From the Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, Vermont 05405
Received for publication, March 26, 2003 , and in revised form, April 21, 2003.
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ABSTRACT |
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INTRODUCTION |
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One way to gain insight into the nature of potential head-head interactions is to study myosins containing two heads that differ functionally. If each head works independently, then the activity of such a heterodimer should be halfway between that of the faster homodimer and the slower homodimer. On the other hand, if there is an interaction between the heads, the properties of the heterodimer might be closer to that of one of the homodimeric species. To study heavy meromyosin (HMM) molecules with different heads, we developed an expression strategy that involves differential labeling of the constituent heavy chains with FLAG and His tags, co-infection in the Sf9 cell system, and sequential affinity chromatography columns to isolate homogeneous preparations.
We assessed two types of smooth muscle HMM heterodimer using enzymatic and mechanical assays. The first was composed of two naturally occurring smooth muscle heavy chain isoforms that differ in vitro by a factor of two in their actin-activated ATPase activity and actin filament motility (5, 6). This difference is due to the presence or absence of a 7-amino acid insert in the surface loop near the nucleotide-binding pocket (7, 8), which has been called loop 1 (9). Such heterodimers almost certainly exist in nature, because the mRNAs for the +insert and insert heavy chains are co-expressed in single smooth muscle cells (10). The second heterodimer contained one wild type (WT) heavy chain and a second head with a mutation in switch 2 (E470A) that prevents the formation of a salt bridge between this residue and R247 in switch 1 of the active site. This mutation slows the ATPase activity of smooth myosin by two orders of magnitude, essentially "locking" it in a weak binding configuration (11, 12). Thus, the heterodimeric E470A/WT molecule has a much greater disparity in function between the two heads.
Enzymatic assays indicated that the two heads of both heterodimers function independently. However, the in vitro motility assay showed that the heterodimeric (+insert/insert) HMM moved actin filaments 17% more slowly than a 50:50 mixture of the corresponding homodimeric HMMs. In contrast, the heterodimeric E470A/WT HMM showed motility that was not significantly different from WT HMM. These results are consistent with the notion that myosin employs only one head at a time to perform work on the actin filament and, furthermore, suggest that one head of a heterodimer can exert a disproportionate effect on mechanical properties in molecules where both heads are actively cycling.
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EXPERIMENTAL PROCEDURES |
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Each construct in pAcSG2 was transfected and amplified in Sf9 cells by established methods (14). To produce protein, myosin heavy chain viruses were co-infected with a recombinant virus encoding both the smooth muscle myosin essential and regulatory light chains (15). In the case of heterodimers, an N-terminally His-tagged heavy chain virus was co-infected with the appropriate C-terminally FLAG-tagged heavy chain, along with the dual light-chain virus. Preliminary trials were conducted to optimize relative viral ratios so that the expression levels of the FLAG- and His-tagged heavy chains were nearly equal.
Purification of HMM ProteinsAfter 3 days of infection, Sf9 cells were harvested and lysed, and proteins were fractionated by two successive ammonium sulfate precipitations, 040% and 4070%. For the homodimers of C-terminally FLAG-tagged +insert and E470A HMM, as well as for the two types of heterodimer, the 4070% pellet was dialyzed overnight versus a buffer containing 90 mM NaCl, 10 mM imidazole-HCl (pH 7.4 at 4 °C), 1 mM EGTA, 1 mM NaN3, 1 mM DTT, and 1 µg/ml leupeptin. MgATP was added to a final concentration of 4 mM, the material was clarified by centrifugation, and the supernatant was applied to an anti-FLAG affinity column (M2 antibody, Sigma-Aldrich Chemical). After washing, HMM was eluted using a large molar excess of FLAG peptide (0.1 mg/ml), and peak fractions were pooled. This was the end point for the preparation of FLAG-labeled homodimers. In the case of the two heterodimer preparations, NaCl was added to the FLAG eluate to give a final concentration of 300 mM, and the pH was increased to 8.0 by adding 35 mM MOPS, pH 8.55. This material was then applied to a nickel-charged poly-histidine binding column (Probond, Invitrogen). Nonspecifically bound proteins were washed away using 300 mM NaCl, 10 mM imidazole, 10 mM MOPS (pH 8.0 at 4 °C); then heterodimeric HMM was eluted using 300 mM NaCl, 300 mM imidazole, 10 mM MOPS (pH 8.0 at 4 °C). Peak fractions were pooled and dialyzed overnight versus a buffer containing 50 mM NaCl, 20 mM MOPS (pH 7.4 at 4 °C), and 0.2 mM DTT. The protein was then precipitated by dialysis versus saturated ammonium sulfate, collected by centrifugation, re-suspended, and finally dialyzed into 50 mM NaCl, 10 mM HEPES (pH 7.0 at 4 °C), 5 mM MgCl2, 1 mM EGTA, 1 mM NaN3, 1 mM DTT.
