Experimental Investigation of the Seesaw Mechanism of the Relay Region That Moves the Myosin Lever Arm
2008; Elsevier BV; Volume: 283; Issue: 49 Linguagem: Inglês
10.1074/jbc.m805848200
ISSN1083-351X
AutoresBálint Kintses, Zhenhui Yang, András Málnási‐Csizmadia,
Tópico(s)Cellular Mechanics and Interactions
ResumoA seesaw-like movement of the relay region upon the recovery step of myosin was recently simulated in silico. In this model the relay helix tilts around its pivoting point formed by a phenylalanine cluster (Phe481, Phe482, and Phe652), which moves the lever arm of myosin. To study the effect of the elimination of the proposed pivoting point, these phenylalanines were mutated to alanines in two Dictyostelium myosin II motor domain constructs (MF481A, F482A and MF652A). The relay movement was followed by the fluorescence change of Trp501 located in the relay region. The steady-state and transient kinetic fluorescence experiments showed that the lack of the phenylalanine fulcrum perturbs the formation of the "up" lever arm state, and only moderate effects were found in the nucleotide binding, the formation of the "down" lever arm position, and the ATP hydrolysis steps. We conclude that the lack of the fulcrum decouples the distal part of the relay from the nucleotide binding site upon the recovery step. Our molecular dynamics simulations also showed that the conformation of the motor is not perturbed by the mutation in the down lever arm state, however, the lack of the pivoting point rearranges the dynamic pattern of the kink region of the relay helix. A seesaw-like movement of the relay region upon the recovery step of myosin was recently simulated in silico. In this model the relay helix tilts around its pivoting point formed by a phenylalanine cluster (Phe481, Phe482, and Phe652), which moves the lever arm of myosin. To study the effect of the elimination of the proposed pivoting point, these phenylalanines were mutated to alanines in two Dictyostelium myosin II motor domain constructs (MF481A, F482A and MF652A). The relay movement was followed by the fluorescence change of Trp501 located in the relay region. The steady-state and transient kinetic fluorescence experiments showed that the lack of the phenylalanine fulcrum perturbs the formation of the "up" lever arm state, and only moderate effects were found in the nucleotide binding, the formation of the "down" lever arm position, and the ATP hydrolysis steps. We conclude that the lack of the fulcrum decouples the distal part of the relay from the nucleotide binding site upon the recovery step. Our molecular dynamics simulations also showed that the conformation of the motor is not perturbed by the mutation in the down lever arm state, however, the lack of the pivoting point rearranges the dynamic pattern of the kink region of the relay helix. Myosins are ATP-driven molecular motors that generate force and move along actin filaments. When ATP binds to the actomyosin complex, the first switch 1 loop closes, which opens the actin-binding cleft, causing actomyosin dissociation (1Conibear P.B. Bagshaw C.R. Fajer P.G. Kovacs M. Malnasi-Csizmadia A. Nat. Struct. Biol. 2003; 10: 831-835Crossref PubMed Scopus (78) Google Scholar, 2Holmes K.C. Angert I. Kull F.J. Jahn W. Schroder R.R. Nature. 2003; 425: 423-427Crossref PubMed Scopus (311) Google Scholar, 3Kintses B. Gyimesi M. Pearson D.S. Geeves M.A. Zeng W. Bagshaw C.R. Malnasi-Csizmadia A. EMBO J. 2007; 26: 265-274Crossref PubMed Scopus (41) Google Scholar, 4Reubold T.F. Eschenburg S. Becker A. Kull F.J. Manstein D.J. Nat. Struct. Biol. 2003; 10: 826-830Crossref PubMed Scopus (142) Google Scholar). It is followed by a rapid equilibrium step called the recovery step (5Malnasi-Csizmadia A. Pearson D.S. Kovacs M. Woolley R.J. Geeves M.A. Bagshaw C.R. Biochemistry. 