From the Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Komaba 3-8-1, Tokyo 153, Japan
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ABSTRACT |
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A loop comprising residues 454-459 of Dictyostelium myosin II is structurally and functionally equivalent to the switch II loop of the G-protein family. The consensus sequence of the "switch II loop" of the myosin family is DIXGFE. In order to determine the functions of each of the conserved residues, alanine scanning mutagenesis was carried out on the Dictyostelium myosin II heavy chain gene. Examination of in vivo and in vitro motor functions of the mutant myosins revealed that the I455A and S456A mutants retained those functions, whereas the D454A, G457A, F458A and E459A mutants lost them. Biochemical analysis of the latter myosins showed that the G457A and E459A mutants lost the basal ATPase activity by blocking of the isomerization and hydrolysis steps of the ATPase cycle, respectively. The F458A mutant, however, lost the actin-activated ATPase activity without loss of the basal ATPase activity. These results are discussed in terms of the crystal structure of the Dictyostelium myosin motor domain.
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INTRODUCTION |
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In the Dictyostelium motor domain designated as S1dC
(1), a bound nucleotide is surrounded by three loops whose sequences are highly conserved among the myosin family (2): the P-loop (residues
179-186 of Dictyostelium myosin II) and the two loops in
the 50K segment (residues 233-240 and 454-459 of
Dictyostelium myosin II) (see Fig. 1A). One of
the loops in the 50K segment (residues 233-240) is homologous to a
loop in the switch I region of GTPases judging from the topological
similarity (3) and has the consensus sequence NXNSSRFG
(NNNSSRFG in Dictyostelium myosin II). Residues in the loop
are aligned along the ATPase pocket, and some of the side chains form
hydrogen bonds with the bound nucleotide. The other loop in the 50K
segment has the consensus sequence, DIXGFE (DISGFE in
Dictyostelium myosin II) and is functionally and
structurally equivalent to a loop in the switch II region of GTPases
(3). In GTPases, the switch II loop connects the GTPase site and the
switch II -helix, which is part of the effector binding region.
Information on the nucleotide state at the GTPase site is transmitted
to the effector binding region partially through this switch II loop.
In myosin, the switch II loop connects the ATPase pocket and a long
conserved
-helix embedded in the lower 50K subdomain (4, 5).
Recent x-ray crystallographic studies on Dictyostelium S1dC
complexed with various nucleotides and nucleotide analogs revealed that
the switch II loop undergoes a significant conformational change during
ATP hydrolysis (Fig. 1B) like
the loop in GTPases. When S1dC is complexed with MgADP/Vi
or MgADP/AlFx (the Vi structure), the switch II loop is
closer to the ATPase pocket, although the loop moves away from the
ATPase pocket when S1dC is complexed with MgADP/BeFx, MgAMPPNP,
MgATPS,1 or MgADP (the
BeFx structure) (5-7). The observed changes in the switch II loop
arise from the main chain rotation at the two pivoting residues,
Ile-455 and Gly-457. In the Vi structure, Gly-457 and
Glu-459 are close to the bound nucleotide. Gly-457 forms a hydrogen
bond with an oxygen atom of the Vi moiety of the bound MgADP/Vi. Glu-459 coordinates with a water molecule that is
expected to attack the bound ATP (6) and also forms an ionic bond with R238 in such a way as to close the exit ("backdoor") through which Pi may be released on ATP hydrolysis (8) (Fig.
1B). In the BeFx structure, however, Gly-457 and Glu-459 are
located away from the bound nucleotide. Moreover, the ionic bond
between Arg-238 and Glu-459 is broken, and thus the exit is open (Fig.
1B). This conformational change of the loop is accompanied
by rigid body motion of the upper and lower 50K subdomains to open and
close the 50K cleft. This opening and closure of the cleft may then trigger the swinging of the lever arm, a long
-helix with bound light chains. Thus, the switch II loop of myosin seems to plays a
critical role in the conversion of energy derived from ATP hydrolysis into sliding and force generation.
