Requirement of Domain-Domain Interaction for Conformational Change and Functional ATP Hydrolysis in Myosin
2003; Elsevier BV; Volume: 278; Issue: 33 Linguagem: Inglês
10.1074/jbc.m304138200
ISSN1083-351X
AutoresKohji Ito, Taro Q.P. Uyeda, Yoshikazu Suzuki, Kazuo Sutoh, Keiichi Yamamoto,
Tópico(s)Viral Infections and Immunology Research
ResumoCoordination between the nucleotide-binding site and the converter domain of myosin is essential for its ATP-dependent motor activities. To unveil the communication pathway between these two sites, we investigated contact between side chains of Phe-482 in the relay helix and Gly-680 in the SH1-SH2 helix. F482A myosin, in which Phe-482 was changed to alanine with a smaller side chain, was not functional in vivo. In vitro, F482A myosin did not move actin filaments and the Mg2+-ATPase activity of F482A myosin was hardly activated by actin. Phosphate burst and tryptophan fluorescence analyses, as well as fluorescence resonance energy transfer measurements to estimate the movements of the lever arm domain, indicated that the transition from the open state to the closed state, which precedes ATP hydrolysis, is very slow. In contrast, F482A/G680F doubly mutated myosin was functional in vivo and in vitro. The fact that a larger side chain at the 680th position suppresses the defects of F482A myosin suggests that the defects are caused by insufficient contact between side chains of Ala-482 and Gly-680. Thus, the contact between these two side chains appears to play an important role in the coordinated conformational changes and subsequent ATP hydrolysis. Coordination between the nucleotide-binding site and the converter domain of myosin is essential for its ATP-dependent motor activities. To unveil the communication pathway between these two sites, we investigated contact between side chains of Phe-482 in the relay helix and Gly-680 in the SH1-SH2 helix. F482A myosin, in which Phe-482 was changed to alanine with a smaller side chain, was not functional in vivo. In vitro, F482A myosin did not move actin filaments and the Mg2+-ATPase activity of F482A myosin was hardly activated by actin. Phosphate burst and tryptophan fluorescence analyses, as well as fluorescence resonance energy transfer measurements to estimate the movements of the lever arm domain, indicated that the transition from the open state to the closed state, which precedes ATP hydrolysis, is very slow. In contrast, F482A/G680F doubly mutated myosin was functional in vivo and in vitro. The fact that a larger side chain at the 680th position suppresses the defects of F482A myosin suggests that the defects are caused by insufficient contact between side chains of Ala-482 and Gly-680. Thus, the contact between these two side chains appears to play an important role in the coordinated conformational changes and subsequent ATP hydrolysis. Myosin is an actin-based motor, which converts chemical energy liberated by the ATP hydrolysis into directed movement of actin filaments. Myosin head consists of a globular motor domain and an extended α-helical carboxyl-terminal domain (so-called "lever arm domain"), to which two light chains bind. In the currently prevailing swinging lever arm model for the mechanism of force generation, the lever arm domain tilts relative to the motor domain while the motor domain is bound to an actin filament, resulting in net displacement between actin and myosin backbone (1Cooke R. Crowder M.S. Wendt C.H. Bamett V.A. Thomas D.D. Adv. Exp. Med. Biol. 1984; 170: 413-427Crossref PubMed Scopus (29) Google Scholar, 2Vibert P. Cohen C. J. Muscle Res. Cell Motil. 