2001; 40: 12727-12737Crossref PubMed Scopus (111) Google Scholar, 6Urbanke C. Wray J. Biochem. J. 2001; 358: 165-173Crossref PubMed Scopus (40) Google Scholar), when the closure of the switch 2 loop drives a 65° rotation of the converter/lever arm region placing the lever arm to the "up" position (7Fisher A.J. Smith C.A. Thoden J.B. Smith R. Sutoh K. Holden H.M. Rayment I. Biochemistry. 1995; 34: 8960-8972Crossref PubMed Scopus (629) Google Scholar, 8Geeves M.A. Holmes K.C. Adv. Protein Chem. 2005; 71: 161-193Crossref PubMed Scopus (294) Google Scholar). Recently, several computer simulations have been published on the analysis of the recovery step (9Fischer S. Windshugel B. Horak D. Holmes K.C. Smith J.C. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 6873-6878Crossref PubMed Scopus (147) Google Scholar, 10Koppole S. Smith J.C. Fischer S. Structure. 2007; 15: 825-837Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 11Mesentean S. Koppole S. Smith J.C. Fischer S. J. Mol. Biol. 2007; 367: 591-602Crossref PubMed Scopus (45) Google Scholar, 12Yu H. Ma L. Yang Y. Cui Q. PLoS Comput. Biol. 2007; 3: e23Crossref PubMed Scopus (71) Google Scholar, 13Yu H. Ma L. Yang Y. Cui Q. PLoS Comput. Biol. 2007; 3: e21Crossref PubMed Scopus (56) Google Scholar). Fischer et al. (9Fischer S. Windshugel B. Horak D. Holmes K.C. Smith J.C. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 6873-6878Crossref PubMed Scopus (147) Google Scholar) composed a model that describes the structural transition between the pre- and post-recovery states (down and up lever arm states, respectively) by simulating the intermediate structures using an unconstrained minimum-energy pathway method. The resulting structural trajectory shows that the relay helix moves in a seesaw-like fashion coupling the movement of switch 2 and the rotation of the lever arm: the closure of switch 2 pulls down the relay helix near its N-terminal end and due to a fulcrum in the middle of the helix, serving as the pivoting point, the C-terminal end of the relay helix swings upwards and finally unwinds in a second phase (Fig. 1). Molecular dynamics simulations confirmed the seesaw-like motion and the pivoting role of this hydrophobic fulcrum (11Mesentean S. Koppole S. Smith J.C. Fischer S. J. Mol. Biol. 2007; 367: 591-602Crossref PubMed Scopus (45) Google Scholar, 12Yu H. Ma L. Yang Y. Cui Q. PLoS Comput. Biol. 2007; 3: e23Crossref PubMed Scopus (71) Google Scholar), however, experimental validation is still lacking. The recovery step is an experimentally well characterized process by the fluorescence change of the conserved Trp501 (Dictyostelium numbering, homologous to Trp510 in rabbit skeletal myosin), which is located at the C-terminal end of the relay helix (5Malnasi-Csizmadia A. Pearson D.S. Kovacs M. Woolley R.J. Geeves M.A. Bagshaw C.R. Biochemistry. 2001; 40: 12727-12737Crossref PubMed Scopus (111) Google Scholar). Scheme 1 shows the reaction steps of the myosin basal ATPase cycle characterized by the use of a mutant motor domain, containing a single Trp501 (MW501+ construct) (14Malnasi-Csizmadia A. Woolley R.J. Bagshaw C.R. Biochemistry. 2000; 39: 16135-16146Crossref PubMed Scopus (103) Google Scholar). Steps 1 and 2 (K1K2 in Scheme 1) represent a two-step inducedfit ATP binding process. M†·ATP, which has a 20% lower fluorescence than the M apo state (Protein Data Bank code 1q5g), corresponds to the down lever arm state (i.e. pre-recovery, open, post-rigor; PDB 1FMW and 1MMD). This state has an open switch 2 and thus the lever arm is in the down orientation. The ATP binding process is followed by the recovery step (step 3a in Scheme 1) when switch 2 loop closes and the relay/converter/lever arm region rotates into the up lever arm state (i.e. post-recovery, pre-power stroke state; M*·ATP in Scheme 1, PDB codes 1VOM and 1MND (7Fisher A.J. Smith C.A. Thoden J.B. Smith R. Sutoh K. Holden H.M. Rayment I. Biochemistry. 