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A lower eukaryote, Dictyostelium discoideum, has a single copy of the heavy chain gene of myosin II (9). Knock-out of this gene generates myosin II-null cells (10), which show myosin-specific defects in growth and development. These myosin-specific phenotypic defects can be reversed by introducing a multicopy plasmid bearing the wild-type heavy chain gene of myosin II (11). Using this Dictyostelium discoideum system established by Spudich and coworkers (10-21), alanine scanning mutagenesis of the switch II loop was carried out to determine how residues in the switch II loop are involved in the energy conversion. The effects of mutations were studied by examining the phenotypes of cells expressing the mutant myosins and also by examining the in vitro motor functions of the purified mutant myosins.
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EXPERIMENTAL PROCEDURES |
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Construction and Expression of Recombinant Myosin and S1dC Genes-- Each residue from Asp-454 to Glu-459 was changed to alanine by site-directed mutagenesis (22, 23) of the Dictyostelium myosin II heavy chain gene (9). The mutant myosin heavy chain genes were ligated to the Dictyostelium actin-15 promoter and Dictyostelium actin-6 terminator to drive their expression in Dictyostelium cells. They were finally inserted into a multicopy extrachromosomal vector, pBIG (24). Plasmids carrying the mutant myosin heavy chain genes were introduced into Dictyostelium myosin-null cells in which the myosin II heavy chain gene had been knocked out by means of homologous recombination (10). Dictyostelium cells transformed by electroporation were selected in a medium supplemented with 20 µg/ml of G418 on plastic dishes for a week. The transformed cells thus obtained expressed the mutant myosin II molecules.
The truncated myosin heavy chain gene corresponding to S1dC (1) was manipulated as above. The C terminus of S1dC was truncated at Arg-761 by introducing a stop codon at the corresponding location. For easier purification of S1dC, a 6-histidine tag (His6) was attached at the N terminus of S1dC by inserting the corresponding DNA sequence between the start codon and the coding sequence of the myosin heavy chain. The resulting transformation vectors were introduced into Dictyostelium AX2 cells.Random Mutagenesis of Glu-459-- Random mutagenesis of the 459th residue was carried out by polymerase chain reaction with the E459A mutant myosin gene as a template. The E459A myosin gene was used as the template instead of the wild-type myosin gene to avoid excess representation of the wild-type gene in the library of mutagenized genes. The random representation of codons in the library was directly confirmed by sequencing some clones. A collection of mutant myosin genes was then inserted into a multicopy Dictyostelium transformation vector bearing the blasticidin-resistance gene, pBIGBsr (25). By means of electroporation, myosin-null cells were transformed with pBIGBsr-based plasmids bearing myosin genes with all possible codons for the 459th residue. The electroporated cells were cultured overnight and then plated onto agar plates including blasticidin (20 µg/ml) together with Escherichia coli cells. After several days at 22 °C, plaques generated by Dictyostelium cells became visible on the E. coli lawns. Starting from 107 myosin-null cells, 672 plaques were obtained. Two types of plaques were easily distinguishable because of the clear difference in their diameters. Cells were cloned from all of the larger plaques (36 plaques). Plasmid DNA was rescued from randomly chosen clones (20 clones) and then sequenced.
Phenotypes of the Transformed Cells-- The growth rates were measured by determining the numbers of cells cultured in suspension. The incubator was shaken at 150 rpm at 22 °C. Development of the transformed cells was examined on agar plates covered with a lawn of E. coli cells. Dictyostelium cells (1.2 × 104) were suspended in 10 mM Tris-Cl, pH 7.5, and then spotted onto the bacterial lawn. When Dictyostelium cells had cleared the bacterial cells, they entered the developmental stage.
Protein Purification-- Phosphorylated myosin was prepared as described previously (32). For experiments involving low concentrations of fluorescent nucleotides (mant-ATP and Cy3-ATP), myosin was purified further to remove the contaminating nucleotides. Myosin eluted from the HPLC column was dialyzed against a solvent comprising 50 mM NaCl, 10 mM MOPS, pH 7.4, and 2 mM MgCl2 to form filaments. The filaments were collected by centrifugation at 560,000 × g for 30 min and then dissolved in 0.35 M NaCl and 10 mM MOPS, pH 7.4.
