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Changes in Mg2+ Ion Concentration and Heavy Chain Phosphorylation Regulate the Motor Activity of a Class I Myosin

2004; Elsevier BV; Volume: 280; Issue: 7 Linguagem: Inglês

10.1074/jbc.m412473200

ISSN

1083-351X

Autores

Setsuko Fujita‐Becker, Ulrike Dürrwang, Muriel Erent, Richard J. Clark, Michael A. Geeves, Dietmar J. Manstein,

Tópico(s)

Cellular Mechanics and Interactions

Resumo

Class I myosins are single-headed motor proteins implicated in various motile processes including organelle translocation, ion channel gating, and cytoskeleton reorganization. Dictyostelium discoideum myosin-ID belongs to subclass 1α, whose members are thought to be tuned for rapid sliding. The direct analysis of myosin-ID motor activity is made possible by the production of single polypeptide constructs carrying an artificial lever arm. Using these constructs, we show that the motor activity of myosin-ID is activated 80-fold by phosphorylation at the TEDS site. TEDS site phosphorylation acts by stabilizing the actomyosin complex and increasing the coupling between actin binding and the release of hydrolysis products. A surprising effect of Mg2+ ions on in vitro motility was discovered. Changes in the level of free Mg2+ ions within the physiological range are shown to modulate motor activity by inhibiting ADP release. Our results indicate that higher concentrations of free Mg2+ ions stabilize the tension-bearing actin myosin ADP state and shift the system from the production of rapid movement toward the generation of tension. Class I myosins are single-headed motor proteins implicated in various motile processes including organelle translocation, ion channel gating, and cytoskeleton reorganization. Dictyostelium discoideum myosin-ID belongs to subclass 1α, whose members are thought to be tuned for rapid sliding. The direct analysis of myosin-ID motor activity is made possible by the production of single polypeptide constructs carrying an artificial lever arm. Using these constructs, we show that the motor activity of myosin-ID is activated 80-fold by phosphorylation at the TEDS site. TEDS site phosphorylation acts by stabilizing the actomyosin complex and increasing the coupling between actin binding and the release of hydrolysis products. A surprising effect of Mg2+ ions on in vitro motility was discovered. Changes in the level of free Mg2+ ions within the physiological range are shown to modulate motor activity by inhibiting ADP release. Our results indicate that higher concentrations of free Mg2+ ions stabilize the tension-bearing actin myosin ADP state and shift the system from the production of rapid movement toward the generation of tension. Class I myosins are produced by a wide range of organisms and cell types (1Mermall V. Post P.L. Mooseker M.S. Science. 1998; 279: 527-533Crossref PubMed Scopus (522) Google Scholar). They share a conserved motor domain, a light chain-binding domain, and a tail region that contains a polybasic region that directly binds to membranes via electrostatic interactions (2Doberstein S.K. Pollard T.D. J. Cell Biol. 1992; 117: 1241-1249Crossref PubMed Scopus (102) Google Scholar, 3Miyata H. Bowers B. Korn E.D. J. Cell Biol. 1989; 109: 1519-1528Crossref PubMed Scopus (79) Google Scholar). Phylogenetic analysis reveals that there are at least four myosin I subclasses. Dictyostelium discoideum myosin-ID is a member of the amoeboid subclass. Despite its name, this is the most widely expressed subclass. Amoeboid class I myosins have two additional domains in their tails that are involved in ATP-insensitive actin binding. The first domain is a region rich in the amino acids glycine, serine, and alanine (or glutamate or serine) (4Lynch T.J. Brzeska H. Miyata H. Korn E.D. J. Biol. Chem. 1989; 264: 19333-19339Abstract Full Text PDF PubMed Google Scholar, 5Jung G. Hammer III, J.A. FEBS Lett. 1994; 342: 197-202Crossref PubMed Scopus (58) Google Scholar, 6Lynch T.J. Albanesi J.P. Korn E.D. Robinson E.A. Bowers