1995; 34: 8960-8972Crossref PubMed Scopus (629) Google Scholar). Note that in the post-recovery state myosin contains ATP, whereas in the pre-power stroke state it has ADP·Pi, however, there structures are similar.). The closed switch 2 is required for the hydrolysis of ATP (15Bauer C.B. Holden H.M. Thoden J.B. Smith R. Rayment I. J. Biol. Chem. 2000; 275: 38494-38499Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 16Holmes K.C. Geeves M.A. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2000; 355: 419-431Crossref PubMed Scopus (121) Google Scholar). The forward direction of the recovery step results in a 110% fluorescence emission intensity increase of Trp501, serving a useful signal to follow the conformational change in the relay/converter region. This article demonstrates experimental investigations on the role of the relay helix fulcrum during the recovery step. This hydrophobic fulcrum is formed mainly by three conserved phenylalanine residues: Phe652 (part of the central ;-sheet), Phe481 and Phe482 (relay helix). Two mutant constructs were produced in the MW501+ construct, in which different parts of the fulcrum were replaced with alanines. MF481A, F482A construct has a reduced fulcrum in the side of the relay helix, whereas MF652A contains a reduced support for the relay helix. The steady-state and transient kinetic measurements show that the three phenylalanines form a real functional unit because the functional properties of the two constructs were very similar to each other. The mutations cause very specific changes in the mechanism of the basal ATPase cycle. No change was detected in the conformations of the apo and the down lever arm states, but a dramatic effect was found in the formation of the up lever arm state (step 3a). K3a is dramatically reduced and the conformation of this state is perturbed. In good agreement with these experimental results our molecular dynamics simulations show that the mutations changed the conformation of the relay region only in the up lever arm state. Interestingly, we also found that the mutations cause site-specific dynamic changes of the relay helix unwinding region in the down lever arm state without significant conformational change. This effect could be an indication of the reaction trajectory perturbation in the recovery step. All chemical reagents were purchased from Sigma, except nucleotides (Roche Applied Sciences) and 3′-(N-methyl-anthraniloyl)-2′-deoxy-ATP (Jena Bioscience GmbH, Germany) and [;-32P]ATP (Izinta Ltd., Hungary). Protein Engineering, Expression, and Purification—F481A/F482A or F652A mutations were introduced into the single tryptophan containing W501+ Dd myosin motor domain II M761 cDNA fragment (14Malnasi-Csizmadia A. Woolley R.J. Bagshaw C.R. Biochemistry. 2000; 39: 16135-16146Crossref PubMed Scopus (103) Google Scholar). Recombinant proteins were expressed in Dd AX2-ORF+ cells and purified using His-tagged chromatography as described previously (14Malnasi-Csizmadia A. Woolley R.J. Bagshaw C.R. Biochemistry. 2000; 39: 16135-16146Crossref PubMed Scopus (103) Google Scholar). Preparations were dialyzed against an assay buffer (40 mm NaCl, 20 mm HEPES, pH 7.3, 2 mm MgCl, and 2 mm mercaptoethanol), in which all experiments were performed. Actin preparation and pyrene labeling were done as described previously (17Gyimesi M. Tsaturyan A.K. Kellermayer M.S. Malnasi-Csizmadia A. Biochemistry. 