The homodimeric N-terminally His-tagged insert HMM was isolated as follows. Following ammonium sulfate fractionation as above, equimolar F-actin was added to the pelleted material, which was then dialyzed overnight at 4 °C versus a phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, pH 7.4), containing 7% sucrose (w/v), 1 mM DTT, and 1 µg/ml leupeptin. The actin-bound material was sedimented, then washed with 300 mM NaCl, 10 mM MOPS (pH 8.0 at 4 °C). The HMM was then eluted into the same buffer containing 1 mM MgATP, and loaded onto a Probond column. The insert HMM was then washed and eluted from this column as described above for the heterodimer.
After purification, aliquots of the various HMM preparations were phosphorylated by the addition of the following reagents (concentrations in parentheses): CaCl2 (0.75 mM); calmodulin (7.5 µg/ml); MgATP (1.5 mM); and myosin light chain kinase (36 µg/ml). Except for assessments of ADP release rates, all assays were conducted exclusively on phosphorylated proteins.
Probes Used in Western BlotsTwo 38% acrylamide gradient, Tris acetate-buffered NuPAGE gels (Invitrogen) were loaded and run with identical samples from different steps of the heterodimer preparation then transferred to nitrocellulose. One filter was reacted with the anti-FLAG monoclonal antibody diluted to 0.025 µg/ml. The filter was then reacted with horseradish peroxidase-conjugated goat anti-mouse secondary antibody (Bio-Rad) diluted 1:3000. To probe for the proteins labeled with the His tag, the duplicate filter was incubated with a horseradish peroxidase-conjugated His-chelating group at a dilution of 1:500 (India His-Probe, Pierce Chemical). Both filters were labeled using diaminobenzamidine in the presence of hydrogen peroxide.
Steady-state ATPase AssaysActin-activated ATPase activity
was measured at 37 °C as described previously
(16).
-activated ATPase was determined at
37 °C, using a buffer containing 400 mM NH4Cl, 2
mM EDTA, 25 mM Tris base (pH 8.0 at 37 °C), 200
mM sucrose, 1 mM DTT, and 1 mg/ml BSA
(15).
Motility and Average Force MeasurementsFor all motility experiments, 1.23.5 µM HMM was mixed with a 2.5-fold molar excess of F-actin in the presence of 1 mM MgATP, and centrifuged for 25 min at 350,000 x g to remove ATP-insensitive cross-bridge heads. The protein concentration of the supernatant was determined by Bradford (17), and dilutions were made to the appropriate concentrations for either measurement of unloaded maximum velocity, or average force (see below). Polyacrylamide SDS gels run on these supernatants confirmed the accuracy of the concentrations and indicated that the amount of contaminating actin was less than 5%. HMMs were anchored to the coverslip using antibody S2.2 (18), and actin filament movement was measured as described previously (16, 19). The basic buffer typically used for actin treatment and washing in the flow cell (see below) contained 60 mM KCl, 25 mM imidazole-HCl (pH 7.4 at 30 °C), 4 mM MgCl2, 1 mM EGTA, and 10 mM DTT (buffer A). The final assay buffer additionally contained 0.7% methylcellulose, an oxygen scavenger mixture (containing 0.1 mg/ml glucose oxidase, 0.018 mg/ml catalase, and 2.3 mg/ml glucose), and 1 mM MgATP. In some experiments, the concentration of KCl in the buffer was changed to 25 or 90 mM.