B. Fujisaki H. J. Biol. Chem. 1986; 261: 17156-17162Abstract Full Text PDF PubMed Google Scholar), and the second domain is a Src homology 3 domain (7Titus M.A. Wessels D. Spudich J.A. Soll D. Mol. Biol. Cell. 1993; 4: 233-246Crossref PubMed Scopus (117) Google Scholar, 8Geli M.I. Lombardi R. Schmelzl B. Riezman H. EMBO J. 2000; 19: 4281-4291Crossref PubMed Scopus (97) Google Scholar).The regulation of class I myosins from a wide range of organisms appears to be mediated by the phosphorylation of a serine or threonine residue in the motor domain that is located 16 residues upstream of the highly conserved DALAK sequence. Vertebrate class I myosins, like nearly all other myosins, have negatively charged glutamate or aspartate residues at the corresponding position. Therefore the phosphorylation site is generally referred to as the TEDS site (9Bement W.M. Mooseker M.S. Cell Motil. Cytoskel. 1995; 31: 87-92Crossref PubMed Scopus (147) Google Scholar). The TEDS site resides in a surface loop that forms part of the actin-binding site. The heavy chain of myosin-ID is an in vitro substrate for members of the p21-activated kinase/STE20 family of protein kinases (10Brzeska H. Young R. Tan C. Szczepanowska J. Korn E.D. J. Biol. Chem. 2001; 276: 47468-47473Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar, 11Wu C. Lee S.F. Furmaniak-Kazmierczak E. Cote G.P. Thomas D.Y. Leberer E. J. Biol. Chem. 1996; 271: 31787-31790Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 12Brzeska H. Young R. Knaus U. Korn E.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 394-399Crossref PubMed Scopus (37) Google Scholar, 13Lee S.F. Mahasneh A. de la Roche M. Cote G.P. J. Biol. Chem. 1998; 273: 27911-27917Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). p21-activated kinases become activated by interaction with lipids and the GTP-bound forms of Rac and Cdc42, leading to reduced inhibition of the catalytic domain by the regulatory domain (14Buchwald G. Hostinova E. Rudolph M.G. Kraemer A. Sickmann A. Meyer H.E. Scheffzek K. Wittinghofer A. Mol. Cell. Biol. 2001; 21: 5179-5189Crossref PubMed Scopus (88) Google Scholar). Although the importance of TEDS site phosphorylation for the kinetic behavior and in vivo function of class I myosins is well documented, the direct effect of TEDS site phosphorylation on the motile activity of class I myosins is less well understood. Here, we generated three types of recombinant constructs to analyze the functional properties of D. discoideum myosin-ID. Motor domain constructs were used to determine steady-state and transient kinetic parameters. To directly measure the motor activity of myosin-ID, we generated a motor domain fragment with an artificial lever arm consisting of D. discoideum α-actinin repeats 1 and 2 (15Anson M. Geeves M.A. Kurzawa S.E. Manstein D.J. EMBO J. 1996; 15: 6069-6074Crossref PubMed Scopus (135) Google Scholar, 16Kliche W. Fujita-Becker S. Kollmar M. Manstein D.J. Kull F.J. EMBO J. 2001; 20: 40-46Crossref PubMed Scopus (54) Google Scholar). The terminology used (D692 and D692–2R) refers to the type of myosin-I and the site of motor domain truncation. 2R serves as abbreviation for the attachment of two α-actinin repeats to the motor domain. To study the effects of the TEDS site phosphorylation on the motor properties, we generated the dephosphorylated and phosphorylated forms of D692–2R by treatment with λ-phosphatase and Acanthamoeba castellanii myosin-I heavy chain kinase (17Brzeska H. Szczepanowska J. Hoey J. Korn E.D. J. Biol. Chem. 1996; 271: 27056-27062Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Additionally, we generated mutant versions of D692–2R, which facilitated the combined characterization of motor and kinetic properties. The serine at the TEDS site of these constructs was replaced by either an alanine residue, to mimic the unphosphorylated state, or a glutamate residue, to mimic the phosphorylated state (18Wang Z.Y. Wang F. Sellers J.R. Korn E.D. Hammer III, J.