2008; 47: 283-291Crossref PubMed Scopus (9) Google Scholar). Steady-state Fluorescence Measurements—These measurements were carried out with a Fluoromax Spex-320 fluorimeter equipped with a 150-watt Xe lamp. Tryptophan was excited at 296 nm with 2-nm bandwidth excitation and emission slits and fluorescence was detected in 310–420 nm range at 20 and 6 °C. During acrylamide quenching experiments the time courses of tryptophan fluorescence were detected at 340 nm and the 3 ;m motor domains were titrated with acrylamide in a 0.05–0.4 mm concentration range. Stopped-flow Measurements—The stopped-flow measurements were carried out on a KinTek SF-2004 (KinTec Corporation) or on a BioLogic SFM-300/400 (BioLogic SAS, France) stopped-flow fluorimeter, both equipped with 150-watt Superquiet Hg-Xe lamps (Hamamatsu Photonics, UK). Tryptophan was excited at 297 nm, slits were 4 nm and a 340-nm interference filter (Corion CFS-001999 9L134) was used on the emission side. Pyrene was excited at 365 nm and fluorescence was detected with a WG420 cut-off filter (Comar Instruments, UK). The dead time of the KinTec SF-2004 was determined to be 1 ms at 18 ml/s flow rate, the BioLogic stopped-flow has 0.2- and 2-ms dead time with the ;FC-08 cuvette and FC-15 cuvette, respectively, at the same flow rate. All concentrations noted are post-mix concentrations, and all experiments were carried out at 20 °C, unless otherwise stated. Quench-flow Experiments—The quench-flow experiments were carried out on a RQF-3 quench-flow apparatus (KinTec Corporation) using [;-32P]ATP radioactive nucleotide. Samples were handled as in Ref. 18Toth J. Varga B. Kovacs M. Malnasi-Csizmadia A. Vertessy B.G. J. Biol. Chem. 2007; 282: 33572-33582Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar and the radioactivity of the ;-32P-hydrolytic product was measured with a Wallac 1409 Liquid Scintillation Counter (PerkinElmer Life Sciences). The indicated concentrations are post-mix concentrations. Actin-activated ATPase—The actin-activated ATPase were determined by the use of a pyruvate kinase/lactate dehydrogenase-coupled assay as described earlier (19Trentham D.R. Bardsley R.G. Eccleston J.F. Weeds A.G. Biochem. J. 1972; 126: 635-644Crossref PubMed Scopus (139) Google Scholar) in buffer containing 5 mm HEPES, pH 7.2, 1 mm MgCl, 1 mm KCl. Molecular Dynamics Simulation—For the molecular dynamics simulations two Dictyostelium myosin II motor domain crystal structures were prepared. InsightII 2000 software was used for generating the missing parts of 1MMD (pre-recovery (up lever arm) structure). The ADP·BeFx located in the nucleotide binding site was replaced with ATP by changing the BeFx to phosphate group. Then equilibrium molecular dynamics simulation was performed to achieve relaxed conformations and by averaging the coordinates of 125 structures picked up at each 2-ps time point of the equilibrium phase, we visualized a relaxed conformation. For the up lever arm state we used a structure from Jon Kull, 2J. Kull, unpublished data. which has exactly the same conformation as 1VOM, and just contains all of the residues. In this structure the BeFx was also replaced by the phosphate group. Mutations F481A, F482A, or F652A were introduced into these structures and further molecular dynamics simulation were done in the same way. Constant volume periodic boundaries were used with the box dimensions of 134.7 Á. Then the structures were solvated by TIP3P water with a 12-Á cut-off value. Finally, the system was mechanically minimized with parm03 parameters and equilibrated for 2 ns at 300 K by the SHAKE algorithm with 2-fs time steps in the AMBER9 program. Temperature control parameters were set up based on the method of Berendsen (26Berendsen H.J.C. Postma J.P.M. Gunsteren W.F. DiNola A. Haak J.R. J. Chem. Phys. 1984; 81: 3684Crossref