1988; 9: 296-305Crossref PubMed Scopus (75) Google Scholar). This model has been supported by many experimental data, including small angle x-ray scattering measurements (3Wakabayashi K. Tokunaga M. Kohno I. Sugimoto Y. Hamanaka T. Takezawa Y. Wakabayashi T. Amemiya Y. Science. 1992; 258: 443-447Crossref PubMed Scopus (126) Google Scholar), x-ray crystallography (4Rayment I. 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Spudich J.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4459-4464Crossref PubMed Scopus (392) Google Scholar, 12Ruff C. Furch M. Brenner B. Manstein D.J. Meyhofer E. Nat. Struct. Biol. 2001; 8: 226-229Crossref PubMed Scopus (106) Google Scholar, 13Anson M. Geeves M.A. Kurzawa S.E. Manstein D.J. EMBO J. 1996; 15: 6069-6074Crossref PubMed Scopus (136) Google Scholar). Structural changes in myosin motor domain during ATP hydrolysis were investigated by x-ray crystallography using crystals of motor domains bound to different nucleotide analogs (6Smith C.A. Rayment I. Biochemistry. 1996; 35: 5404-5417Crossref PubMed Scopus (515) Google Scholar, 14Fisher A.J. Smith C.A. Thoden J.B. Smith R. Sutoh K. Holden H.M. Rayment I. Biochemistry. 1995; 34: 8960-8972Crossref PubMed Scopus (637) Google Scholar, 15Dominguez R. Freyzon Y. Trybus K.M. Cohen C. Cell. 1998; 94: 559-571Abstract Full Text Full Text PDF PubMed Scopus (591) Google Scholar). Two structures were obtained using the crystals of Dictyostelium myosin motor domain. One is called "open state," because the nucleotide-binding pocket is open. Another is called "closed state," because the nucleotide-binding pocket is closed. Intensive spectroscopic studies using several nucleotides and nucleotide analogs showed that the open state corresponds to M†·ATP and the closed state corresponds to M*·ATP and M*·ADP·Pi (Scheme I), where the dagger (†) represents the quenched fluorescence and the asterisk (*) represents the enhanced fluorescence of conserved Trp-501 (amino acid residue numbers are of Dictyostelium myosin II throughout this article) (16Malnasi-Csizmadia A. Woolley R.J. Bagshaw C.R. Biochemistry. 2000; 39: 16135-16146Crossref PubMed Scopus (103) Google Scholar, 17Malnasi-Csizmadia A. Pearson D.S. Kovacs M. Woolley R.J. Geeves M.A. Bagshaw C.R. Biochemistry. 2001; 40: 12727-12737Crossref PubMed Scopus (112) Google Scholar). Comparison of the structure of the open state with the closed state demonstrated a substantial conformational change during ATP hydrolysis (18Vale R.D. J. Cell Biol. 1996; 135: 291-302Crossref PubMed Scopus (244) Google Scholar, 19Sablin E.P. Fletterick R.J. Curr. Opin. Struct. Biol. 2001; 11: 716-724Crossref PubMed Scopus (49) Google Scholar, 20Geeves M.A. Holmes K.C. Annu. Rev. Biochem. 1999; 68: 687-728Crossref PubMed Scopus (640) Google Scholar). Namely, the converter region rotates by about 70° and the lever arm swings concomitantly. This shows that the converter region communicates with the nucleotide-binding site, although the two regions are apart. How do the two regions communicate with each other? At least two communication pathways are suggested. One is through the interaction between the relay helix and the converter (15Dominguez R. Freyzon Y. Trybus K.M. Cohen C. Cell. 1998; 94: 559-571Abstract Full Text Full Text PDF PubMed Scopus (591) Google Scholar). Recently, Sasaki et al. (21Sasaki N. Ohkura R. Sutoh K. Biochemistry. 2003; 42: 90-95Crossref PubMed Scopus (42) Google Scholar) have provided clear evidence for this interaction. They have shown that disruption of a hydrophobic linkage between Ile-499 in the relay helix and the converter region uncouples the converter swing from the ATP hydrolysis cycle. They have also shown that disruption of a hydrophobic linkage between Phe-692 in the SH1-SH2 helix and the converter region uncouples alike (21Sasaki N. Ohkura R. Sutoh K. Biochemistry. 