The wild-type or mutant S1dC bearing an N-terminal histidine tag was extracted from the transformed Dictyostelium cells, and precipitated as an actoS1dC complex by dialysis against a solvent comprising 50 mM NaCl, 10 mM MOPS, pH 7.4, and 2 mM MgCl2. S1dC was then extracted from the precipitate with a solvent comprising 10 mM MOPS, pH 7.4, 0.25 M NaCl, 7 mM MgCl2, and 5 mM ATP. The extract was directly applied to an NTA-Ni column (Qiagen), an affinity column for histidine-tagged proteins. After washing the column with a column volume of the above solvent containing 1 mM ATP, S1dC was eluted with a linear gradient of imidazole, pH 7.4, from 10 mM to 0.5 M. The eluted protein was dialyzed against a solvent comprising 50 mM NaCl, 10 mM MOPS, pH 7.4, and 2 mM MgCl2. Since the majority of the expressed E459A S1dC remained unbound to F-actin during the formation of actoS1dC, the supernatant obtained on high speed centrifugation of the actoS1dC complex was passed through the NTA-Ni column. The bound E459A S1dC was eluted with imidazole as above. In this alternative way, the major portion of the expressed E459A S1dC was recovered. The amount of E459A S1dC purified with the standard procedure was ~10% of that of the protein recovered with the alternative procedure. Relative concentrations of the proteins were determined by Coomassie Protein Assay Reagent (Pierce), whereas their exact values were calculated photometrically by the method of Gill and von Hippel (36). Both methods gave consistent results.Single Turnover of ATP Hydrolysis-- Cy3-ATP (1 µM) was added to the wild-type (0.2 µM), G457A (0.5 µM), or E459A (0.5 µM) myosin in 50 mM NaCl, 10 mM MOPS, pH 7.4, and 1 mM MgCl2 at 25 °C. After various times, a part of the reaction mixture was taken out and mixed with a 0.01 volume of PCA to stop the reaction. The resulting solution was centrifuged at 10,000 × g for 10 min to remove insoluble materials. The supernatant was applied directly to a reverse-phase HPLC column (Waters, Nova-pack C18). Elution was carried out with 100 mM potassium phosphate buffer, pH 6.8, and 11% acetonitrile. Under these elution conditions, Cy3-ATP and Cy3-ADP were well resolved. Both nucleotides were detected with a fluorimeter. The ratio of the concentrations of Cy3-ATP and Cy3-ADP was calculated to determine the amount of hydrolyzed Cy3-ATP. To compensate for the small amount of Cy3-ADP present in the Cy3-ATP preparation as a contaminant, myosin was first mixed with PCA and then mixed with Cy3-ATP. The resulting solution was treated as above. As for the D454A myosin, reliable measurement of the single turnover of Cy3-ATP was repeatedly hampered by its low yield.
Fluorescence Measurements-- All fluorescence measurements were performed with a Perkin-Elmer LS50B Luminescence Spectrophotometer. The binding of mant-ATP to the D454A, G457A, or E459A myosin (0.25 µM) was determined in 150 mM NaCl, 20 mM MOPS, pH 7.4, and 5 mM MgCl2. Under these conditions, Dictyostelium myosin II remained in a soluble state. Since the myosins used for the measurements had lost their ATPase activities, mant-ATP was not hydrolyzed to an appreciable extent during the measurements. The nucleotide concentration was increased by stepwise addition of mant-ATP. The base line was determined using the above solvent without myosin. Fluorescence intensity was measured with excitation at 365 nm and emission at 440 nm. The actin-S1dC interaction was followed using pyrene-labeled F-actin (0.25 µM) (28) and S1dC (0.25 µM) in a solvent comprising 50 mM NaCl, 10 mM MOPS, pH 7.4, and 2 mM MgCl2. Excitation was at 365 nm and emission at 410 nm. Intrinsic tryptophan fluorescence spectra were recorded using the wild-type or mutant S1dC (0.5 µM) in a solvent comprising 50 mM NaCl, 10 mM MOPS, pH 7.4, 2 mM MgCl2, and in the presence and absence of 0.1 mM ATP. Excitation was at 290 nm. Tryptophan fluorescence spectra of completely denatured proteins were also recorded after denaturing them in 6 M GuHCl, and normalized to each other to confirm that observed difference in the tryptophan fluorescence actually arose from different conformations.
