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A Point Mutation in the SH1 Helix Alters Elasticity and Thermal Stability of Myosin II

2006; Elsevier BV; Volume: 281; Issue: 41 Linguagem: Inglês

10.1074/jbc.m605365200

1083-351X

Sosuke Iwai, Daisuke Hanamoto, Shigeru Chaen,

Muscle Physiology and Disorders

Movement generated by the myosin motor is generally thought to be driven by distortion of an elastic element within the myosin molecule and subsequent release of the resulting strain. However, the location of this elastic element in myosin remains unclear. The myosin motor domain consists of four major subdomains connected by flexible joints. The SH1 helix is the joint that connects the converter subdomain to the other domains, and is thought to play an important role in arrangements of the converter relative to the motor. To investigate the involvement of the SH1 helix in elastic distortion in myosin, we have introduced a point mutation into the SH1 helix of Dictyostelium myosin II (R689H), which in human nonmuscle myosin IIA causes nonsyndromic hereditary deafness, DFNA17. The mutation resulted in a significant impairment in motile activities, whereas actin-activated ATPase activity was only slightly affected. Single molecule mechanical measurements using optical trap showed that the step size was not shortened by the mutation, suggesting that the slower motility is caused by altered kinetics. The single molecule measurements demonstrated that the mutation significantly reduced cross-bridge stiffness. Motile activities produced by mixtures of wild-type and mutant myosins also suggested that the mutation affected the elasticity of myosin. These results suggest that the SH1 helix is involved in modulation of myosin elasticity, presumably by modulating the converter flexibility. Consistent with this, the mutation was also shown to reduce thermal stability and induce thermal aggregation of the protein, which might be implicated in the disease process. Movement generated by the myosin motor is generally thought to be driven by distortion of an elastic element within the myosin molecule and subsequent release of the resulting strain. However, the location of this elastic element in myosin remains unclear. The myosin motor domain consists of four major subdomains connected by flexible joints. The SH1 helix is the joint that connects the converter subdomain to the other domains, and is thought to play an important role in arrangements of the converter relative to the motor. To investigate the involvement of the SH1 helix in elastic distortion in myosin, we have introduced a point mutation into the SH1 helix of Dictyostelium myosin II (R689H), which in human nonmuscle myosin IIA causes nonsyndromic hereditary deafness, DFNA17. The mutation resulted in a significant impairment in motile activities, whereas actin-activated ATPase activity was only slightly affected. Single molecule mechanical measurements using optical trap showed that the step size was not shortened by the mutation, suggesting that the slower motility is caused by altered kinetics. The single molecule measurements demonstrated that the mutation significantly reduced cross-bridge stiffness. Motile activities produced by mixtures of wild-type and mutant myosins also suggested that the mutation affected the elasticity of myosin. These results suggest that the SH1 helix is involved in modulation of myosin elasticity, presumably by modulating the converter flexibility. Consistent with this, the mutation was also shown to reduce thermal stability and induce thermal aggregation of the protein, which might be implicated in the disease process. Class II myosins are actin-based motor proteins that drive cell motile processes including cytokinesis, migration, and morphogenesis, as well as muscle contraction (1Sellers J.R. Biochim. Biophys. Acta. 2000; 1496: 3-22Crossref PubMed Scopus (622) Google Scholar). Myosin II molecules consist of two identical heavy chains (HC) 3The abbreviations used are: HC, heavy chain; S1, subfragment 1; DTT, dithiothreitol; BSA, bovine serum albumin; Ni-NTA, nickel nitrilotriacetic acid; MOPS, 3-(N-morpholine)propanesulfonic acid; mhc, myosin heavy chain; N, newton. and two pairs of light chains. The carboxyl-terminal tail