Scopus (22550) Google Scholar). We determined the amplitude (;) of the torsional mobility of the :, : angles according to Equation 1, δ=Σ(x−x¯)2n−1 (Eq. 1) where x is the actual torsion angle, [overbar]x is the average of all the : or : angles of the given residue, and n the number of data points. The experiments were repeated three times and averaged, each based on 125 collected structures picked up at each 2-ps time point from 250-ps long equilibrium phases. Steady-state Fluorescence of MF481A, F482A and MF652A—The effects of the mutations on the steady-state fluorescence emission spectra of Trp501 were studied by comparing MF481A, F482A and MF652A to the MW501+ in the presence of different nucleotides at 20 °C (Fig. 2). In the absence of nucleotide the three constructs have similar emission spectra (emission maximum at 342 nm). Also, the fluorescence spectra of the mutants were identical to that of MW501+ in the presence of ADP (15% quenches and 2-nm blue shifts compared with the apo states). On addition of ATP the mutants show decreased fluorescence enhancements compared with the MW501+. Although the fluorescence of MW501+ increases by 100% compared with the ADP-bound fluorescence level (14Malnasi-Csizmadia A. Woolley R.J. Bagshaw C.R. Biochemistry. 2000; 39: 16135-16146Crossref PubMed Scopus (103) Google Scholar), the fluorescence intensity increases of MF481A, F482A and MF652A were only 10 and 5%, respectively. These observations have raised the question, whether the smaller fluorescence enhancements of the mutants are due to the formation of new fluorescent states, or just K3a of the mutants are pulled to the M†ATP states. The comparison of the fluorescence intensities of the ADP·AlF4-bound forms answers this question, because the ADP·AlF4 induces the up lever arm state of myosin, which is the high fluorescence state of MW501+. Fig. 2 shows that, whereas the fluorescence level of the MW501+·ADP·AlF4 state is 110% higher than the MW501+·ADP state, that of the MF481A,F482A·ADP·AlF4 and MF652A·ADP·AlF4 are just 40 and 35% higher, respectively. To demonstrate that the ADP·AlF4-bound mutant constructs are also single fluorescent states just as MW501+·ADP·AlF4 (5Malnasi-Csizmadia A. Pearson D.S. Kovacs M. Woolley R.J. Geeves M.A. Bagshaw C.R. Biochemistry. 2001; 40: 12727-12737Crossref PubMed Scopus (111) Google Scholar) and not a mixture of the low and high fluorescent states of MW501+, the temperature dependence of the fluorescence emissions were determined (see supplemental Fig. S1). Because the relative fluorescence intensities of the apo, ADP-, and ADP·AlF4-bound forms were not influenced by temperature and the former states are known to be single fluorescent states, MF481A,F482A·ADP·AlF4 and MF652A·ADP·AlF4 populate predominantly a single fluorescent state, which is structurally different from the wild type up lever arm state (MW501+·ADP·AlF4). Acrylamide Quenching Experiment—To explore the possible structural differences between the ADP·AlF4-bound forms of the mutants and the MW501+ in the Trp501 environment, we performed acrylamide fluorescence quenching experiments. The effect of the acrylamide collisional quenching on the intensity of the fluorescence emission depends on the exposure of the fluorophore to the solvent. We titrated all three constructs with up to 0.4 m acrylamide in the absence and presence of ADP, ATP, and ADP·AlF4 (supplemental Fig. S2). MW501+·ATP and MW501+·ADP·AlF4 show significantly decreased Stern-Volmer constants compared with the apo and ADP-bound forms, indicating the more buried tryptophan fluorophore, which is a characteristic of the up lever arm conformation (14Malnasi-Csizmadia A. Woolley R.J. Bagshaw C.R. Biochemistry. 