Incorporation of mant-ATP into E459A S1dC-- E459A S1dC purified by the standard or alternative procedure (1 µM) in 50 mM NaCl, 10 mM MOPS, pH 7.4, and 2 mM MgCl2 was incubated with 20 µM mant-ATP. The mixture was passed through a gel filtration HPLC column (Ashahipack) equilibrated with a solvent comprising 0.5 M NaCl and 10 mM MOPS, pH 7.4. The fluorescence intensities of the bound and free mant-ATP (excitation, 365 nm; emission, 440 nm) as well as the absorption of S1dC (at 280 nm) were monitored using two tandemly arranged flow cells.
After 2 days of incubation with mant-ATP, the mixture was passed through the gel filtration column to purify the S1dC·mant-ATP complex. At various times after the purification, the purified E459A S1dC·mant-ATP complex was again passed through the column to determine the amount of the fluorescent nucleotide released.ATPase and in Vitro Motility Assays-- Actin-activated and basal MgATPase activities were measured as described (32). In vitro motility assays were carried out as described (29-31).
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RESULTS |
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Cell Phenotypes Expressing Recombinant Myosins-- Dictyostelium myosin II-null cells could not undergo normal cytokinesis and only slowly grew in suspension up to the density of ~1 × 106 cells/ml, becoming multinucleated cells (Fig. 2, Null), consistent with the previous reports (10, 12). When a multicopy plasmid bearing the wild-type heavy chain gene of myosin II was introduced into myosin-null cells, the defect in cytokinesis was reversed so that the resulting transformants (designated as "wild-type cells") could grow in suspension as mononucleated cells up to the density of ~2 × 107 cells/ml (Fig. 2, WT). The other transformants expressing the mutant myosins could be grouped into two types according to their behavior in suspension culture (Fig. 2). I455A and S456A cells grew like the wild-type cells, although D454A, G457A, F458A, and E459A cells did not grow or grew only slowly, like myosin-null cells. When transformed Dictyostelium cells were allowed to develop on agar plates covered with E. coli cells, the I455A and S456A cells, which grew well in suspension, formed fruiting bodies like the wild-type cells. However, the D454A, G457A, F458A, and E459A cells, which had a defect in suspension culture, could not develop beyond the mound stage, like myosin-null cells.
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Motor Functions of the Purified Myosins-- The actin-activated and basal MgATPase activities of the purified myosins were measured (Fig. 3). The D454A, G457A, F458A, and E459A myosins exhibited very low Vmax values for the actin-activated ATPase activity. Among them, the D454A, G457A, and E459A myosins also exhibited very low basal ATPase activities, suggesting that these three mutant myosins had almost completely lost their ability to hydrolyze ATP. In contrast to these myosins, the F458A myosin retained high basal activity similar to that of the I455A or S456A myosin. The Vmax value for the actin-activated MgATPase activity of the F458A myosin was, however, almost the same as that of its basal activity, indicating the complete loss of the actin-activated ATPase activity. The I455A and S456A myosins, which complemented the defects of myosin-null cells, retained their actin-activated and basal ATPase activities.
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Determination of the Step Blocked by Mutations-- As described above, the D454A, G457A, and E459A mutations almost completely abolished the ATPase activity. To determine which step of the ATPase cycle was blocked by the mutations, the binding of mant-ATP to these myosins was measured. Since all these myosins lost their ATPase activities, it was possible to carry out stepwise titration without appreciable hydrolysis of mant-ATP during the measurements. As shown in Fig. 5, the G457A myosin bound mant-ATP tightly, whereas the D454A and E459A myosins bound it more weakly. As shown below, however, it seems that the weak binding of mant-ATP to the E459A myosin was because of the slow binding of the fluorescent nucleotide to the mutant, not to its intrinsically low affinity. These results suggest that the D454A, G457A, and E459A myosins could bind mant-ATP with various affinities, implying that these mutations did not block the ATP binding to the mutant myosins, but blocked the ATP hydrolysis step, like the R238A mutation in the switch I loop (32).