region of each HC forms an α-helical coiled-coil rod with the tail of the adjacent HC. Myosin II molecules associate, through the tail region, to assemble into bipolar filaments, which are required for their cellular functions. The amino-terminal head region of the HC forms a globular motor domain with ATPase and motile activities. The myosin motor domain is followed by an extended α-helical neck region, the so-called lever arm domain, to which the two light chains bind. Structural studies revealed that the myosin motor domain consists of four major subdomains that are linked by three flexible, highly conserved joints (2Rayment I. Rypniewski W.R. Schmidt-Base K. Smith R. Tomchick D.R. Benning M.M. Winkelmann D.A. Wesenberg G. Holden H.M. Science. 1993; 261: 50-58Crossref PubMed Scopus (1877) Google Scholar, 3Houdusse A. Kalabokis V.N. Himmel D. Szent-Györgi A.G. Cohen C. Cell. 1999; 97: 459-470Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). A comparison of crystal structures showed conformational changes in the motor with rearrangements of the subdomains during ATP hydrolysis. In particular, the converter subdomain rotates ∼70° during the prestroke-poststroke transition (4Fisher 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, 5Smith C.A. Rayment I. Biochemistry. 1995; 34: 8973-8981Crossref PubMed Scopus (94) Google Scholar). This rotation is thought to drive a long-distance swing of the lever arm domain (6Geeves M.A. Holmes K.C. Annu. Rev. Biochem. 1999; 68: 687-728Crossref PubMed Scopus (640) Google Scholar). In a widely accepted model, the driving force for motion is generated as a result of conformational changes in the myosin head while attaching to the actin filaments. Such conformational changes cause distortion of an elastic element within the myosin molecule and allow strain to develop before movement (7Huxley A.F. Prog. Biophys. Biophys. Chem. 1957; : 255-318Crossref PubMed Google Scholar). Relief of this strain can drive relative displacement of the myosin and the actin filaments. It has been suggested that the conformational changes involve the swing of the lever arm domain relative to the motor domain. Nevertheless, the precise location of the elastic element in myosin remains unclear. Recently, Köhler et al. (8Köhler J. Winkler G. Schulte I. Scholz T. McKenna W. Brenner B. Kraft T. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3557-3562Crossref PubMed Scopus (73) Google Scholar) have studied the mechanical properties of muscle fibers from patients with familial hypertrophic cardiomyopathy, and suggested that the main elastic distortion occurs within the converter subdomain or associated structures. The SH1 helix is the joint that links the converter to the NH2-terminal subdomain (Fig. 1A), and is thought to play an important role in arrangements of the converter relative to the motor (3Houdusse A. Kalabokis V.N. Himmel D. Szent-Györgi A.G. Cohen C. Cell. 1999; 97: 459-470Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar, 9Himmel D.M. Gourinath S. Reshenikova L. Shen Y. Szent-Györgi A.G. Cohen C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12645-12650Crossref PubMed Scopus (101) Google Scholar). Therefore, the SH1 helix may play an important role in distortion of the elastic element in myosin motor. To investigate the involvement of the SH1 helix in the elastic distortion of myosin, we have introduced a point mutation in the SH1 helix of Dictyostelium myosin II. Dictyostelium discoideum contains a single myosin II gene (10De Lozanne A. Lewis M. Spudich J.A. Leinwand L.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 6807-6810Crossref PubMed Scopus (27) Google Scholar), which has been previously disrupted by means of homologous recombination to generate myosin II-null cells (11De Lozanne A. Spudich J.A. Science. 1987; 236: 1086-1091Crossref PubMed Scopus (763) Google Scholar). Wild-type and mutated myosins can readily be expressed in myosin II-null cells and characterized in vivo as well as in vitro (12Ruppel K.M. Uyeda T.Q. Spudich J.A. J. Biol. Chem. 1994; 269: 18773-18780Abstract Full Text PDF PubMed Google Scholar). As shown in Fig. 1B, the SH1 helix region is highly conserved in class II myosins and in Dictyostelium myosin II. The mutation replaces a conserved arginine in the SH1 helix with a histidine (Fig. 1B, bold). This mutation in human nonmuscle myosin IIA (R705H) is linked to nonsyndromic hereditary deafness, DFNA17 (13Lalwani A.K. Goldstein J.A. Kelley M.J. Luxford W. Castelein C.M. Mhatre A.N. Am. J. Hum. Genet. 