2003; 42: 90-95Crossref PubMed Scopus (42) Google Scholar). The second candidate for the communication pathway is through the interaction between the relay helix and the SH1-SH2 helix (22Houdusse A. Kalabokis V.N. Himmel D. Szent-Gyorgyi A.G. Cohen C. Cell. 1999; 97: 459-470Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar, 23Houdusse A. Szent-Gyorgyi A.G. Cohen C.U. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11238-11243Crossref PubMed Scopus (285) Google Scholar). In this paper we investigated the latter possibility, namely communication from the relay helix to the SH1-SH2 helix. The small conformational change of the switch region at the open-closed transition is accompanied by a rotational movement of the relay helix around its axis (14Fisher A.J. Smith C.A. Thoden J.B. Smith R. Sutoh K. Holden H.M. Rayment I. Biochemistry. 1995; 34: 8960-8972Crossref PubMed Scopus (637) Google Scholar, 15Dominguez R. Freyzon Y. Trybus K.M. Cohen C. Cell. 1998; 94: 559-571Abstract Full Text Full Text PDF PubMed Scopus (591) Google Scholar, 22Houdusse A. Kalabokis V.N. Himmel D. Szent-Gyorgyi A.G. Cohen C. Cell. 1999; 97: 459-470Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar) (Fig. 1a). The rotational movement of the relay helix is accompanied with a contact between several side chains in the relay helix and the SH1-SH2 helix. In this paper we focused analyses to the contact between the side chain of Phe-482 in the relay helix and the side chain of Gly-680 in the SH1-SH2 helix. This contact is observed only in the closed structure (6Smith C.A. Rayment I. Biochemistry. 1996; 35: 5404-5417Crossref PubMed Scopus (515) Google Scholar, 14Fisher A.J. Smith C.A. Thoden J.B. Smith R. Sutoh K. Holden H.M. Rayment I. Biochemistry. 1995; 34: 8960-8972Crossref PubMed Scopus (637) Google Scholar), suggesting the possibility that this contact plays an important role in the closed structure. Specifically, we hypothesized that this contact is one of the central mechanisms by which the nucleotide-binding site communicates with the converter region (Fig. 1b). To examine this hypothesis, we changed Phe-482 of Dictyostelium myosin II to alanine whose side chain is much smaller than the side chain of phenylalanine (F482A mutant) and examined whether this mutation abolished the transmission of conformational changes. We also made the F482A/G680F double mutant to see if the G680F mutation rescued the defect of the F482A mutant. Our results have clearly shown that the contact between Phe-482 and Gly-680 mediates communication between the nucleotide-binding site and the converter region and is essential for the conformational change and the subsequent ATP hydrolysis in myosin. Reagents—N-Methylanthraniloyl derivatives of 2-deoxy-ADP (mant-ADP) was obtained from Molecular Probes (Portland, OR). Restriction enzymes and modifying enzymes were purchased from New England Biolabs (Beverly, MA). EDTA, EGTA, DTT, 1The abbreviations used are: DTT, dithiothreitol; FRET, fluorescence resonance energy transfer; GFP, green fluorescent protein. and ATP were purchased from Wako Chemicals (Osaka, Japan). Protease inhibitors and phalloidin were purchased from Sigma. Construction and Expression of Mutant Myosin and S1—Mutations of F482A and G680F were made by site-directed mutagenesis using ExSite PCR-based site-directed mutagenesis kit (Stratagene). The sequences of the oligonucleotides used to create the mutations were 5′-GCAATTTGCTAATCACCATATGTTCAAATTGGAACAAG-3′ and 5′-TGGAGTTTTTCATTGGTATAATTGATACATAATTG-3′ for F482A, and 5′-TTCGTATTACGCGTAAAGGT-3′ and 5′-TTCCTTCGAGGACAAAATTG-3′ for G680F. G680V myosin was kindly provided by B. Patterson. Double mutant myosins (F482A/G680F and F482A/G680V) were made by inserting the BglII/NcoI-digested fragment (0.64-kb) of G680F or G680V into BglII/NcoI-digested pMyDap (24Egelhoff T.T. Manstein D.J. Spudich J.A. Dev. Biol. 1990; 137: 359-367Crossref PubMed Scopus (37) Google Scholar) carrying the F482A mutation. After verifying their sequences, the mutant products were subcloned into pTIKLMyDAP (25Liu X. Ito K. Lee R.J. Uyeda T.Q. Biochem. Biophys. Res. Commun. 2000; 271: 75-81Crossref PubMed Scopus (21) Google Scholar) for the expression of myosin or into pTIKLOE S1 for the expression of S1 forms (26Uyeda T.Q. Tokuraku K. Kaseda K. Webb M.R. Patterson B. Biochemistry. 2002; 41: 9525-9534Crossref PubMed Scopus (23) Google Scholar). The resultant pTIKLMyDAP and pTIKLOE S1 carrying each mutation were electroporated into Dictyostelium cells that lack the endogenous copy of mhc A (27Ruppel K.M. Uyeda T.Q. Spudich J.A. J. Biol. Chem. 1994; 269: 18773-18780Abstract Full Text PDF PubMed Google Scholar, 28Egelhoff T.T. Titus M.A. Manstein D.J. Ruppel K.M. Spudich J.A. Methods Enzymol. 1991; 196: 319-334Crossref PubMed Scopus (72) Google Scholar) and transformants were selected in the presence of 15 μg/ml G418 in the HL5 medium containing 60 μg/ml each of penicillin and streptomycin. Protein Purification—Mutant and wild-type myosins were prepared by the method of Ruppel et al. (27Ruppel K.M. Uyeda T.Q. Spudich J.A. J. Biol. Chem. 1994; 269: 18773-18780Abstract Full Text PDF PubMed Google Scholar). After purification, all myosins were phosphorylated using bacterially expressed myosin light chain kinase that carried a T166E mutation (29Smith J.L. Silveira L.A. Spudich J.A. EMBO J. 1996; 15: 6075-6083Crossref PubMed Scopus (27) Google Scholar), according to the method of Ruppel et al. (27Ruppel K.M. Uyeda T.Q. Spudich J.A. J. Biol. Chem. 1994; 269: 18773-18780Abstract Full Text PDF PubMed Google Scholar). Phosphorylation of each myosin was checked by urea-SDS-glycerol polyacrylamide gel electrophoresis (27Ruppel K.M. Uyeda T.Q. Spudich J.A. J. Biol. Chem. 1994; 269: 18773-18780Abstract Full Text PDF PubMed Google Scholar). Mutant and wild-type S1 were prepared by the method of Manstein and Hunt (30Manstein D.J. Hunt D.M. J. Muscle Res. Cell Motil. 1995; 16: 325-332Crossref PubMed Scopus (72) Google Scholar). Rabbit skeletal muscle actin was prepared using the method of Spudich and Watt (31Spudich J.A. Watt S. J. Biol. Chem. 1971; 246: 4866-4871Abstract Full Text PDF PubMed Google Scholar). Labeling of actin with pyrene was carried out according to the method of Kouyama and Mihashi (32Kouyama T. Mihashi K. Eur. J. Biochem. 1981; 114: 33-38Crossref PubMed Scopus (720) Google Scholar). The concentrations of actin, S1, and myosin were determined spectrophotometrically using extinction coefficients of 0.62 cm2/mg at 290 nm for actin (33Gordon D.J. Yang Y.Z. Korn E.D. J. Biol. Chem. 1976; 251: 7474-7479Abstract Full Text PDF PubMed Google Scholar), 0.80 cm2/mg at 280 nm for S1 (26Uyeda T.Q. Tokuraku K. Kaseda K. Webb M.R. Patterson B. Biochemistry. 