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Actin-S1dC Interaction-- The ATP-dependent association and dissociation of the Dictyostelium myosin motor domain, S1dC (1), with pyrene-labeled F-actin was studied, the pyrene fluorescence being followed (28). S1dC was used mainly because more reproducible data were obtained using this soluble, single-headed fragment. As previously shown, the pyrene fluorescence decreased when the wild-type S1dC formed a rigor complex with the pyrene-labeled actin (34). On the addition of ATP, S1dC was transiently dissociated from the F-actin, and then reassociated with it after ATP had been completely hydrolyzed to ADP and Pi. Thus, the pyrene fluorescence transiently increased and then decreased (Fig. 7A). F458A S1dC also formed a rigor complex with F-actin in the absence of ATP, as indicated by the decrease in the pyrene fluorescence (Fig. 7B). The rigor complex was transiently dissociated on the addition of ATP and was formed again after ATP had been exhausted. Complete dissociation of the rigor complex was achieved only when a large excess of ATP was added because F458A S1dC retained high basal ATPase activity and, therefore, quickly consumed ATP. Thus, F458A S1dC exhibited normal ATP-dependent dissociation-association with F-actin even though it had lost its actin-activated ATPase activity. When G457A S1dC was mixed with the pyrene-labeled F-actin in the absence of ATP, a rigor complex was formed, as judged from the decrease in the pyrene fluorescence. On the addition of a small amount of ATP (even 1 mol/mol of S1dC), almost complete dissociation of the rigor complex occurred (Fig. 7C), indicating that G457A S1dC entered in a weak-binding state when it bound ATP. Because of the lack of ATPase activity, G457A S1dC remained in this weak-binding state. Unlike these mutants, however, E459A S1dC, purified by either the standard or the alternative procedure (see "Experimental Procedures"), failed to form a rigor complex with F-actin even in the absence of ATP, as judged from the fact that the pyrene fluorescence of F-actin never decreased on the addition of the purified S1dC.
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Tryptophan Fluorescence-- The intrinsic tryptophan fluorescence of the wild-type S1dC increased on the addition of excess ATP (Fig. 8A), as previously reported for a similar fragment of Dictyostelium myosin (34). On the addition of MgADP, a slight decrease in the fluorescence intensity was observed. Unlike that of the wild-type S1dC, however, the tryptophan fluorescence of G457A S1dC did not change on the addition of ATP or ADP (Fig. 8B). The fluorescence intensity remained at the same level as that of the wild-type S1dC in the absence of ATP. The tryptophan fluorescence of E459A S1dC also did not respond to ATP or ADP. The fluorescence intensity remained higher than that of G457A S1dC, being similar to that of the wild-type S1dC in the presence of ATP (Fig. 8B). On the addition of ADP to F458A S1dC, the tryptophan fluorescence slightly decreased, as in the case of the wild-type S1dC (Fig. 8C). However, on the addition of ATP, the tryptophan fluorescence did not increase, but decreased further (Fig. 8C), indicating that F458A S1dC took on a unique steady state in the presence of ATP.
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Trapping of ATP in E459A S1dC-- As mentioned above, the purified E459A S1dC did not form a rigor complex with F-actin. One possibility for this unexpected result is that the E459A mutation induced large conformational changes that made the E459A myosin and S1dC unable to form the rigor complex. Another possibility is that the ATPase site of the E459A mutant was occupied by a tightly trapped endogenous ATP. The latter possibility is much more likely because the intensity of the tryptophan fluorescence of E459A S1dC remained higher than that of G457A S1dC in the presence and absence of ATP, as if it bound ATP or ADP·Pi (35).
When ATP was tightly trapped at the ATPase site, it would be chased only slowly by the free nucleotide. To test if this was the case, E459A S1dC was incubated with a 20-fold molar excess of mant-ATP. After various times, the mixture was passed through a gel filtration HPLC column to separate the bound and free nucleotides. As shown in Fig. 9A, the amount of the fluorescent nucleotide incorporated into the protein very slowly increased with increasing incubation time. After a week, ~60% of the protein had incorporated the fluorescent ATP. Thus, it seems that the mant-ATP slowly chased ATP which was tightly trapped at the ATPase site of E459A S1dC and then was bound there. It must be noted that the binding of mant-ATP to other mutants such as G457A S1dC took place within several seconds under the same conditions (data not shown). To further confirm the tight trapping of the bound nucleotide, the rate of release of mant-ATP trapped in E459A S1dC was measured. At various times after the purification, the purified complex of E459A S1dC and mant-ATP was passed through the HPLC column again to separate the bound from the released mant-ATP. As shown in Fig. 9B, the bound fluorescent nucleotide was only slowly released, confirming the notion that ATP (or its analog) was tightly trapped at the ATPase site of the E459A mutant, once it had been incorporated there.