2000; 67: 1121-1128Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). Recently, Hu et al. (14Hu A. Wang F. Sellers J.R. J. Biol. Chem. 2002; 277: 46512-46517Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar) showed that mutations such as N93K and R702C, which are located within the motor domain of myosin IIA and linked to diseases, result in impairment of in vitro enzymatic and motile activities. The R705H mutation may also affect myosin II activity, whereas its effects on the in vitro activities have not been examined. In this study, we engineered the mutation at an equivalent site in the Dictyostelium myosin II (R689H), and studied motile activities and mechanical properties of the mutant protein. The mutation resulted in a significant impairment in motile activities. Single molecule mechanical measurements using optical trap revealed that the elastic property of myosin is modulated by the mutation. The mutation was also shown to affect thermal stability of the protein, which might be implicated in the disease process. Construction of Plasmids—The R689H mutation was introduced into the Dictyostelium myosin heavy chain (mhc) gene (10De Lozanne A. Lewis M. Spudich J.A. Leinwand L.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 6807-6810Crossref PubMed Scopus (27) Google Scholar) using two-step PCR-based mutagenesis (15Higuchi R. Krummel B. Saiki R.K. Nucleic Acids Res. 1988; 16: 7351-7367Crossref PubMed Scopus (2104) Google Scholar). In the first PCR, two fragments containing the mutation were generated separately. The 5′ fragment was amplified using the 5′ primer 5′-GTCATGTATGAGATTCAAGATTGG-3′ and the 3′ mutagenic primer 5′-TGGGAAACCTTTATGAGTAATACGA-3′. The 3′ fragment was amplified using the 3′ primer 5′-GAGTCGGTGAGATTAGATTTGAGT-3′ and the 5′ mutagenic primer 5′-TCGTATTACTCATAAAGGTTTCCCA-3′. The mutagenic primers shared an overlapping region, such that these two fragments could be recombined and the whole fragment was amplified with the 5′ and 3′ primers through a second round of PCR. The resulting fragment was digested with BglII/NcoI, and was used to replace the corresponding region of the mhc gene in pTIKLMyDAP (16Liu X. Ito K. Lee R.J. Uyeda T.Q. Biochem. Biophys. Res. Commun. 2000; 271: 75-81Crossref PubMed Scopus (21) Google Scholar). For expression of the S1 fragment of the mutant, the BglII-NcoI fragment of pTIKLOES1 (17Uyeda T.Q. Tokuraku K. Kaseda K. Webb M.R. Patterson B. Biochemistry. 2002; 41: 9525-9534Crossref PubMed Scopus (23) Google Scholar) was replaced with that of the mutant. Preparation of Proteins—Plasmids carrying either the mutant or wild-type mhc gene were introduced into Dictyostelium cells lacking the endogenous mhc gene (11De Lozanne A. Spudich J.A. Science. 1987; 236: 1086-1091Crossref PubMed Scopus (763) Google Scholar). Plasmids carrying S1 were introduced into Dictyostelium Ax2 cells. All transformants were selected in HL5 medium containing 10 μg/ml G418. Myosins were prepared by combining the methods of Clarke et al. (18Clarke M. Spudich J.A. J. Mol. Biol. 1974; 86: 209-222Crossref PubMed Scopus (129) Google Scholar) and Ruppel et al. (12Ruppel K.M. Uyeda T.Q. Spudich J.A. J. Biol. Chem. 1994; 269: 18773-18780Abstract Full Text PDF PubMed Google Scholar), with some modifications. In brief, washed cells were resuspended in 2.5-4 volumes/g cells in lysis buffer (20 mm HEPES (pH 7.4), 100 mm NaCl, 40 mm sodium pyrophosphate, 2 mm EDTA, 30% sucrose, 1 mm DTT, and a mixture of protease inhibitors). The cell suspension was sonicated on ice until >95% of the cells were lysed. Cell extracts were clarified by sequential centrifugation at 15,000 and 300,000 × g. The resultant supernatant was dialyzed against a buffer containing 10 mm MOPS (pH 6.8), 50 mm KCl, 1 mm EDTA, and 0.5 mm DTT. After the addition of 0.1% Triton X-100 to solubilize membranes, the cytoskeletal fraction was collected by centrifugation at 15,000 × g for 30 min. The pellet was washed with extraction buffer (10 mm HEPES (pH 7.4), 250 mm NaCl, 7 