2000; 39: 16135-16146Crossref PubMed Scopus (103) Google Scholar). MF481A, F482A and MF652A do not show such differences in the Stern-Volmer constants when ATP or ADP·AlF4 is bound compared with the ADP-bound state (Table 1).TABLE 1Stern-Volmer constants of the acrylamide quenching experiments 3 ;m MW501+, MF481A, F482A, and MF652A were titrated with up to 0.4 m acrylamide in the absence and presence of different nucleotides at 20 °C (supplemental Fig. S2). In MW501+ the difference in the Stern-Volmer constants of the "up" (ADP·AlF4) and the "down" (ADP) lever arm states is significantly larger than the mutants'.NucleotideStern-Volmer constant (M–1)MW501+MF481A, F482AMF652ANone4.11 ± 0.094.10 ± 0.083.77 ± 0.08ADP3.73 ± 0.073.70 ± 0.083.34 ± 0.12ATP3.20 ± 0.083.74 ± 0.083.26 ± 0.10ADP·AlF42.98 ± 0.043.58 ± 0.073.15 ± 0.07 Open table in a new tab Transient Kinetic Characterization of Trp501 Signal Changes— The fluorescence changes of Trp501 allowed us to characterize the kinetics of the nucleotide binding steps of MF481A, F482A and MF652A in a stopped-flow device and compare them to MW501+. By mixing them with different concentrations of ADP under pseudo-first order conditions, an ∼10–15% fluorescence quench was observable in all three constructs. Single exponentials were fitted to the records and the rate constants were plotted as a function of ADP concentration (supplemental Fig. S3). We found that the quenches of the mutants show two-step binding kinetics of an induced fit mechanism as it was described earlier using MW501+ (steps 6 and 7 in Scheme 1) (14Malnasi-Csizmadia A. Woolley R.J. Bagshaw C.R. Biochemistry. 2000; 39: 16135-16146Crossref PubMed Scopus (103) Google Scholar). The second-order rate constants of ADP binding to the mutants are 5-fold less compared with the wild type (Table 2). Because the ADP off-rates of the mutants also decrease ∼10 times as measured by mant-ADP chasing experiments (Table 2 and supplemental Fig. S4), the ADP affinities are only slightly affected by the mutations.TABLE 2Rate constants and equilibrium constants of some reaction steps in Scheme 1 K3a = M*·ATP/M†·ATP and K3b = M*·ADP·Pi/M*·ATP were calculated from the amplitude of the Pi burst (Kapphydrolysis = M*·ADP·Pi/(M†·ATP + M*·ATP)) and the amplitude of the fluorescence enhancement upon ATP binding ( Kapprecovery step = (M*·ATP + M*·ADP·Pi)/M†ATP). and M*·ATP = M*(total) – M*·ADP·Pi). All parameters were measured 20 °C, otherwise stated.Parameters of Scheme 1NucleotideMW501+MF481A, F482AMF652AK1k+2 (;m–1s–1)ADP1.500.330.30k+6 (s–1)mant-ADP3.50.360.4K1k+2 (;m–1s–1) (6 °C)ATP0.800.150.13k+2 (s–1) (6 °C)400170126Kapprecovery step5.250.270.14(Kapphydrolysis0.430.110.05K3a (recovery step)2.70.140.09K3b (hydrolysis)0.550.910.66kobserved hydrolysis (s–1)25.05.32.2k4+ (s–1)0.050.140.07 Open table in a new tab ATP binding to MW501+ (steps 1 and 2 in Scheme 1) is associated with the same fluorescence quench of Trp501 as ADP binding. At 20 °C the subsequent fast recovery step coupled to the fluorescence enhancement does not allow the detection of the transient fluorescence quench. At 6 °C the recovery step slows down compared with the binding event, therefore the fluorescence quench transiently appears in the stopped-flow record (5Malnasi-Csizmadia A. Pearson D.S. Kovacs M. Woolley R.J. Geeves M.A. Bagshaw C.R. Biochemistry. 