50 kDa lower domains was suppressed The 50 kDa upper and 50 kDa lower domains are connected by the end of the nucleotide-binding cleft through the protein matrix that links the 50 kDa fragment to the light-chain binding domain Our results suggest that this protein region becomes more rigid in the closed conformation The conformational transition underlying the change in the dynamic properties could reflect the relocation of the converter domain and probably plays a role in transferring the energy from the catalytic site to the lever arm Conclusions The structural basis for the interaction of skeletal S1 with actin is provided, at least partly, by the 50 kDa lower domain, which was found to maintain substantial rigidity in the different nucleotide states (Figs and 3) The conformation of the S1 in the apo-enzyme and in S1.ADP.Vi set the two extremes amongst the nucleotide states studied here Considering the atomic structures and the results of rapid kinetic experiments, we assume that S1 was predominantly in the open conformation in the apo-form and in the closed conformation in S1.ADP.Vi The changes in the flexibility of the S1 during the open-to-closed transition are complex; we observed contrasting tendencies on comparison of different protein regions This complexity is probably attributed to the different roles played by the protein regions in the function of S1 In the open conformation, the flexibility of the 50 kDa upper domain was the greatest of the three directions studied here and this large flexibility further increased during the open-to-closed transition The flexible nature of this protein region can be essential in providing the structural conditions for the rapid motion and reorientation of the phosphate group and for its interaction with surrounding water molecules, and may become important in the actin–myosin complex for the dissociation of the phosphate product The solvent accessibility of the Ser181 was reduced, and the amplitude of the relative fluctuations of the upper 50 kDa and lower 50 kDa domains was suppressed in the closed conformation as compared to that of the open one The suppressed amplitude suggests that the protein region near the bottom of the nucleotide-binding cleft, which links the two domains together, becomes more rigid The more rigid conformation adapted in the closed conformation can provide the Ó FEBS 2003 ´ 4844 E Bodis et al (Eur J Biochem 270) mechanical basis of the transfer of the information or energy from the catalytic site to the light-chain binding domain 15 Acknowledgements The authors gratefully acknowledge Dr Michael A Geeves’s continuous support and suggestions during the preparation of the manuscript, ´ ´ ´ and the insightful comments from Andras Lukacs and from Dr Jozsef ´ Belagyi during the course of this work This work was supported by grants from the National Research Foundation (OTKA grants: T32700, T34442, T43103), from the Ministry of Education (0252/ 2000), and from the Hungarian Academy of Sciences NKFP 1/026/ 2001 M Nyitrai is an EMBO/HHMI Scientist 16 17 18 References Goodno, C.C (1979) Inhibition of myosin ATPase by vanadate ion Proc Natl Acad Sci USA 76, 2620–2624 Maruta, S., Henry, G.D., Sykes, B.D & Ikebe, M (1993) Formation of the stable myosin-ADP-aluminum fluoride and myosinADP-beryllium fluoride complexes and their 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