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15200-15205Crossref PubMed Scopus (36) Google Scholar, 19De La Cruz E.M. Ostap E.M. Sweeney H.L. J. Biol. Chem. 2001; 276: 32373-32381Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). These constructs are referred to as S332A or S332E mutants in the following text.EXPERIMENTAL PROCEDURESReagents—Standard chemicals were purchased from Sigma, and restriction enzymes, polymerases, and DNA-modifying enzymes were from Roche Applied Science. TRITC-labeled 1The abbreviations used are: TRITC, tetramethylrhodamine isothiocyanate; MlcD, D. discoideum myosin-ID light chain; MOPS, 4-morpholinepropanesulfonic acid. phalloidin was a gift from Dr. H. Faulstich (20Faulstich H. Trischmann H. Mayer D. Exp. Cell Res. 1983; 144: 73-82Crossref PubMed Scopus (96) Google Scholar).Cell Growth and Transformation—Dictyostelium cells were grown in HL-5C medium (21Watts D.J. Ashworth J.M. Biochem. J. 1970; 119: 171-174Crossref PubMed Scopus (835) Google Scholar). The cells were transformed by electroporation (22de Hostos E.L. Bradtke B. Lottspeich F. Guggenheim R. Gerisch G. EMBO J. 1991; 10: 4097-4104Crossref PubMed Scopus (242) Google Scholar). G418 was used as selectable marker at 10 μg/μl.Plasmid Constructs and Mutagenesis—Genomic DNA was isolated from AX2 cells according to Ref. 23Bain G. Tsang A. Mol. Gen. Genet. 1991; 226: 59-64Crossref PubMed Scopus (13) Google Scholar. PCR-directed mutagenesis was used to isolate myoD gene fragments encoding the motor domains with a unique BamHI site at the 5′-end of the coding region and a unique XhoI site at position 692. The PCR products were digested with BamHI and XhoI and cloned into pDXA-3H (24Manstein D.J. Schuster H.P. Morandini P. Hunt D.M. Gene (Amst.). 1995; 162: 129-134Crossref PubMed Scopus (192) Google Scholar), which carries sequences for the fusion of a C-terminal His8 tag. The resulting plasmids were analyzed by sequencing. For the production of motor domain constructs fused to two D. discoideum α-actinin repeats (2R), a DNA fragment encoding 2R, a Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly linker, enhanced yellow fluorescent protein, and a His8 tag was obtained as XhoI/SphI fragment from pM790–2R-EYFP (25Knetsch M.L. Tsiavaliaris G. Zimmermann S. Ruhl U. Manstein D.J. J. Mus. Res. Cell Motil. 2002; 23: 605-611Crossref PubMed Scopus (29) Google Scholar) and inserted in the XhoI/SphI-digested myosin-ID motor domain expression plasmid.Direct Functional Assays—Actin sliding motility was measured as described previously (15Anson M. Geeves M.A. Kurzawa S.E. Manstein D.J. EMBO J. 1996; 15: 6069-6074Crossref PubMed Scopus (135) Google Scholar, 26Kron S.J. Spudich J.A. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 6272-6276Crossref PubMed Scopus (704) Google Scholar). TEDS site phosphorylation was performed by mixing 1 mg/ml D692–2R with 0.027 mg/ml activated kinase and incubation in the presence of 1 mm EGTA, 3 mm MgCl2, and 2 mm ATP at 30 °C for 20 min. A. castellanii myosin-I heavy chain kinase was activated by autophosphorylation at 30 °C for 20 min in a buffer containing 100 mm imidazole, pH 7.0, 4 mm ATP, 6 mm MgCl2, and 2 mm EGTA (12Brzeska H. Young R. Knaus U. Korn E.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 394-399Crossref PubMed Scopus (37) Google Scholar). A. castellanii myosin-I heavy chain kinase was generously provided by Drs. E. D. Korn and H. Brzeska (Laboratory of Cell Biology, NHLBI, National Institutes of Health). Dephosphorylation was performed by incubation of 1 mg/ml D692–2R with 4000 units/ml λ-protein phosphatase in the presence of 4 mm dithiothreitol, 2 mm MnCl2, and 0.01% Brij 35 at 30 °C for 30 min.Kinetic Measurements—Stopped flow measurements were performed at 20 °C with a Hi-tech Scientific SF-61 DX2 double mixing stopped flow system using the procedures and kinetic models described previously (27Cremo C.R. Geeves M.A. Biochemistry. 1998; 37: 1969-1978Crossref PubMed Scopus (148) Google Scholar, 28Furch M. Fujita-Becker S. Geeves M.A. Holmes K.C. Manstein D.J. J. Mol. Biol. 1999; 290: 797-809Crossref PubMed Scopus (70) Google Scholar, 29Batra R. Geeves M.A. Manstein D.J. Biochemistry. 