2001; 40: 12727-12737Crossref PubMed Scopus (111) Google Scholar). Fig. 3A shows the ATP binding stopped-flow records of the mutants and that of the MW501+ at 6 °C. The fluorescence quenches can be seen in all three cases. The observed rate constants of the fitted exponentials plotted against ATP concentration show that the mutants bind ATP 5 times slower than the MW501+ just like in the case of ADP (supplemental Fig. S5A and Table 2). The subsequent recovery step coupled to the fluorescence enhancement is detected only in the case of MW501+. At 20 °C, where the high fluorescent state is more populated (based on the emission spectra), both mutants show a small increase (MF481A, F482A and MF652A, 3 and 2%, respectively) after the quench (Fig. 3B). At higher ATP concentrations, when the accelerating fluorescent quench has already separated from the enhancement phase, the enhancement phase does not show ATP concentration dependence, the rate constants for MF481A, F482A and MF652A are 5 and 2 s-1, respectively. In MW501+ the amplitude of the ATP-induced fluorescent enhancement is larger by almost 2 orders of magnitude compared with the mutants. In contrast to the mutants this enhancement has two phases. The first fast enhancement phase is the recovery step (>150 s-1), whereas the second phase is represented by the hydrolysis step (kobs = (K3a/(1 + K3a)) × k3b + k-3b = 25 s-1), which pulls the previous equilibrium toward the high fluorescent M*ATP state (supplemental Fig. S5B). Characterization of the ATP Hydrolysis Steps—The hydrolytic activities of the mutant motor domains were determined by quenched-flow experiments using 32P-labeled ATP (Fig. 4). The experiments were carried out under multiple turnover conditions. All three constructs show a burst phase of the phosphate (Pi) production followed by a steady-state phase, indicating that the observed rate of the ATP hydrolysis step is faster than the rate-limiting step of the enzyme cycle. The amplitude of the burst is three and six times smaller in the case of MF481A, F482A and MF652A, respectively, than that of the MW501+ (Fig. 4). The steady-state ratio of the pre- and post-hydrolytic states gives the apparent equilibrium constant of the hydrolysis step (Kapphydrolysis = M*·ADP·Pi/(M†·ATP + M*·ATP)), which is 4 and 9 times smaller than that of the MW501+ (Table 2). The rate constants of the exponentials fitted to the Pi burst phases of the mutants (kobserved hydrolysis = 5.3 s-1 of MF481A, F482A, kobserved hydrolysis = 2.2 s-1 of MF652A) are identical to the rate constants of the single phase fluorescence enhancements upon adding ATP. In the case of MW501+ the observed rate of the Pi burst phase is identical to that of the second phase of the fluorescence enhancement upon ATP binding (25 s-1). The rate constants of the steady-state phases of the quenched-flow experiments were slightly faster in the case of the mutants than in that of MW501+ (Table 2) and identical to the steady-state turnover rate (k4+ in Scheme 1) measured by pyruvate kinase/lactate dehydrogenase-coupled assay. Determination of K3a and K3b—The fluorescence experiments prove the existence of the down lever arm state of the mutants and a subsequent conformational change that is analogous with the recovery step in the wild type. Furthermore, the quench-flow experiments indicate that the mutants can hydrolyze ATP in this structurally distorted up lever arm state. These findings indicate that the mutations do not change basically the reaction mechanism represented by Scheme 1. According to this scheme three states (M†·ATP, M*·ATP, and M*·ADP·Pi) are populated in the steady-state before the rate-limiting conformational change (step 4) (20Gyimesi M. Kintses B. Bodor A. Perczel A. Fischer S. Bagshaw C.R. Malnasi-Csizmadia A. J. Biol. Chem. 2008; 283: 8153-8163Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). To calculate the steady-state fraction of the low (M†·ADP) and the high (M*(total) = M*·ATP + M*·ADP·Pi) fluorescent states we used the fluorescence intensities of the spectra at the emission maximum (F): (M† = (FADP·AlF4 - FATP)/(FADP·AlF4 - FADP) and (M* = (FATP - FADP)/(FADP·AlF4 - FADP)). The