2002; 41: 9525-9534Crossref PubMed Scopus (23) Google Scholar), and 0.53 cm2/mg at 280 nm for myosin (27Ruppel K.M. Uyeda T.Q. Spudich J.A. J. Biol. Chem. 1994; 269: 18773-18780Abstract Full Text PDF PubMed Google Scholar). In Vitro Motility Assay—Movement of F-actin labeled and stabilized by rhodamine-phalloidin over nitrocellulose surfaces coated with phosphorylated myosin was observed at 24 °C according to the method described by Uyeda et al. (34Uyeda T.Q. Kron S.J. Spudich J.A. J. Mol. Chem. 1990; 214: 699-710Google Scholar). Phosphorylated myosin in 25 mm Hepes, pH 7.4, 250 mm KCl, 3 mm MgCl2, 2 mm DTT was mixed with 0.5 mg/ml (final concentration) of rabbit skeletal muscle F-actin and incubated on ice for 10 min. After an addition of 2 mm ATP, the mixture was centrifuged for 10 min at 4 °C and 200,000 × g to remove denatured myosin that bound irreversibly to actin (35Kron S.J. Toyoshima Y.Y. Uyeda T.Q. Spudich J.A. Methods Enzymol. 1991; 196: 399-416Crossref PubMed Scopus (347) Google Scholar). After centrifugation, the supernatants were introduced into a chamber having a depth of 0.12–0.17 mm. Before introduction of F-actin labeled with rhodamine-phalloidin, myosin-coated flow cells were treated with unlabeled F-actin and Mg2+-ATP to block residual denatured myosin. The velocities were determined by measuring displacements of smoothly moving actin filaments over a period of 4 s. Concentrations of myosin solutions used on the surfaces were 0.3–0.5 mg/ml for wild-type and F482A/G680F myosins because smooth movements of actin filaments were observed within this concentration. For F482A myosin, motility assay was carried out at concentrations between 0.3 and 2.0 mg/ml. ATPase Assays—Steady state ATPase activities were determined by measuring released phosphate using the method of Kodama et al. (36Kodama T. Fukui K. Kometani K. J. Biochem. 1986; 99: 1465-1472Crossref PubMed Scopus (274) Google Scholar) under the conditions described by Ruppel et al. (27Ruppel K.M. Uyeda T.Q. Spudich J.A. J. Biol. Chem. 1994; 269: 18773-18780Abstract Full Text PDF PubMed Google Scholar). The reaction mixtures for the assay of basal Mg2+-ATPase activity contained 25 mm Hepes (pH 7.4), 25 mm KCl, 4 mm MgCl2, 1 mm DTT, 1 mm ATP, and 0.13 mg/ml S1. The reaction mixtures for the assay of actin-activated Mg2+-ATPase activity contained 25 mm Hepes (pH 7.4), 25 mm KCl, 4 mm MgCl2, 1 mm DTT, 1 mm ATP, and 0.1 mg/ml myosin with F-actin. The reactions were started by the addition of ATP and performed at 30 °C. Cosedimentation Assays in the Presence of ATP—The affinity of S1 for actin in the presence of ATP was measured using cosedimentation assays as described previously (37DasGupta G. Reisler E. Biochemistry. 1992; 31: 1836-1841Crossref PubMed Scopus (39) Google Scholar, 38Giese K.C. Spudich J.A. Biochemistry. 1997; 36: 8465-8473Crossref PubMed Scopus (33) Google Scholar). Phalloidin-actin (3 μm) and S1 at concentrations of 1.5–15 μm were mixed in the assay buffer (25 mm Hepes, 25 mm KCl, 4 mm MgCl2, and 1 mm DTT) and incubated at 22 °C for 10 min before adding 3 mm ATP and spinning at 200,000 × g for 10 min. Pellets were resuspended in the assay buffer, and the concentration of S1 was determined by scanning Coomassie Blue-stained gels using an EPSON GT-7000S image scanner and by analyzing the band densities using the NIH Image software. Transient Kinetic Experiments—All kinetic experiments were done in 25 mm Hepes (pH 7.4), 25 mm KCl, 5 mm MgCl2, 1 mm DTT at 22 °C using a KinTek SF-2001 or an Applied Photophysics SX18MV stopped-flow spectrophotometer. Release of mant-ADP from acto-S1 was monitored by the decrease of its fluorescence accompanying dissociation of mant-ADP from the acto-S1·mant-ADP complex (39Murphy C.T. Spudich J.A. Biochemistry. 