From the
Departments of Neurology and
¶Biochemistry and Molecular Genetics, University
of Alabama at Birmingham, Birmingham, Alabama 35294-3293 and the
||Department of Physiology, University of
Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
Received for publication, March 13, 2003 , and in revised form, April 22, 2003.
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ABSTRACT |
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INTRODUCTION |
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However, vertebrate myosin II is in fact widely distributed and readily
detectable in non-muscle cells as well
(58).
Sequence studies reveal three classes of non-muscle myosin II isoforms named
A, B, and C (9,
10). Myosin II is necessary
for cell motility, maintenance of normal cell-surface receptor distribution,
and stress fiber formation
(1216).
Whereas myosin IIA is found in nearly all tissues, the distribution of myosin
IIB appears to be more limited
(810).
Myosin IIB composes 70% of the myosin II found in the central nervous
system and 100% of cardiac non-muscle myosin II. Myosin IIB is required for
normal development of the brain and heart
(17), and it also plays an
essential role in directed growth cone motility
(14,
18,
19). In particular, studies of
cultured neurons from myosin IIB knockout mice have led to the suggestion that
this motor is required for maintenance of cortical tension, a key component in
directed cell motility
(18).
Non-muscle myosin II thick filaments are 2030-fold smaller than their smooth or skeletal muscle counterparts, containing <28 myosin molecules (20). This is not surprising because the 12-µm long filaments of skeletal or smooth muscle would be far too large to effectively generate force in the presence of the tightly packed meshwork that characterizes the actin cytoskeleton (13). Although the smaller size of non-muscle myosin II-containing thick filaments represents an adaptation to the demands of cytoplasmic contractility, it also presents a challenge for a motor designed to maintain cortical tension. Conventional myosins typically have duty ratios in the range of 34%. This low ratio ensures that myosin heads that have completed their power stroke do not resist those heads beginning theirs. For a large thick filament such as myosin II from smooth or skeletal muscle, this does not present a problem because the large number of heads within a thick filament would ensure that at least a few would be strongly attached at any given time, and they would prevent the thick filament from diffusing away. However, a low duty ratio for non-muscle myosin II would mean that there would be an appreciable chance that, at any given time, no heads would be strongly attached to actin. This could be problematic for a myosin designed to generate cortical tension because even brief loss of contact of a myosin IIB filament with actin would lead to immediate loss of tension and elastic recoil of the cytoskeleton.
Given these considerations, we would therefore predict that myosin IIB has a high duty ratio, a feature generally thought to be more characteristic of the unconventional myosins (21, 22). However, a high duty ratio for a filament-forming myosin such as myosin IIB would present another problem: the resistance of strongly bound heads to force generation by actively contracting heads, which has been discussed above. To get around this problem, we would therefore also predict that myosin IIB has an additional feature generally thought to be characteristic of unconventional myosins: a strain-dependent release mechanism (23, 29). In such a mechanism, release of a strongly bound head would occur only when it experienced strain induced by another actively contracting head.
In this study, we have systematically examined the steps that constitute the myosin IIB mechanochemical cycle. This work establishes that, although myosin IIB resembles smooth muscle myosin II in some aspects of its enzymology, its kinetics also share features that have heretofore been thought to be characteristic of unconventional myosins such as myosins V and VI. This study also provides insight into how the physiologic demand for maintenance of cortical tension is translated into specific changes in the kinetics of the actomyosin ATPase cycle.
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MATERIALS AND METHODS |
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Expression of recombinant myosin IIB subfragment-1 was accomplished via infection of Sf9 insect cells with a baculovirus expression vector capable of driving high level expression of foreign proteins. The Sf9 cells were co-infected with recombinant virus expressing the myosin IIB heavy chain and with recombinant viruses for the non-muscle essential and regulatory light chains. Details of the protein expression and purification have been published (25). Myosin IIB was made nucleotide-free by incubation in 10 mM EDTA for 20 min at room temperature, followed by gel filtration on Sephadex G-25 (PD-10, Amersham Biosciences).
Fluorescence and Kinetic MethodologiesSteady-state and transient-state fluorescence measurements were made as previously described (25) in samples that had been equilibrated in 20 mM KCl, 25 mM HEPES, 1 mM MgCl2, 1 mM EGTA, and 1 mM dithiothreitol (pH 7.50). Kinetic measurements were made using an Applied Photophysics SX.18MV stopped-flow spectrophotometer with an instrument dead time of 1.2 ms as described (21, 25, 26).
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RESULTS |
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ATP-induced Formation of a Weakly Bound StateATP binding to
actomyosin can be monitored by quenching of a pyrene fluorophore on actin.
Formation of a weak binding state occurs as a two-step reaction, with initial
formation of a collision complex followed by a first-order quenching of pyrene
fluorescence: AM + T AM(T)
A*M·T, where the asterisk refers to a state of
enhanced pyrene fluorescence. Scheme
1 predicts a fluorescence rise that obeys a first-order process
whose rate varies hyperbolically with ATP concentration.
Fig. 1 confirms this and
reveals a maximum rate of 409 ± 13 s1 and
a dissociation constant of 646 ± 41 µM
(Table I). Similar results were
obtained using the turbidity change at 350 nm as a measure of the dissociation
step (data not shown).