Measurements of average force were made on the motility surface using
-actinin as a load, which impeded the movement of actin
(20).
-Actinin (Sigma)
was dialyzed into buffer A with 1 rather than 10 mM DTT and
clarified for 25 min at 350,000 x g. Its concentration was
determined using BSA as a standard
(17). The following components
were added sequentially to the flow cell: 1) 25 µg/ml S2.2 antibody for 1
min, wash with buffer A; 2)
-actinin for 1 min, wash with buffer A,
including 0.5 mg/ml BSA; 3) HMM for 1 min, wash with buffer A; and finally 4)
actin for 2x 30 s. Finally, buffer A containing methylcellulose, oxygen
scavengers, and ATP was added, and the filaments were observed under
fluorescent illumination.
The concentration of -actinin which just stopped filament movement
on the motility surface was determined for different HMM concentrations. The
"stopping concentration" of
-actinin was defined by the
long, straight, "taut" appearance of the filaments, as well as
their failure to move. The precision of these measurements was defined as
one-half the concentration difference between the stopping point as defined
above and the closest lesser
-actinin concentration at which long, taut
filaments were not observed.
ADP Release from Acto-HMMThe rate of ADP release was determined by mixing acto-HMM·(100 µM ADP) with 2 mM MgATP, and measuring acto-HMM dissociation either by the decrease in light scattering or the increase in pyrene actin fluorescence. Actin was labeled with pyrene-iodoacetamide as described previously (16). Experiments were done in 10 mM HEPES (pH 7.0), 0.1 M NaCl, 5 mM MgCl2, 1 mM EGTA, 1 mM NaN3, 1 mM DTT at 20 °C using a Kin-Tek stopped-flow spectrophotometer and a 100-watt mercury lamp. For 90° light scattering, the exciting beam was passed through a 294-nm interference filter, and the emission was detected with a 294-nm interference filter. Pyrene actin was excited using a 360-nm interference filter, and emission was detected with a 400-nm cutoff filter. Transients are the average of at least three independent mixings. The signal averaging and fitting was done using Kin-Tek software.
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RESULTS |
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The flowchart in Fig. 1 illustrates the stages of this purification for the (+insert/insert) heterodimer, along with Western blots depicting the amounts of FLAG- and His-reactive material in each step. The sensitivities of our anti-FLAG and anti-His probes were adjusted to give approximately the same amount of staining when reacted with equal amounts of the respective control proteins (Fig. 1B, lane S). The protein that was loaded onto the FLAG column (Fig. 1B, lane 1) contained nearly equal amounts of FLAG-labeled (+insert) heavy chain and His-labeled (insert) heavy chain. As expected, the flowthrough from the anti-FLAG column contained predominantly His-tagged molecules (Fig. 1B, lane 2). The small amount of FLAG immunoreactivity in this fraction indicated that the column was loaded with more FLAG-labeled material than it had the capacity to bind. Protein eluted from this column contained both FLAG-labeled homodimers of the +insert heavy chain and heterodimers (Fig. 1A, step 3 and Fig. 1B, lane 3). After loading this mixture onto the poly-histidine binding column, a small amount of the His-reactive material was seen in the flowthrough due to supersaturation of the column, along with a large excess of FLAG-tagged protein (Fig. 1B, lane 4). After washing with loading buffer, a more stringent wash with buffer containing 10 mM imidazole-HCl was employed to release any nonspecifically bound FLAG-FLAG homodimer (data not shown). Elution with 300 mM imidazole buffer liberated the final, homogeneous preparation of heterodimeric HMM (Fig. 1B, lane 5), which had equal amounts of FLAG- and His-reactive material in it. Western blots conducted on preparations of the heterodimeric E470A/WT HMM gave the same results (21).