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Random Mutagenesis of the 459th Residue-- The 459th residue was randomly mutagenized using the E459A myosin gene as a template. Then the mixture of mutagenized myosin genes was introduced into myosin-null cells. When transformed cells were allowed to grow in the presence of E. coli cells, they formed two types of plaques that were easily distinguishable from their diameters. The diameter of the larger plaques was ~2-fold larger than that of the smaller ones, which was almost the same as that of myosin-null cells. Among the 672 plaques generated on the transformation of 107 cells, 36 plaques were of the larger type. The diameter of a plaque is a good indicator of the in vitro motor functions of myosin (20, 24). Plasmids bearing the myosin genes were retrieved from Dictyostelium cells isolated from the larger plaques. The sequencing of randomly chosen plasmids thus rescued (20 plasmids) showed that the codon of the 459th residue was either GAG or GAA (14 GAG and 6 GAA), indicating that the 459th residue of a functional myosin must be glutamic acid.
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DISCUSSION |
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Mutant myosins generated by alanine scanning mutagenesis of the switch II loop can be classified into two groups according to their in vivo phenotypes. One group, comprising the I455A and S456A myosins, fully reverse the myosin-specific defects of myosin-null cells. The other group, comprising the D454A, G457A, F458A, and E459A myosins do not reverse any of the defects.
Although I455 is a highly conserved residue in almost all myosins (2) and functions as a pivoting residue for the main chain rotation of the switch II loop during the transition from the Vi structure to the BeFx structure (5, 6), the I455A myosin retained most of its motor functions. It must be noted that when the other pivoting residue, G457, was replaced with alanine, the motor functions were completely lost. It seems that the I455A mutation did not block the main chain rotation of the switch II loop, unlike the G457A mutation. Another mutant in the first group, the S456A myosin, exhibited normal motor functions, as expected from the facts that this residue is not conserved among myosins and that alanine occupies this position in some myosins (2).
The mant-ATP titration and single turnover measurements showed that the G457A mutant was unable to hydrolyze ATP because the ATP hydrolysis step (Fig. 10) was blocked. The G457A mutant could bind ATP tightly, however, as judged from the mant-ATP binding to the mutant myosin. Consistent with the result, the actin·G457A S1dC complex was readily dissociated on addition of an equivalent amount of ATP, whereas the tryptophan fluorescence of the mutant S1dC did not change. These results lead us to conclude that G457A S1dC tightly binds ATP and enters in a state (M'·ATP in Fig. 10) distinguishable from the M·ATP or M*·ATP state (35). The simple collision complex (MATP) is expected to bind ATP much more weakly, and the M*·ATP complex is expected to exhibit more enhanced tryptophan fluorescence than the G457A S1dC·ATP complex.
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The position of Gly-457 relative to the -phosphate of ATP in the
ATPase pocket may change on rotation of the main chain of the switch II
loop, depending on the state of the nucleotide. Thus, Gly-457 in
Dictyostelium myosin seems to function like the "
-phosphate sensor" glycine in GTPases (for example, Gly-60 in Ras). It is likely that the G457A mutation blocked this rotation of the
main chain because of steric hindrance. Given the fact that the G457A
mutant was trapped before the isomerization step when it bound ATP, it
is tempting to speculate that the isomerization step is coupled with
the rotation of the main chain of the switch II loop (33), which occurs
on the transition from the BeFx structure to the Vi
structure, and that G457A S1dC·ATP takes on the BeFx structure. Consistent with this notion, mant-ADP/BeFx was trapped in
G457A S1dC, whereas mant-ADP/Vi was not (data not
shown).