1999; 38: 6126-6134Crossref PubMed Scopus (53) Google Scholar, 30Kurzawa S.E. Geeves M.A. J. Mus. Res. Cell Motil. 1996; 17: 669-676Crossref PubMed Scopus (69) Google Scholar). The binding and hydrolysis of ATP by myosin-ID head fragments were analyzed in terms of the seven-step model (see Scheme 1) described by Bagshaw et al. (31Bagshaw C.R. Eccleston J.F. Eckstein F. Goody R.S. Gutfreund H. Trentham D.R. Biochem. J. 1974; 141: 351-364Crossref PubMed Scopus (227) Google Scholar). Transients in the presence of actin were analyzed in terms of Schemes 2 and 3 (32Millar N.C. Geeves M.A. FEBS Lett. 1983; 160: 141-148Crossref PubMed Scopus (66) Google Scholar, 33Siemankowski R.F. White H.D. J. Biol. Chem. 1984; 259: 5045-5053Abstract Full Text PDF PubMed Google Scholar). Steady-state ATPase activities were measured at 25 °C with the NADH-coupled assay (34Furch M. Geeves M.A. Manstein D.J. Biochemistry. 1998; 37: 6317-6326Crossref PubMed Scopus (147) Google Scholar) in a buffer containing 25 mm HEPES, 25 mm KCl, and 4 mm MgCl2. The myosin concentration was 0.25–1 μm. NADH oxidation was followed using the change in absorption at 340 nm in a Beckman DU-650 spectrophotometer. The values for kcat and Kapp were calculated from fitting the data to the Michaelis-Menten equation. The apparent second order rate constant for actin binding (kcat/Kapp) was obtained from the calculated ratio of both values. Additionally, at concentrations of actin much lower than Kapp, the data were fitted to a straight line, and kcat/Kapp was determined from the slope of this line. The transient kinetics data were interpreted as described previously (27Cremo C.R. Geeves M.A. Biochemistry. 1998; 37: 1969-1978Crossref PubMed Scopus (148) Google Scholar, 29Batra R. Geeves M.A. Manstein D.J. Biochemistry. 1999; 38: 6126-6134Crossref PubMed Scopus (53) Google Scholar, 34Furch M. Geeves M.A. Manstein D.J. Biochemistry. 1998; 37: 6317-6326Crossref PubMed Scopus (147) Google Scholar, 35Kurzawa S.E. Manstein D.J. Geeves M.A. Biochemistry. 1997; 36: 317-323Crossref PubMed Scopus (76) Google Scholar).Scheme 2View Large Image Figure ViewerDownload Hi-res image Download (PPT)Scheme 3View Large Image Figure ViewerDownload Hi-res image Download (PPT)RESULTSActin Activation of ATPase and Motor Activity Is Dependent on TEDS Phosphorylation—To investigate the regulation of myosin-ID by TEDS site phosphorylation, we used mutant constructs in which the serine at the TEDS site was mutated to either glutamate or alanine to mimic the phosphorylated and dephosphorylated states of the protein.The steady-state ATPase activity was measured using motor domain construct D692 with TEDS site mutations S332A and S332E. Wild-type and mutant constructs could be purified in sufficient quantities for detailed kinetic analysis. In the absence of actin filaments, all of the constructs displayed similar ATPase activity. To determine the maximum values of the ATPase activity and the efficiency of coupling between actin and nucleotide binding, we measured the ATPase rates with actin concentrations in the range of 0–60 μm F-actin (Fig. 1A). The parameters kcat, Kapp, and kcat/Kapp were obtained by fitting the data to the Michaelis-Menten equation. They are presented in Table I. Kapp is the apparent dissociation equilibrium constant for actin binding in the presence of ATP, and kcat gives the maximum value of the ATPase activity. The apparent second order rate constant for actin binding (kcat/Kapp) indicates the coupling efficiency between actin and nucleotide binding. The ATPase activity of D692(S332A) was only slightly activated by the addition of F-actin. In contrast the ATPase activity of D692(S332E) was strongly activated by actin and showed a hyperbolic dependence on actin concentration. The coupling efficiency of D692(S332E) is 80 times higher than that of the Ser-to-Ala mutants.Fig. 1Interaction of myosin-ID with