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-ATPase activity was used to
quantitatively assess the homogeneity of the heterodimeric E470A/WT HMM
preparation, because one of the two heads is virtually inactive
(Table I). Homodimeric WT HMM
had
-ATPase activity of 24.2
± 2.8 s1, whereas the activity of the
homodimeric E470A was barely measurable, even though 50100 times as
much of this protein was used in the assay as the other two species
(12). The activity of
heterodimeric E470A/WT HMM was half that for the homodimeric WT HMM,
supporting our contention that this preparation contains exclusively
heterodimeric molecules.
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Actin-activated ATPase of the Heterodimer Is Comparable to a 50:50 Mix
of HomodimersThe actin-activated ATPase activity of the
heterodimeric (+insert/insert) HMM was compared with the corresponding
homodimers, as well as an equal mix of these two isoforms
(Fig. 2). Measurements were
made at an actin concentration of 40 µM, which is greater than
the Km for each of these species
(6). The ATPase activity of the
homodimeric (insert) HMM was approximately half that of homodimeric
(+insert) HMM, as we have shown previously
(6). The rate of the
heterodimer was the same as an equal mixture of the two homodimers, at a value
intermediate between the rates shown by the homodimers. Taken together with
the ATPase results of
Table I, these data indicate
that each of the heads in our heterodimers have steady-state ATPase activities
that are unaffected by the other head, both in the presence or absence of
actin.
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Rate of ADP Release from Acto-HMM by Two Techniques The
release of ADP from the nucleotide-binding pocket is thought to be the step in
the cross-bridge cycle that limits the velocity of actin movement by a given
type of myosin (22).
Pyrene-labeled actin was used to measure the rate of ADP release from
acto-HMM. When pyrene actin-HMM·ADP is mixed with excess ATP, pyrene
fluorescence increases at the rate at which ADP leaves the active site and is
replaced with ATP (23). The
time course of this fluorescence increase was best fit by a single exponential
that was 4-fold faster for the +insert HMM than the insert HMM
(Table II). Phosphorylation had
little effect on this rate. When measured over a range of temperatures, the
Q10 value for this kinetic step was
3. Although our ADP release
measurements showed a 4-fold difference between the +insert and insert
species, our current and previous
(6) comparisons of velocities
by the motility assay indicate a 2-fold difference between these isoforms.
These results suggest that other steps in the cross-bridge cycle in addition
to ADP release may limit the velocity of actin filament movement.
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The difference in ADP release rates of the homodimers allowed us to probe
the properties of heterodimeric (+insert/insert) HMM
(Table II). As expected, the
fluorescence time course of the 50:50 mixture of dephosphorylated homodimers
was better fit by an equation with two exponential terms than one, yielding
rate constants of about 10 and 40 s1, similar to
the values determined from the homodimers
(Table II). The heterodimer,
independent of its phosphorylation state, also yielded two rates that were
4-fold different and nearly equal in amplitude, suggesting independent
head action.
ADP release rates were also measured by monitoring the decrease in light scattering of acto-HMM·ADP caused by rapid mixing with excess ATP (Table III and Fig. 3; see "Experimental Procedures"). This technique gave essentially the same results as had been obtained by pyrene fluorescence quenching for the homodimeric HMMs (Fig. 3, A and B), as well as the mixtures of homodimers (Fig. 3C and Table III). However, in contrast to the pyrene data, the light scattering signals for six different preparations of the heterodimeric HMM were better fit by a single exponential with a rate of 1415 s1, closer to the value obtained for the homodimeric (insert) HMM (Fig. 3D and Table III). This observation is the first demonstration that the light scattering signal measures the movement of the entire double-headed HMM molecule away from the mass of the actin filament, whereas pyrene fluorescence gives an independent assessment of the rate of ADP release from each head. Because the slower head will dictate the rate at which the entire HMM can move away from actin, a rate dominated by the slower head is expected and was observed.
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Motility of the HeterodimersBoth the E470A/WT HMM and the +insert/insert HMM heterodimers were assayed for their ability to move actin in the in vitro motility assay. Homodimeric E470A HMM does not support motility (Fig. 4A). Surprisingly, heterodimeric E470A/WT HMM showed an average velocity (cross-hatched bar, 1.50 ± 0.19 µm/s) in 60 mM KCl buffer that was nearly 95% as great as that of WT HMM (black bar, 1.58 ± 0.20 µm/s). These values were not significantly different when assessed by unpaired t tests at a p < 0.001 level of significance. Thus, the presence of a non-cycling, weak-binding cross-bridge head had little impact on actin movement by the WT head.