ratio of the high and low fluorescent states gives the apparent equilibrium constant of the recovery step Kapprecovery step = M*(total)/M†·ADP). In the case of MF481A, F482A and MF652A, Kapprecovery step is 19 and 38 times smaller than that of MW501+ (Table 2). As the steady-state fraction of the M*·ADP·Pi state can be estimated by the relative amplitude of the Pi burst of the quench-flow experiment, the relative population of the three states (M†·ATP, M*·ATP, and M*·ADP·Pi), the equilibrium constants of the recovery step (K3a), and the hydrolysis step (K3b) can be calculated as it shown in Table 2. The greatest perturbation was observed in K3a, which is 20 and 30 times smaller in MF481A, F482A and MF652A, respectively, compared with that of MW501+. Although, the Pi bursts of the mutants are also decreased several times, K3b do not show significant differences. It is the consequence of the changed conformation of the up lever arm state that pulls back K3a and hence decreases the Pi burst. Effects of the Eliminated Pivoting Point in the Presence of Actin—We also investigated the effect of the reduced fulcrum on actin binding. In a stopped-flow device, we determined the observed rate constants of actin binding by mixing pyrene-labeled actin with myosin up to 2 ;m in the absence and presence of ADP. The observed rate constants as a function of the myosin concentration were plotted (supplemental Fig. S6) and the on- and off-rates of actin binding were determined (k+A and k-A, respectively, in Table 3) and compared with MW501+ (17Gyimesi M. Tsaturyan A.K. Kellermayer M.S. Malnasi-Csizmadia A. Biochemistry. 2008; 47: 283-291Crossref PubMed Scopus (9) Google Scholar). They show that the actin affinity (Kd,A) of MF481A, F482A and MF652A compared with MW501+ decreased by 10 and 5 times, respectively. As the actin affinity of the mutants weakened similarly in the presence of ADP (Kd,DA), the thermodynamic coupling ratios did not change significantly.TABLE 3Kinetic and thermodynamic parameters of the actin-myosin interaction and actin activationExperimentParameterMW501+MF481A, F482AMF652AATP induced actin-myosin dissociationK1k+2 (;m–1s–1) of ATP0.181.31.5kmax (s–1)121600700Actin bindingk+A (;m–1s–1)1.60 ± 0.04aPublished in Ref. 170.41 ± 0.050.61 ± 0.04k–A (s–1)0.047 ± 0.002aPublished in Ref. 170.14 ± 0.010.1 ± 0.03Kd,A (;m)0.03aPublished in Ref. 170.340.16k+DA (m–1s–1)0.22 ± 0.02aPublished in Ref. 170.07 ± 0.0020.12 ±k–DA (s–1)0.027 ± 0.002aPublished in Ref. 170.11 ± 0.0030.108 ± 0.008Kd,DA (;m)0.12aPublished in Ref. 171.570.90Actin-activated ATPase activity;max (s–1)3.81.21.2Km (;m)676198a Published in Ref. 17Gyimesi M. Tsaturyan A.K. Kellermayer M.S. Malnasi-Csizmadia A. Biochemistry. 2008; 47: 283-291Crossref PubMed Scopus (9) Google Scholar Open table in a new tab The ATP-induced actin-myosin dissociation also reveals that the mutations weaken the strengths of the actin-myosin interaction. Myosin preincubated with pyrene-labeled actin was mixed with up to 1 mm ATP in the stopped-flow. The observed rate constants were plotted as a function of the ATP concentration (supplemental Fig. S7). We found that the second-order rate constants of ATP binding and the maximum rate constants of the mutants were several times larger than those of the MW501+ (Table 3). We also measured the actin-activated ATPase activities by protein kinase/lactate dehydrogenase-coupled assay (supplemental Fig. S8). We found that the Vmax values of the mutants are one-third of that of the MW501+, whereas half-saturation (Km) does not differ significantly (Table 3). Molecular Dynamics Simulations of the Mutant Motor Domains—Equilibrium molecular dynamics simulations were performed both on the down and up lever arm state s
Referência(s)