1998; 37: 6738-6744Crossref PubMed Scopus (83) Google Scholar). Mant-ADP was excited at 295 nm and emission was observed after passing through a 389-nm cutoff filter. Acto-S1·mant-ADP complex was mixed with excess ATP to suppress the reassociation of mant-ADP to S1. The concentration of S1, mant, ADP, and actin stabilized by phalloidin and ATP was 1 μm, 2.5 μm, and 5 μm, and 4 mm, respectively. The decrease in mant fluorescence was fitted to a single exponential, which expresses a k –AD. Dissociation of acto-S1 by ATP was monitored through changes in fluorescence intensities of pyrene-labeled actin stabilized by phalloidin. The concentration of acto-S1 was 0.5 μm. Pyrene-actin was excited at 365 nm and the fluorescence was detected after passing through a 389-nm cutoff filter (40Batra R. Geeves M.A. Manstein D.J. Biochemistry. 1999; 38: 6126-6134Crossref PubMed Scopus (53) Google Scholar). Tryptophan Fluorescence—Tryptophan fluorescence spectra of 0.2 mg/ml S1 were recorded at room temperature using a Hitachi F-4500 fluorescence spectrophotometer in a medium containing 25 mm Hepes (pH 7.4), 25 mm KCl, 4 mm MgCl2, 0.3 mm DTT and in the presence and absence of 0.1 mm ATP. Excitation wavelength was 293 nm. Tryptophan fluorescence spectra of completely denatured proteins were also recorded after treating them in 6 m guanidine HCl, and normalized each other to confirm that the observed difference in the tryptophan fluorescence arose from conformation change. Fluorescence Resonance Energy Transfer (FRET)—FRET assays were done as described previously (41Suzuki Y. Yasunaga T. Ohkura R. Wakabayashi T. Sutoh K. Nature. 1998; 396: 380-383Crossref PubMed Scopus (156) Google Scholar). Initial Burst in ATP Hydrolysis—The initial burst of phosphate liberation from ATP was measured at 23 °C using the method of Kodama et al. (36Kodama T. Fukui K. Kometani K. J. Biochem. 1986; 99: 1465-1472Crossref PubMed Scopus (274) Google Scholar). The reaction mixtures for the assay of Mg2+-ATPase activity contained 25 mm Hepes (pH 7.4), 25 mm KCl, 4 mm MgCl2, 1 mm DTT, 0.3 mm ATP, and 3 μm S1. After 15, 30, 45, or 60 s of incubation, the ATPase reaction was stopped by adding perchloric acid. The size of the initial phosphate burst was determined by extrapolating to zero time. A straight line was drawn by linear regression. Phenotypes of Dictyostelium Cells Expressing Mutant Myosins—Dictyostelium myosin II-null cells could not undergo normal cytokinesis and grew only slowly in suspension culture up to the density of ∼1 × 106 cells/ml, becoming multinucleated cells (Fig. 2, Null). This is consistent with previous reports (42Sasaki N. Shimada T. Sutoh K. J. Biol. Chem. 1998; 273: 20334-20340Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 43DeLozanne A. Methods Cell Biol. 1987; 28: 489-495Crossref PubMed Scopus (10) Google Scholar, 44Manstein D.J. Ruppel K.M. Spudich J.A. Science. 1989; 246: 656-658Crossref PubMed Scopus (67) Google Scholar). When a plasmid containing the wild-type myosin gene was introduced into the myosin II-null cells, they regained the ability to undergo cytokinesis and grow in suspension up to a density of ∼2 × 107 cells/ml with a doubling time of 11 h (Fig. 2, WT). The growth curve of the transformed cells was almost the same as that of wild-type cells having an endogenous copy of the myosin heavy chain gene. However, myosin II-null cells expressing F482A myosin could not grow in suspension (Fig. 2, F482A), suggesting that F482A myosin is not functional. We reasoned that if this functional defect of F482A myosin is caused by the lack of the collision between the side chains of Ala-482 and Gly-680, it should be possible to rescue this defect by changing Gly-680 to an amino acid having a larger side chain that would touch Ala-482. Indeed, myosin II-null cells expressing F482A/G680F myosin could divide and grow in suspension culture up to the density ∼2 × 107 cells/ml with a doubling time of 11 h (Fig. 2, F482A/G680F). This