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The heterodimer containing cycling heads with 2-fold different kinetics behaved differently, suggesting head-head interactions. The velocity of the homodimeric (+insert) HMM (black bar, 1.60 ± 0.19 µm/s) was twice that of the insert HMM (white bar, 0.76 ± 0.10 µm/s), as we previously showed (6) (Fig. 4B). The cumulative average of a number of different 50:50 mixtures of the two homodimeric species assessed on different dates was 1.13 ± 0.13 µm/s (gray bar, Fig. 4B). Six different heterodimeric (+insert/insert) HMM preparations (cross-hatched bars) moved more slowly on any given date than the 50:50 mixture they were directly compared with and gave velocities ranging from 7% to 26% slower than the cumulative mixture average (Fig. 4B). These differences were in every case significant at the p < 0.001 level. Additionally, the cumulative average actin filament velocity of the six heterodimeric preparations (0.94 ± 0.14 µm/s, n = 454 filaments) was 17% less than that of the 50:50 mixtures (1.13 ± 0.13 µm/s, n = 292 filaments), which was also highly significant (p < 0.0001). These results suggest that the presence of the kinetically slower insert head within the same molecule exerts a disproportionate slowing effect on the performance of the molecule as a whole as it moves actin.
Because the heterodimers had been exposed to two different types of affinity column and therefore endured more manipulation than either of the homodimer preparations, it could be argued that their decreased velocities were due to damage sustained during preparation. To refute this idea, we isolated a control HMM, which had an N-terminal His tag on one (+insert) heavy chain and a C-terminal FLAG tag on the other (+insert) heavy chain (i.e. homodimeric with respect to HMM sequence and heterodimeric with respect to tags). The control HMM was subjected to the same purification scheme as our experimental heterodimers. An aliquot of HMM eluted from the FLAG column was saved for functional comparison with the elution from the His column, which contained exclusively FLAG/His heterodimers. As shown in Fig. 4C, the velocity of control HMM purified on a single FLAG column (black bar, 1.60 ± 0.19 µm/s) was not significantly different from the material that bound to and was eluted from the FLAG column (white bar, 1.68 ± 0.28 µm/s) or the material that bound to and was eluted from both columns (gray bar, 1.62 ± 0.17 µm/s). This control showed that the decreased velocity exhibited by our +insert/insert heterodimer is an inherent property of this molecule, not an artifact due to the purification procedure.
Average Forces Produced by the +insert and insert HMM Are the SameTwo methods were used to determine whether the relative amounts of force generated by the homodimeric +insert and insert HMMs differed, which would allow us to characterize this parameter in the heterodimer. First, the motility velocity of mixtures of varying ratios of the +insert and insert homodimers was quantified (Fig. 5A). To assess the relative amounts of force generated by the two smooth HMMs, the data were fit using a model for the interaction of two different myosin isoforms on the motility surface (24). The value derived for the ratio of maximum force generated by the insert to the +insert isoform (P0(insert)/P0(+insert)), was 1.34, showing little difference in average force, as reported previously (25).
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The second method used a variation of the motility assay in which
-actinin acts as a load that the HMM works against to create movement
(20). The concentration of
-actinin needed to stop movement was assayed as a function of HMM
concentration, and data were fit by a simple linear regression
(Fig. 5B). The forces
produced by the homodimeric +insert and insert HMMs were
indistinguishable by this assay, negating our ability to assess this
mechanical property of heterodimeric HMM.
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DISCUSSION |
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Our approach to further addressing the role of myosin's two heads was to
develop an expression and purification scheme to isolate pure populations of
heterodimeric double-headed HMM and to compare their performance with
molecules containing two identical heads. To ensure that our isolated
heterodimer was a homogeneous preparation, we employed separate epitope tags
for each type of heavy chain, and used two successive affinity columns, one
for each of the tags (Fig. 1).