mm MgCl2, and 1 mm DTT), and centrifuged again. The resultant pellet was resuspended in extraction buffer containing 5 mm ATP, and centrifuged at 300,000 × g for 30 min. The supernatant was recovered and diluted five times with a buffer containing 10 mm HEPES (pH 7.4), 10 mm MgCl2, 2 mm ATP, and 1 mm DTT. For phosphorylation of the regulatory light chains of myosins, the solution was incubated overnight on ice with myosin light chain kinase according to Ruppel et al. (12Ruppel K.M. Uyeda T.Q. Spudich J.A. J. Biol. Chem. 1994; 269: 18773-18780Abstract Full Text PDF PubMed Google Scholar). The phosphorylated myosin was recovered by centrifugation at 110,000 × g for 12 min. The pellet was dissolved in a buffer containing 10 mm HEPES (pH 7.4), 250 mm KCl, 5 mm MgCl2, 2 mm ATP, and 1 mm DTT, and finally centrifuged at 300,000 × g for 20 min to remove insoluble materials. S1 fragments were prepared as described previously (19Sasaki N. Ohkura R. Sutoh K. Biochemistry. 2003; 42: 90-95Crossref PubMed Scopus (42) Google Scholar), with slight modifications. They were extracted from the cytoskeletal fraction with a buffer containing 20 mm HEPES (pH 7.4), 100 mm NaCl, 7 mm MgCl2, 5 mm ATP, and 1 mm β-mercaptoethanol. Once purified using a Ni-NTA-agarose column (Qiagen), the proteins were dialyzed against an assay buffer (25 mm HEPES (pH 7.4), 25 mm KCl, 4 mm MgCl2, and 1mm DTT). F-actin was extracted and prepared from acetone powder of rabbit skeletal muscle as described previously (20Pardee J.D. Spudich J.A. Methods Cell. Biol. 1982; 24: 271-289Crossref PubMed Scopus (340) Google Scholar). Actin concentration was determined spectrophotometrically using an extinction coefficient of 0.63 cm2 mg-1 at 290 nm (21Houk Jr., T.W. Ue K. Anal. Biochem. 1974; 62: 66-74Crossref PubMed Scopus (298) Google Scholar). Concentrations of myosins and S1s were determined by the Bradford method using BSA as standard. In Vitro Motility Assay—Phosphorylated myosins were incubated with F-actin, and centrifuged after addition of ATP to remove denatured myosin, as described previously (22Ito K. Uyeda T.Q. Suzuki Y. Sutoh K. Yamamoto K. J. Biol. Chem. 2003; 278: 31049-31057Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Myosins were introduced at a concentration of 0.1 mg/ml into a flow chamber with a nitrocellulose-coated coverslip. The surface was subsequently blocked with 1 mg/ml BSA. To assay thermally inactivated proteins, the flow chamber, containing a BSA solution, was incubated in a block incubator at 40 °C for 2.5-25 min. Finally, a motility solution (25 mm imidazole-HCl (pH 7.5), 25 mm KCl, 4 mm MgCl2, 1 mm EGTA, 1% β-mercaptoethanol, and oxygen scavengers (4 mg/ml glucose, 0.2 mg/ml glucose oxidase, and 30 μg/ml catalase)) containing 0.2% methylcellulose, 5 nm F-actin labeled with rhodamine-phalloidin, and 1 mm or 50 μm ATP was applied to the flow chamber. The sliding of actin filaments was observed at 25 °C, and analyzed as described previously (23Chaen S. Nakaya M. Guo X.F. Watabe S. J. Biochem. (Tokyo). 1996; 120: 788-791Crossref PubMed Scopus (28) Google Scholar). Average velocity was determined by measuring displacement of actin filaments moving smoothly for 5 s or longer. Analysis of Motility Generated by Myosin Mixtures—Under the "Appendix" of their paper, Cuda et al. (24Cuda G. Pate E. Cooke R. Sellers J.R. Biophys. J. 1997; 72: 1767-1779Abstract Full Text PDF PubMed Scopus (88) Google Scholar) have proposed an analytical expression for the sliding velocity produced by myosin mixtures. Here, based on a formula obtained by Howard (25Howard J. Mechanics of Motor Proteins and the Cytoskeleton. Sinauer Associates, Sunderland, MA2001Google Scholar), we provide a slightly modified expression for the sliding velocity generated by mixtures of wild-type and mutant myosins. According to a simple two-state cross-bridge model that was originally proposed by Huxley (7Huxley A.F. Prog. Biophys. Biophys. Chem. 1957; : 255-318Crossref PubMed Google Scholar), Howard gave an expression for the average force as, 〈F〉=1/2(κΔ+2/d){1-exp(-kmaxd/V)}(1-2V2t-2Δ+2) where κ is the cross-bridge stiffness, δ+ is the powerstroke distance, d is the path distance, kmax is the maximum ATPase rate, t- is the drag time, and V is the sliding velocity. In a mixture