result demonstrates that doubly mutated F482A/G680F myosin is functional and the G680F mutation suppressed the defect of the F482A mutant. In contrast, myosin II-null cells expressing F482A/G680V myosin could not grow in suspension culture (Fig. 2, F482A/G680V), indicating that the G680V mutation could not suppress the defect of the F482A mutant. This result suggests that there is a minimum size of the side chain at this position to suppress the F482A mutation. Myosin II is also required for the development of fruiting bodies (45Springer M.L. Patterson B. Spudich J.A. Development. 1994; 120: 2651-2660Crossref PubMed Google Scholar). Myosin II-null cells were arrested at the mound stage (Fig. 3, Null) under the starvation condition, whereas myosin II-null cells expressing wild-type myosin II made fruiting bodies (Fig. 3, WT). When F482A myosin was expressed in myosin II-null cells, it did not suppress the defect of the phenotype of myosin II-null cells (Fig. 3, F482A), consistent with the results of growth experiments in suspension culture. In contrast, myosin II-null cells expressing F482A/G680F myosin made fruiting bodies, although they had slightly deformed sorocarps (Fig. 3, F482A/G680F). Thus, the experiments of the development of fruiting bodies confirmed that F482A myosin is non-functional and F482A/G680F myosin is functional. ATPase Activities—Yields and purities of all the recombinant myosins and S1s prepared were almost the same as those of wild-type. Basal Mg2+-ATPase in the absence of actin of F482A S1 was similar to that of wild-type S1 (Table I). Addition of 24 μm actin to wild-type myosin enhanced its Mg2+-ATPase activity by 15-fold. However, the Mg2+-ATPase of F482A myosin was hardly activated by actin (only 1.2-fold activation in the presence of 24 μm actin). In contrast, Mg2+-ATPase activity of F482A/G680F myosin was activated up to 25-fold by 24 μm actin.Table ISteady-state ATPase activitiesMg2+-ATPaseMg2+-ATPase + 24 μm actinWT0.17 ± 0.022.5 ± 0.3F482A0.11 ± 0.010.13 ± 0.006F482A/G680F0.054 ± 0.011.4 ± 0.2 Open table in a new tab V max and K app values of the actin-activated Mg2+-ATPase of the mutant myosins were determined from the dependence of the activation on actin concentration (Fig. 4). Each value shown in Fig. 4 is net actin-activated Mg2+-ATPase activity obtained by subtracting the basal-Mg2+-ATPase activity from the measured value of the actin-activated Mg2+-ATPase. V max of wild-type, F482A, and F482A/G680F myosin were 2.7 s–1, 0.025 s–1, and 1.5 s–1, respectively, and K app were 5.1, 0.79, and 2.2 μm, respectively. In Vitro Motility Assays—As expected from the phenotype of cells expressing F482A myosin, F482A myosin could not drive the sliding of actin filaments in vitro (Fig. 5). Although actin filaments attached to the F482A myosin-coated surface in the absence of ATP, the majority of the actin filaments dissociated on addition of ATP. Some stayed near the surface and exhibited random, lateral motion without making noticeable unidirectional axial movement. Up to 2.0 mg/ml F482A myosin did not move actin, whereas 0.3 mg/ml wild-type myosin caused continuous actin movement (see also "Experimental Procedures"). Addition of methylcellulose to the motility assay buffer reduces the critical concentration of surface density of myosin necessary for the actin movement (34Uyeda T.Q. Kron S.J. Spudich J.A. J. Mol. Chem. 1990; 214: 699-710Google Scholar, 46Uyeda T.Q. Warrick H.M. Kron S.J. Spudich J.A. Nature. 