F-actin.A, actin stimulation of the steady-state ATPase activities of D692(S332A), D692, and D692(S332E). B, interaction of the actin-bound protein with nucleotides and ATP-induced dissociation of pyr-acto·D692. At low ATP concentrations kobs was linearly dependent upon [ATP], and the slopes define the second order binding K1k+2. In each case the intercept was not significantly different from zero. The symbols correspond to the following constructs D692(S332A) (filled circle), D692 (half-filled circle), and D692(S332E) (open circle). C, stopped flow records of the increase in pyrene fluorescence during the pyr-actin displacement of 0.5 μm of the pyr-acto·D692 complex with 20 μm unlabeled actin. The best fits to a single exponential are superimposed with kobs = 0.0027, 0.0430, and 0.0872 s-1 for D692(S332A), D692, and D692(S332E), respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IActin activation of ATPase activity The experimental conditions were 25 mm HEPES, pH 7.4, 25 mm KCl, and 4 mm MgCl2 at 25 °C.Myosin constructATPase activityaActin-activated ATPase activity was measured in the presence of 20 μm rabbit skeletal muscle F-actin. ATPase activation = (actin-activated ATPase - basal ATPase)/basal ATPaseMichaelis-Menten parametersbThe values for kcat and Kapp were calculated from fitting the data to the Michaelis-Menten equation,cThe apparent second order rate constant for actin binding (kcat/Kapp) was obtained from the calculated ratio of both values. Alternatively the data at concentrations of actin much lower than Kapp could be fit to a straight line, and kcat/Kapp was determined from the slope of this lineBasalActivatedActivationkcatKappkcat/Kapps-1s-1s-1μmμm-1 s-1D692(S332A)0.09 ± 0.010.10 ± 0.020.1NDdND, not determinedND0.001D6920.12 ± 0.020.17 ± 0.030.3NDND0.004D692(S332E)0.10 ± 0.021.34 ± 0.1012.410.0 ± 3.0130 ± 500.077M7650.08 ± 0.010.68 ± 0.097.52.6 ± 0.473 ± 200.036a Actin-activated ATPase activity was measured in the presence of 20 μm rabbit skeletal muscle F-actin. ATPase activation = (actin-activated ATPase - basal ATPase)/basal ATPaseb The values for kcat and Kapp were calculated from fitting the data to the Michaelis-Menten equationc The apparent second order rate constant for actin binding (kcat/Kapp) was obtained from the calculated ratio of both values. Alternatively the data at concentrations of actin much lower than Kapp could be fit to a straight line, and kcat/Kapp was determined from the slope of this lined ND, not determined Open table in a new tab Motor function was analyzed directly by measuring the average gliding velocity of actin filaments in an in vitro motility assay (26Kron S.J. Spudich J.A. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 6272-6276Crossref PubMed Scopus (704) Google Scholar). To investigate the regulation of myosin-ID by TEDS site phosphorylation, we treated the purified D692–2R motor domain constructs with λ-phosphatase to generate the dephosphorylated form or with myosin-I kinase (12Brzeska H. Young R. Knaus U. Korn E.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 394-399Crossref PubMed Scopus (37) Google Scholar) to generate the phosphorylated form. Additionally, we used D692–2R constructs in which the serine at the TEDS site was mutated to either glutamate or alanine.For each construct, the movement of at least 50 filaments was followed, and the velocity was determined. The average sliding velocities are summarized in Table II. The phosphorylated form of D692–2R showed an average velocity of 890 nm s-1, whereas the dephosphorylated protein displayed no detectable motile activity. Most filaments stayed on the assay surface after the addition of Mg2+ATP. Similar changes in motile activity were observed for the TEDS site mutants. The Ser-to-Glu mutant moved actin filaments with an average velocity of 670 nm s-1, 13-fold faster than the Ser-to-Ala mutant.Table IIMotile activity of myosin-ID constructsAverage sliding velocityaThe uncertainties represent standard deviations of the mean values,bThe experimental