of a fraction, σ, of the wild-type myosin and the corresponding fraction, 1 - σ, of the mutant myosin, the unloaded sliding velocity, V, will be the velocity for which σ 〈Fw 〉 + (1 - σ) 〈Fm 〉 = 0, where subscripts w and m denote the contributions from wild-type and mutant myosins, respectively. As described below, a steady-state ATPase assay showed that the difference in kmax between the wild-type and mutant myosins is small. Similarly, optical trap measurements showed that δ+ is similar for the wild-type and mutant myosins. Given that d is invariable, the balance of forces is given by, σ(1/2κwΔ+2-κwV2t-w2)+(1-σ)(1/2κmΔ+2-κmV2t-m2)=0 where κw and κm are elastic constants for the wild-type and mutant myosins, respectively. Solving for the unloaded sliding velocity V, V=Vw{σ+η(1-σ)}1/2/{σ+η(1-σ)(Vw/Vm)2}1/2 where (Vw=d+/(v2t-w) and (Vm=d+/(v2t-m) are the sliding velocity generated by the wild-type and mutant myosins alone, respectively, and η = κm/κw is the ratio of the elastic constants of the two myosins. Velocity data of the mixtures was fitted to Equation 3 using non-linear least-squares fitting. ATPase Assays—Basal and actin-activated Mg-ATPase activities were determined by measuring the release of phosphate at 25 °C using the methods of Kodama et al. (26Kodama T. Fukui K. Kometani K. J. Biochem. (Tokyo). 1986; 99: 1465-1472Crossref PubMed Scopus (274) Google Scholar). Reaction mixtures contained S1s (25-200 μg/ml) and various concentrations of F-actin (0-50 μm) in the assay buffer. Reactions were started by adding 1/5 volume of 6 mm ATP, and were stopped by adding 4 volumes of 0.3 m perchloric acid. To assay thermally inactivated proteins, reaction mixtures were incubated in a block incubator at 40 °C for 5-30 min before the addition of ATP. Laser Trap Experiments—The flow chamber used in the experiments was constructed from a 24 × 32-mm2 coverslip coated with 2.3-μm silica beads (Bangs Lab) suspended in 0.1% nitrocellulose in amyl acetate, and a 18 × 18-mm2 coverslip that were separated with a layer of silicon grease and two strips of adhesive tape. The chamber was first incubated with 0.25 μm Ni-NTA horseradish peroxidase conjugate (Qiagen) for 3 min, followed by an addition of 1 mg/ml BSA for 3 min. Purified S1 proteins were then applied to the chamber at a concentration of 1 μg/ml. At this concentration, the probability that myosinactin interaction was detected was <0.25, so that the probability that two or more myosins was bind to actin filaments would be <0.05. Following a 3-min incubation to allow the S1s to bind to the Ni-NTA horseradish peroxidase conjugate, the chamber was washed with 1 mg/ml BSA in the motility solution. Polystyrene beads (1 μm, Molecular Probes) were coated with N-ethylmaleimide-myosin according to Veigel et al. (27Veigel C. Bartoo M.L. White D.C. Sparrow J.C. Molloy J.E. Biophys. J. 1998; 75: 1424-1438Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). Finally, the chamber was filled with the motility solution containing 5 nm F-actin labeled with rhodamine-phalloidin, N-ethylmaleimidemyosin-coated beads, and 1 μm ATP, before the measurements were made. Single molecule mechanical measurements of S1s were performed using optical trap with the "three-bead" configuration as described by other investigators (27Veigel C. Bartoo M.L. White D.C. Sparrow J.C. Molloy J.E. Biophys. J. 1998; 75: 1424-1438Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 28Finer J.T. Simmons R.M. Spudich J.A. Nature. 1994; 368: 113-119Crossref PubMed Scopus (1583) Google Scholar, 29Molloy J.E. Burns J.E. Kendrick-Jones J. Tregear R.T. White D.C. Nature. 1995; 378: 209-212Crossref PubMed Scopus (527) Google Scholar). In brief, an infrared laser (1064 nm, 1000 milliwatts, IRCL-1W-1064; CrystaLaser) was split into two beams, using polarizing beam-splitters, to trap the two beads. A N-ethylmaleimide-myosin-coated bead was caught in each of the two laser traps, and each bead was attached to the end of the fluorescently labeled actin filaments. The actin filament was pulled taut (2 pN), and positioned over a silica bead. A ×60 oil immersion objective lens (PlanApo, NA 1.45; Olympus) was used for the optical trapping procedures and observations of the fluorescent actin filaments. The epifluorescence images