Control experiments showed that protein purified over two columns was
functionally equivalent to protein purified by the standard one-column method
(Fig. 4C). These
results not only refuted the possibility of damage, but also showed that the
alternative locations of the two tags at either the amino or carboxyl termini
had no impact upon the activity of the molecule. Moreover, quantitative
evidence for the homogeneity of the heterodimeric preparation was obtained by
showing that the ATPase activity of
a heterodimer with one non-functional head was exactly half that of the
homodimer with two functional heads. Western blots of the final species were
also consistent with equal mixtures of the two species.
The Two Heads of HMM Are Enzymatically IndependentAs
expected, the actin-activated ATPase activity of the heterodimeric
(+insert/insert) HMM was the same as the activity of an equal mixture
of the two homodimeric HMMs, indicating that the two heads hydrolyze ATP
independently. This conclusion was strengthened by the results from the
ATPase comparison of the
heterodimeric E470A/WT HMM with its two cognate homodimers. Our measurements
of ADP release obtained by pyrene fluorescence were also consistent with the
idea of independent heads, as the heterodimer revealed distinct fast and slow
rates that were quite similar to those of the homodimers
(Table II). In contrast, when
light scattering was used to measure the rate of ADP release, there was a bias
toward the rate of the slower head. This result suggests that the light
scattering signal remains the same whether one or two heads are bound and only
changes when both heads are dissociated from actin. The rate at which the
whole HMM molecule dissociates from actin could only occur as fast as the
slowest head comes off.
Contribution of the Two Heads to Actin MovementSmooth
muscle S1 (21) and
single-headed smooth muscle HMM
(30) move actin at least
2-fold more slowly than WT HMM. Even a construct lacking only the motor domain
on one of the two heads, but retaining both neck regions, showed lower rates
of motility than WT HMM (30).
Moreover, a recent laser trap study showed that the unitary step size of
double-headed myosin (10 nm) was twice that of single-headed myosin
(
6 nm), although the length of time that the myosin was attached to actin
following the power stroke was the same for both species
(4). Here we show that a second
weak binding, non-cycling head can restore motility to WT HMM levels, whereas
concurrent single molecule studies showed that the E470A/WT HMM heterodimer
can achieve the same working stroke of
10 nm as WT HMM
(21). One interpretation of
these data is that the ability of the mutant head to interact weakly with
actin allows the stroking head to achieve maximal performance. Thus, a
reasonable model is that only one head of HMM moves actin, while the second
head optimally orients the working head with respect to the actin filament.
Alternatively, a second head may be necessary simply to maintain the
structural integrity of the head-rod junction and/or to minimize flexibility
and unfavorable orientations of the working head. In this case, the ability of
the second head to bind actin is not essential. The current experiments cannot
distinguish between these two mechanisms, but it is clear that the presence of
the second head is necessary for optimal mechanical performance.
In contrast to the E470A/WT HMM heterodimer, the +insert/insert HMM was always slower than the comparable mixture of homodimers, by an average of 17%. This heterodimer differs from the one described above in that both heads are cycling. If only one head of the pair is involved in motion generation at any given time, the simplest interpretation of these data is that kinetic differences between the two species cause the insert head to interact with actin slightly more frequently than the +insert head. This assumption is reasonable, because it is known that changes at the active site can be propagated to the actin-binding interface and vice versa. The most obvious example of communication between the nucleotide and actin-binding sites is the dissociation of myosin from actin upon ATP binding. Thus, it is conceivable that changes to loop 1 can cause insert HMM to interact differently with actin than the +insert HMM (31). Alternatively, it is conceivable that a second, slower head in the same molecule exerts a disproportionate mechanical hindrance to movement.
In summary, our motility data indicate that two cross-bridge heads are necessary for optimal actin filament movement but that the contribution of the two heads to movement is not equal. A non-cycling head paired with an active head is sufficient to support motility at the same rate as seen with WT HMM. However, when the second head can also cycle, the observed motility can deviate from that expected from an equimolar mixture of the two types of heads, implying that the ability of both heads to bind strongly to actin is an important feature affecting myosin's mechanical properties.