conditions were 2 mm ATP, 4 mm MgCl2, 25 mm imidazole, pH 7.4, 25 mm KCl, 1 mm EGTA, and 10 mm dithiothreitol at 30 °CD692-2RM765-2Rμm/sUntreated0.13 ± 0.040.82 ± 0.07Phosphatase-treatedNo motilityNAeNA, not applicableKinase-treated0.89 ± 0.10NAS332A0.05 ± 0.02NAS332E0.67 ± 0.09NAHigh [ATP]cThe experimental conditions were 10 mm ATP, 4 mm MgCl2, 25 mm imidazole, pH 7.4, 25 mm KCl, 1 mm EGTA, and 10 mm dithiothreitol at 30 °C1.78 ± 0.20dMeasured for D692-2R(S332E)0.83 ± 0.08a The uncertainties represent standard deviations of the mean valuesb The experimental conditions were 2 mm ATP, 4 mm MgCl2, 25 mm imidazole, pH 7.4, 25 mm KCl, 1 mm EGTA, and 10 mm dithiothreitol at 30 °Cc The experimental conditions were 10 mm ATP, 4 mm MgCl2, 25 mm imidazole, pH 7.4, 25 mm KCl, 1 mm EGTA, and 10 mm dithiothreitol at 30 °Cd Measured for D692-2R(S332E)e NA, not applicable Open table in a new tab TEDS Site Phosphorylation Stabilizes the Acto·Myosin-ID Complex—The rate of actin binding was measured following the exponential decrease in pyrene fluorescence observed on binding of an excess of pyrene-actin to the myosin-ID constructs. The observed rate constants were plotted against pyrene-actin concentration, and the kobs values were linearly dependent upon actin concentration over the range studied (Fig. 1B). The second order rate constants of pyrene-actin binding (k+A) were obtained from the slope of the plot, and the resulting values are summarized in Table III. A 3-fold decrease in k+A was observed for D692(S332E) compared with the Ser- to-Ala mutant.Table IIIActin affinity Treatment of the TEDS site mutant constructs with apyrase did not result in significant changes in the observed rate constants. KA, KAD, and KD are defined as dissociation equilibrium constants. The experimental conditions were 20 mm MOPS, pH 7.0, 5 mm MgCl2, and 100 mm KCl at 20 °C.Myosin constructk+Ak-AKAμm-1 s-1ms-1nmD6923.5 ± 0.243.0 ± 0.712.3D692(S332A)4.1 ± 0.387.2 ± 1.921.1D692(S332E)1.4 ± 0.12.7 ± 0.21.9M7650.8 ± 0.12.2 ± 0.22.9 Open table in a new tab The rate constant for actin dissociation (k-A) was determined by chasing pyrene-actin with a 40-fold excess of unlabeled actin (Fig. 1C). The observed process could be fitted to a single exponential, where kobs corresponds directly to k-A. In contrast to their similar rates of actin binding, the mutant constructs displayed significant differences in the rates of actin dissociation. Actin dissociates 30 times faster from D692(S332A) than from the Ser-to-Glu mutant. The dissociation equilibrium constant for actin binding (KA), as calculated from the ratio of k-A and k+A, indicates an 11-fold increased actin affinity for D692(S332E) compared with the Ser-to-Ala mutant (Scheme 1).Binding of Nucleotide to Myosin-ID—The rate constants measured for nucleotide binding to wild-type constructs and to TEDS site mutants were mostly identical. Therefore, although all of the measurements were performed with wild-type and mutant constructs, we will refer in most instances only to the wild-type construct.Rates of ATP binding (K1k+2) and ADP binding (k-6/K7) were monitored from the increase in intrinsic protein fluorescence following the addition of excess ATP or ADP (Scheme 2). The observed increases in intrinsic protein fluorescence were 10 and 14% for ADP and ATP binding, respectively. Myosin-ID has the conserved tryptophan in the relay loop, which reports the open to closed transition of Switch II that accompanies ATP hydrolysis (36Batra R. Manstein D.J. Biol. Chem. 1999; 380: 1017-1023Crossref PubMed Scopus (49) Google Scholar). Additionally, we measured the binding of the nucleotide analogues 2′(3′)-O-(N-methylanthraniloyl)-adenosine 5′-triphosphate and 2′(3′)-O-(N-methylanthraniloyl)-adenosine 5′-diphosphate by following the fluorescence enhancement after mixing with the D692 motor domain constructs. The results of these measurements were analyzed as described previously (30Kurzawa S.E. Geeves M.A. J. Mus. Res. Cell Motil. 