of actin filaments and beads, both of which were excited by a green laser (532 nm, 25 milliwatt, GCL-025-L; CrystaLaser) were captured by an image intensifier (VS4-1845; VideoScope) and CCD camera (XC-ST50; Sony). The trapped bead image was illuminated by a halogen lamp and was projected onto a quadrant photodiode (S994-13; Hamamattsu-Photonics) to measure the bead displacement. The output from the quadrant photodiode was sampled at 10 kHz and stored in a personal computer by an AD converter (PowerLab/4sp, AD Instruments) to be analyzed off-line. The calibration of the bead displacement was performed by moving the stage on which the quadrant photodiode was mounted. The trap stiffness, κtrap, was determined by measuring the Brownian motion of the bead (variance, 〈x 〉2) and applying the equipartition law (1/2κ 〈x 〉2trap = 1/2kBT) (27Veigel C. Bartoo M.L. White D.C. Sparrow J.C. Molloy J.E. Biophys. J. 1998; 75: 1424-1438Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 29Molloy J.E. Burns J.E. Kendrick-Jones J. Tregear R.T. White D.C. Nature. 1995; 378: 209-212Crossref PubMed Scopus (527) Google Scholar). The laser trap experiments were performed at 23 °C at a trap stiffness of 0.05 pN nm-1. Light Scattering—Kinetics of thermal aggregation were determined by measuring light scattering of S1 solutions at 500 nm. The absorbance of 0.3 mg/ml S1 proteins in the assay buffer was measured in 1-cm cells using a DU 640 spectrophotometer (Beckman Coulter). Samples were maintained at 40 °C using circulating water. A probe thermometer (Anritsu) confirmed the temperature of cell contents. Apparent first-order rate constants of aggregation were determined from the slope of the Guggenheim plot. In Vivo Functions of Myosins—To examine the effects of mutation at Arg-705 within the SH1 helix on activities of myosin II, we have engineered the mutation at an equivalent site in Dictyostelium myosin II (R689H), and expressed the mutant in Dictyostelium myosin II-null cells. We first assessed the in vivo functions of the mutant myosin. Dictyostelium myosin II-null cells were unable to grow in suspension culture or to form fruiting bodies under starvation, providing evidence that myosin II was involved in cytokinesis and morphogenesis (11De Lozanne A. Spudich J.A. Science. 1987; 236: 1086-1091Crossref PubMed Scopus (763) Google Scholar). Myosin II-null cells expressing the R689H myosin grew only slightly more slowly than cells expressing wild-type myosin (doubling time, 16.6 and 14.3 h). The cells expressing the mutant developed completely to form normal fruiting bodies comparable with those of the cells expressing the wild-type myosin (Fig. 2). Thus, the R689H mutation only slightly affects functions of myosin II for normal growth and development of Dictyostelium cells. These results suggested that the mutant myosin retained some of the motile activities. Motile Activities of Myosins—To reveal in vitro activities of the mutant myosin, we purified wild-type and R689H myosin proteins and determined their motile activities using an in vitro motility assay. At a concentration of 1 mm ATP, R689H myosin moved actin filaments at an average velocity of 0.4 μms-1, whereas wild-type myosin moved actin filaments at 1.9 μms-1 (Fig. 3). The sliding velocity generated by mutant myosin was decreased to ∼20% of that generated by wild-type myosin. This result indicated that the R689H mutation in myosin II resulted in significant impairment in motile activity under physiological conditions. In contrast, at 50 μm ATP, the wild-type and mutant myosins exhibited comparable sliding velocities (Fig. 3). This result suggested that some steps other than ATP-induced actin-myosin dissociation were decelerated by the R689H mutation. ATPase Activities of S1s—To determine whether the mutation affects ATPase activities in solution, we expressed mutant S1, the soluble fragment of myosin. The basal Mg-ATPase activity of R689H S1 was 1.8 times higher than that of wild-type S1 (Table 1), suggesting that phosphate release was accelerated by the mutation. Fig. 4 shows actin-activated Mg-ATPase activities of wild-type and R689H S1s. They showed comparable activities over the range of the actin concentrations used. Fitting the data to the Michaelis-Menten equation yielded