1991; 352: 307-311Crossref PubMed Scopus (158) Google Scholar), but F482A myosin did not slide actin filament even in the presence of 0.8% methylcellulose. For F482A/G680F, continuous and unidirectional movements of actin filaments were observed, although the speeds were only 1/70 of that of wild-type myosin. To examine if mutant myosins have a braking effect on the actin motility supported by wild-type myosin, we performed mixing experiments in which wild-type myosin was mixed with equal amounts of mutant myosins (Fig. 5). The speed of the actin sliding movements by the mixture of wild-type myosin and the F482A/G680F was significantly lower than that by wild-type myosin alone. In contrast, F482A myosin did not block the actin sliding movement powered by wild-type myosin at all. Interaction with Actin in the Absence of ATP—One possible explanation for the defect of F482A myosin is that F482A myosin cannot bind to actin filaments, because the ATPase activity of F482A myosin was scarcely activated by actin and F482A myosin did not impede actin sliding movement generated by wild-type myosin. To test this possibility, actin binding properties of F482A myosin were investigated by actin cosedimentation assays (Fig. 6). Wild-type myosin, F482A myosin, and F482A/G680F myosin efficiently co-sedimented with actin in the absence of ATP, but did not in the presence of ATP. Thus F482A myosin exhibited normal ATP-dependent dissociation-association with actin filaments even though it had lost its actin-activated ATPase activity. When F482A S1 was mixed with pyrene-labeled actin, the pyrene fluorescence decreased, like wild-type S1 (data not shown). These results suggest that F482A myosin can bind to actin strongly in the absence of ATP, like wild-type myosin. Affinity with Actin in the Presence of ATP—Varying concentrations of S1 were cosedimented with 3 μm actin in the presence of 3 mm ATP, and the amounts in the pellets were determined by scanning SDS-PAGE gels (Fig. 7A). The curve fitting showed that the K d value (dissociation constant for actin binding) of wild-type S1 in the presence of ATP is 14 μm, which is similar to the previously reported value (38Giese K.C. Spudich J.A. Biochemistry. 1997; 36: 8465-8473Crossref PubMed Scopus (33) Google Scholar). The K d value of F482A/G680F doubly mutated S1 (17 μm) is similar to that of wild-type S1. In contrast, the K d value of F482A S1 is 42 μm, which is 3-fold higher than that of wild-type S1, showing that the affinity of F482A S1 for actin in the presence of ATP is much lower than that of wild-type S1. ADP Dissociation from Acto-S1—To delineate the biochemical defects underlying the incapability of the F482A mutant to be activated by actin, we measured several kinetic parameters of F482A S1 using a stopped-flow apparatus. First, dissociation of ADP from acto-S1 was followed by monitoring the decrease in fluorescence of mant-ADP accompanying dissociation of mant-ADP from the acto-S1·mant-ADP complex upon addition of excess ATP. The dissociation rate constant of mant-ADP from acto-F482A S1 (k –AD) was 60 ± 5 s–1 (Table II). That value is about half of that of wild-type S1 (123 ± 12 s–1). In contrast, k –AD of F482A/G680F S1 was 3.1 ± 0.2 s–1, which is only 1/40 of that of wild-type S1. The slower actin sliding movement by F482A/G680F myosin (1/70 of that of wild-type myosin) is largely explained by this lower k –AD.Table IITransient kinetic analysisRate constantWTF482AF482A/G680FIn the presence of actink +2 (s-1)aValues are averages ± S.D. of 10-18 independent measurements from at least two independent protein preparations.196 ± 12285 ± 30231 ± 22K 1 k +2 (m-1s-1)bDetermined as described in Fig. 8.1.4 × 1054.0 × 1053.0 × 105K 1 (m-1)cCalculated from K 1 k +2 and k +2.71014001300k -ad (s-1)aVa
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