1996; 17: 669-676Crossref PubMed Scopus (69) Google Scholar) and are summarized in Table IV. Our results show that for myosin-ID the apparent second order association rate constants (K1k+2 or k-6/K7) are similar for ATP, ADP, and the mant analogues. At high ATP concentrations the rate of binding saturates for D692 at 640 s-1. For most myosins this maximum rate constant has been attributed to the rate constant for the ATP hydrolysis step (k+3 + k-3), which is signaled by the fluorescence change of the tryptophan located at the tip of the relay loop (36Batra R. Manstein D.J. Biol. Chem. 1999; 380: 1017-1023Crossref PubMed Scopus (49) Google Scholar).Table IVRate and equilibrium constants of nucleotide binding to myosin and acto·myosin complexes For comparison, the parameters obtained for D. discoideum myosin II motor domain fragment M765 are shown.NucleotideRate constantD692M765Nucleotide binding to myosinATPK1k2 (μm−1 s−1)0.66 ± 0.010.56 ± 0.03kmax (s−1)640 ± 1030 ± 1mantATPK1k2 (μm−1 s−1)0.53 ± 0.020.81 ± 0.02ADPk−6/K7 (μm−1 s−1)0.98 ± 0.02No signalk+6 (s−1)0.60 ± 0.002No signalKD (μm)aThe values are derived from biphasic ADP dissociation reactions at different ADP concentrations for D692—2R(S332E). The value in parentheses is obtained from the calculated k+6/(k−6/K7).1.9 ± 0.3 (0.6)(14)bRef. 29.mantADPk−6/K7 (μm−1 s−1)0.87 ± 0.010.36 ± 0.004Nucleotide binding to acto⋅ myosinATPK1k+2 (μm−1 s−1)0.49 ± 0.010.16 ± 0.002k+2 (s−1)960 ± 20490 ± 20ADPcThe values refer to the Ser-to-Glu mutants of the myosin-I motor domain constructs. The KAD value for D692(S332A) is 118 μm. KAD and KD are defined as dissociation equilibrium constants. The experimental conditions were 20 mm MOPS, pH 7.0, 5 mm MgCl2, and 100 mm KCl at 20 °C.KAD (μm)75 ± 4253 ± 11KAD/KD4018a The values are derived from biphasic ADP dissociation reactions at different ADP concentrations for D692—2R(S332E). The value in parentheses is obtained from the calculated k+6/(k−6/K7).b Ref. 29.c The values refer to the Ser-to-Glu mutants of the myosin-I motor domain constructs. The KAD value for D692(S332A) is 118 μm. KAD and KD are defined as dissociation equilibrium constants. The experimental conditions were 20 mm MOPS, pH 7.0, 5 mm MgCl2, and 100 mm KCl at 20 °C. Open table in a new tab As mentioned above, ATP binding to myosin-ID constructs produced a larger fluorescence increase than the binding of ADP. Therefore, the displacement of ADP by ATP could be followed from the net increase in fluorescence upon displacement of excess ADP from the D692-ADP complex by the addition of a larger excess ATP. The rate of ADP release from D692 (k+6) was 0.60 s-1. At intermediate ADP concentrations ( 2 mm), the observed rate constants saturate, and the dependence on the ATP concentration could be described by a hyperbola, where the maximum value of kobs defines k+2 (Fig. 2A, inset).Fig. 2Transient kinetic analysis of the interaction of myosin-ID with actin and nucleotides.A, ATP-induced dissociation of the actomyosin complex. The observed rate constant for D692 is linearly dependent on the ATP concentration in the range 5–25 μm. The apparent second order rate constant for ATP binding to actomyosin (K1k+2) was determined from the slope of the line. The inset shows data over the range from 0.005 to 8 mm fitted to a hyperbola. The rate constants for the isomerization step are given by the plateau values. B, ADP inhibition of ATP-induced dissociation of the actomyosin complex of D692(S332E). Monophasic dissociation reactions are observed in the presence of different amounts of ADP, compatible with a fast equilibrium for ADP binding. C, the observed rate constants were plotted against the ADP concentration, and the data were fitted with a hyperbola.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The affinity of ADP for the complex formed by the myosin-ID motor domain and F-actin (KAD) was determined from the inhibition of the ATP-induced dissociation of acto·D692 by ADP. The observed rate of dissociation was reduced

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