similar values of Vmax for wild-type and R689H S1s (Table 1). These results suggested that the R689H mutant retained normal actin activation of ATPase activities. This indicated that ATPase activity was not entirely coupled to motile activity for the mutant. Furthermore, R689H showed a somewhat lower Kactin (Table 1), suggesting that the mutant might have a higher affinity for actin in the presence of ATP.TABLE 1Kinetic parameters of steady-state ATPase activities of S1sBasalVmaxKactins−1μMWild type0.11 ± 0.0061.1255.0R689H0.20 ± 0.0061.0230.0 Open table in a new tab Single Molecule Mechanics of S1s—The sliding velocity of actin filaments in an in vitro motility assay is related to the step size and time spent in the strongly actin-bound state (30Spudich J.A. Nature. 1994; 372: 515-518Crossref PubMed Scopus (424) Google Scholar). To determine the mechanism underlying the reduction in motility, we performed single molecule mechanical measurements of wild-type and mutant S1s using an optical trap. Here, we have used S1 fragments to reveal the mechanical properties of a single myosin head. The S1 proteins contained a His tag at the COOH terminus to be attached to the substrate via horseradish peroxidase conjugated with Ni2+-nitrilotriacetic acid. At low surface densities of the S1 molecules and in the presence of 1 μm ATP, both the wild type and R689H behaved similarly to other myosin II derivatives (27Veigel C. Bartoo M.L. White D.C. Sparrow J.C. Molloy J.E. Biophys. J. 1998; 75: 1424-1438Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 28Finer J.T. Simmons R.M. Spudich J.A. Nature. 1994; 368: 113-119Crossref PubMed Scopus (1583) Google Scholar, 29Molloy J.E. Burns J.E. Kendrick-Jones J. Tregear R.T. White D.C. Nature. 1995; 378: 209-212Crossref PubMed Scopus (527) Google Scholar). They showed transient events of reduced thermal noise, presumably due to binding of a myosin to the actin filaments (Fig. 5A). Distributions of individual mean displacements produced by wild-type or mutant S1s are shown in Fig. 5B. These were fitted by single Gaussian distributions with a S.D. matching the combined stiffness of the optical trap (σ = (kBT/κtrap)½ ≈ 9 nm). The average step size of the myosin working stroke can be estimated from the positional shifts of these distributions. For the wild-type and R689H S1s, the average step size was not significantly different (p = 0.32, Student's t test), with values of ∼3 nm. Because our results were uncorrected for system elasticity, the step size may be underestimated. In the absence of ADP and at low concentrations of ATP, the duration of myosin attachments is primarily limited by the ATP-induced actin-myosin dissociation step, with the second-order ATP-induced dissociation rate constant (31Baker J.E. Brosseau C. Joel P.B. Warshaw D.M. Biophys. J. 2002; 82: 2134-2147Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Fig. 5C shows distributions of the duration of attachments for the wild-type and R689H S1s at 1 μm ATP. The distributions were fitted by single exponentials with rate constants of 0.4 and 1.3 s-1 for the wild-type and R689H, respectively. The rate constant of the wild-type dissociation was comparable with that of ATP-induced actin-Dictyostelium S1 dissociation at low ATP concentrations obtained from solution studies (17Uyeda T.Q. Tokuraku K. Kaseda K. Webb M.R. Patterson B. Biochemistry. 2002; 41: 9525-9534Crossref PubMed Scopus (23) Google Scholar, 22Ito K. Uyeda T.Q. Suzuki Y. Sutoh K. Yamamoto K. J. Biol. Chem. 2003; 278: 31049-31057Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 32Ritchie M.D. Geeves M.A. Woodward S.K. Manstein D.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8619-8623Crossref PubMed Scopus (98) Google Scholar). These results suggested that a single myosin molecule was responsible for the individual mechanical events. The dissociation of the mutant was somewhat faster than that of the wild-type S1. This result is consistent with the finding that the wild-type and mutant myosins exhibited comparable sliding velocities at 50 μm ATP in an in vitro motility assay. We also estimated total stiffness during the attachments, by applying the equipartition law to Brownian motion of the beads (27Veigel C. Bartoo M.L. White D.C. Sparrow J.C. Molloy