The side chain of Glu-459 is located close to the bound nucleotide in the Vi structure of Dictyostelium S1dC, forming a hydrogen bond with a water molecule suitably positioned to participate in ATP hydrolysis (6). This strategic location of Glu-459 suggests that the residue is crucial for the hydrolysis step. In fact, the E459A mutant was unable to hydrolyze ATP because the ATP hydrolysis step was blocked (Fig. 10). Once ATP was in the ATPase pocket of the E459A mutant, it was almost irreversibly trapped there without hydrolysis, as observed here. The results suggest that the E459A mutant was trapped possibly at the M*·ATP state (Fig. 10) (35). Besides its role in ATP hydrolysis, Glu-459 may also play a role as a "gatekeeper" of the backdoor for Pi release (8), opening and closing it through the ionic interaction with Arg-238 (Fig. 1B) (32). This notion implies that ATP hydrolysis is tightly coupled with the opening and closing of the backdoor. The crucial importance of Glu-459 was also highlighted by the observation that the motor functions were retained only when glutamic acid occupied the 459th position.
The side chain of Asp-454 faces the ATPase pocket, and is coordinated to an Mg ion of the bound nucleotide through a water molecule (5, 6). Ser-237 in the switch I loop of Dictyostelium myosin is also directly coordinated to the Mg ion from the other side of the ATPase pocket. Unlike the S237A myosin (32), however, the D454A myosin bound mant-ATP, although weakly, indicating that Asp-454 is of secondary importance in retaining the MgATP in the ATPase pocket, whereas Ser-237 is essential for this.
In contrast to the D454A, G457A, and E459A myosins, the F458A myosin in the second group retained the basal MgATPase activity although it completely lost the actin-activated ATPase activity. The observed in vivo defects of the F458A myosin arose from the lack of this essential ability to power the motor. The side chain of Phe-458 points away from the ATPase pocket and is buried in a hydrophobic pocket formed by residues such as Asn-472, Asn-475, His-572, Tyr-573, and Ala-574 in the core of the lower 50K subdomain (Fig. 1C). When the main chain of the switch II loop rotates at Gly-457 and Ile-455, the side chain of Phe-458 swings and rotates. It seems that the hydrophobic side chains surrounding Phe-458 follow this motion through the hydrophobic interaction. Thus, the swinging and rotation of Phe-458 seem to trigger the rigid body motion of the lower 50K subdomain, which opens and closes the 50K cleft (5). Therefore, disruption of the hydrophobic interaction by the F458A mutation blocks some of the structural changes expected to occur during the ATPase cycle, forcing the mutant to bypass some intermediate states. In fact, in the presence of ATP, F458A S1dC was in a unique steady state quite different from M**·ADP·Pi, as judged from the tryptophan fluorescence intensity. The F458A mutant in this unique steady state failed to interact with F-actin in such a way that it stimulated the actin-activated ATPase activity. Further kinetic and structural studies on the F458A myosin would reveal how F-actin triggers the actin-activated ATPase activity.
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ACKNOWLEDGEMENTS |
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We thank Reiko Ohkura for her excellent technical assistance. The coordinates of the motor domain of Dictyostelium myosin II were kindly provided by Dr. Rayment (University of Wisconsin). The myosin II heavy chain gene, myosin-null cells, pBIG vector, and recombinant MLCK gene were provided by Dr. Spudich (Stanford University), Dr. Patterson (University of Arizona), and Dr. Uyeda (National Institute for Advanced Interdisciplinary Research, Japan). Mant-ATP and Cy3-ATP were provided by Dr. Hiratsuka (Asahikawa Medical University) and Dr. Oiwa (Kansai Advanced Research Center, Communication Research Laboratory, Japan), respectively.
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FOOTNOTES |
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* This work was supported by a grant-in-aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan and by a research grant from the Human Frontier Science Program (to K. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel./Fax:
81-3-5454-6751; E-mail: cksutoh{at}komaba.ecc.u-tokyo.ac.jp.
The abbreviations used are:
MgATPS, adenosine
5'-O-(thiotriphosphate)MOPS, 4-morpholinepropanesulfonic
acidNTA, nitriloacetic acidHPLC, high performance liquid
chromatography.
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REFERENCES |
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