Average Force of Myosin IsoformsThere are a number of
instances of heterodimeric molecules in nature. The best described of these is
the V2 isoform in mammalian cardiac muscle, which is a heterodimer of the
and
heavy chain gene products. Like the smooth muscle +insert
and insert myosins, the homodimeric
-heavy chain (V1) isoform
has significantly higher ATPase activity and motility than the
-heavy
chain (V3) isoform (24,
32,
33). Thus, based upon our
results here, it might be expected that the in vitro actin filament
velocity supported by V2 would be different than that of an equal mixture of
V1 and V3 isoforms.
One potentially key difference between the cardiac and smooth muscle heterodimers has to do with the relative amounts of force that the two isoforms in these tissues can produce. We showed here by two mechanical assays that the +insert and insert HMMs generate approximately the same average force. This is consistent with a recent report showing no correlation between the amount of force developed by single skinned smooth muscle cells and their content of +insert mRNA (34). Conversely, a recent study on mice in which the +insert isoform was specifically knocked out demonstrated a decrease in the force as well as the rate of tension production in these animals, suggesting that the faster +insert isoform exerts more force than the slower insert HMM (35). However, in the case of cardiac V1 and V3, data from the motility mixture assay (24), and direct measurements with a microneedle (33) clearly demonstrate that the slower V3 cardiac myosin from larger mammals produces at least twice as much force as V1. The two smooth muscle isoforms differ only at loop 1, but the cardiac isoforms also vary at other locations such as loop 2 and subfragment 2 (36), suggesting that structural differences in regions of the molecule other than loop 1 are necessary to obtain different levels of force generation.
Implications for Heterodimeric Myosins in Disease States
Here we showed that, when the two heads of myosin cycle at different rates,
one head can exert a disproportionate effect on the mechanical properties of
the molecule. Although this situation might occur in mammalian hearts in the
context of the V2 isoform, it could have even more profound effects in
diseases such as familial hypertrophic cardiomyopathy and dilated
cardiomyopathy, where in many cases mutations in the -cardiac myosin
gene are thought to be the genetic basis for the disease
(37). Because the mutation
occurs most often in a single allele, heterodimeric myosins with one WT and
one mutant head should be prevalent, and there is considerable evidence that
some of these mutations enhance the kinetics of the mutant myosin molecule
(38,
39). Thus, it is reasonable to
suggest that the mutant head would mechanically predominate in these
molecules, altering the contractile kinetics of the heart and exacerbating the
phenotype. Our ability to isolate heterodimeric HMMs will position us to more
closely examine the properties of these mutant molecules to better understand
the processes that give rise to the disease.
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FOOTNOTES |
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Both authors contributed equally to this work.
To whom correspondence may be addressed. Tel.: 802-656-8004; Fax:
802-656-0747; E-mail:
rovner{at}physiology.med.uvm.edu.
¶ To whom correspondence may be addressed. Tel.: 802-656-8750; Fax: 802-656-0747; E-mail: trybus{at}physiology.med.uvm.edu.
1 The abbreviations used are: S1, subfragment 1; HMM, heavy meromyosin; FHC,
familial hypertrophic cardiomyopathy; MOPS,
3-[N-morpholino]propanesulfonic acid; DTT, dithiothreitol; BSA,
bovine serum albumin, FLAG, epitope tag with amino acid sequence DYKDDDDK;
His, epitope tag with amino acid sequence HHHHHH; +insert, smooth muscle
myosin heavy chain containing the 7-amino acid insert in loop 1;
insert, smooth muscle myosin heavy chain lacking the 7-amino acid
insert in loop 1; WT HMM, wild type homodimeric heavy meromyosin; E470A HMM,
E470A homodimeric heavy meromyosin; +insert/insert HMM, heterodimeric
heavy meromyosin with one head containing the 7-amino acid insert and the
other head lacking the insert; E470A/WT HMM, heterodimeric heavy meromyosin
with one wild type head and one carrying the point mutation E470A; AMP-PNP,
adenosine 5'-(,
-imino)triphosphate.
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ACKNOWLEDGMENTS |
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REFERENCES |
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