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ATP Hydrolysis by Myosin IIBATP binding to myosin IIB produced an 11% enhancement of tryptophan fluorescence emission, whereas ADP binding produced a 45% enhancement (data not shown). Tryptophan fluorescence enhancement produced by ATP binding is characteristic of myosins, and the maximum rate of this process has been shown for both smooth and skeletal muscle myosin II, as well as myosin V, to be a measure of the hydrolysis step, k3 + k3 (21). As with these other myosins, the rate of this process for myosin IIB varied hyperbolically with ATP concentration (Fig. 2). This dependence defines a maximum rate of 16.7 ± 1.1 s1 with an apparent ATP dissociation constant of 37 ± 9 µM (Table I).
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Phosphate ReleaseThe kinetics of actin-activated phosphate release from myosin IIB were measured in a sequential mixing experiment. Nucleotide-free myosin IIB was mixed with a 10-fold molar excess of ATP. The complex was allowed to age for 500 ms to allow population of the myosin IIB·ADP·Pi state, and it was then mixed with varying concentrations of actin and 2 mM ADP. ADP was present in the final mixture to block further ATP binding and hydrolysis and to drive the myosin IIB into a strong binding state. Phosphate release was monitored by using MDCC1-labeled phosphate-binding protein at a 10-fold molar excess over active sites (21, 22). The resulting fluorescence enhancement, due to phosphate binding to MDCC-labeled phosphate-binding protein, fit a single exponential process. The rate of this process (k4') varied linearly over the range of actin concentrations examined (Fig. 3, closed triangles), and the slope of this curve (solid line) defines an apparent second-order rate constant of 0.0038 ± 0.0003 µM1 s1.
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Because phosphate release is rate-limiting for other myosin II isoforms (27), we expected this to be the case for myosin IIB as well. To test this, we measured the rate of product release from actomyosin IIB as a function of actin concentration by performing the following experiment. Nucleotide-free myosin IIB was mixed with a 10-fold molar excess of 2'-deoxy-mant-ATP. The mixture was allowed to age for 500 ms and then mixed with varying concentrations of actin and 2 mM ADP. As shown in Fig. 3 (open squares), the rate of mant-ADP release also varied linearly with actin concentration (dashed line), but the apparent second-order rate constant is nearly 2.5-fold lower, at 0.0015 ± 0.0001 µM1 s1. This implies that another step, subsequent to phosphate release, at least partially limits the ATPase rate.
Myosin IIB·ADP and Actomyosin IIB·ADP
StatesIn our previous study of smooth muscle actomyosin, we
utilized the fluorescent nucleotide 2'-dmD to examine the sequence of
conformational changes that the active site undergoes as it releases ADP
(26). Our results could be
interpreted in terms of Scheme
2, in which the affinity of ADP for actomyosin decreases from the
AM·D1 to the AM·D2 state. Binding of
smooth muscle myosin·ADP to actin favors formation of the
AM·D2 state
(26), and we wished to see if
a similar scheme applied to myosin IIB. We utilized the same approach that we
employed in our previous study of smooth muscle myosin II: an examination of
the fluorescence lifetimes of 2'-dmD bound to myosin IIB and actomyosin
IIB. The fluorescence decay of myosin IIB·2'-dmD can be
characterized by two lifetimes of 8.49 and 2.22 ns with fractional
steady-state intensities of f1 and f2,
respectively. The ratio of these fractional intensities
(f2/f1) defines an equilibrium
constant, where for myosin IIB·ADP,
f2/f1 =
[M·D2]/[M·D1], and for actomyosin
IIB·ADP, f2/f1 =
[AM·D2]/[AM·D1]. We measured
f2/f1 as a function of temperature,
and the resulting van't Hoff plots are depicted in
Fig. 4 for myosin
IIB·2'-dmD (open squares, solid line) and actomyosin
IIB·2'-dmD (closed triangles, dashed line). This reveals
H0 and
S0 values of 10.8
kcal/mol and 36.5 cal/degree/mol, respectively, for myosin IIB and 8.4
kcal/mol and 24.8 cal/degree/mol, respectively, for actomyosin IIB.
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These results allow us to make a comparison with smooth muscle myosin and
actomyosin. Whereas f2/f1 for smooth
muscle myosin II·ADP is 0.075 at 20 °C, the corresponding value for
myosin IIB·ADP is 0.58. Thus, a significantly greater fraction of
myosin IIB·ADP is in a weak nucleotide binding state. Binding of smooth
muscle myosin II·ADP to actin increases the value of
f2/f1 by a factor of 8
(26). In the case of myosin
IIB·ADP, just the opposite is seen: actin binding reduces
f2/f1 from 0.58 to 0.12. Thus, whereas
actin binding to smooth muscle myosin II·ADP drives the
AM·D1
AM·D2 equilibrium to the right,
enhancing ADP dissociation, it drives this equilibrium to the left for myosin
IIB and, in doing so, would be expected to increase ADP affinity.
Kinetics of Myosin IIB·ADP Binding to ActinThe fluorescence lifetime data indicate that myosin IIB·ADP and actomyosin IIB·ADP are distributed between two states that differ in their affinity for ADP. However, the data do not provide information on the actin binding affinities of these states. We therefore examined the effect of myosin IIB·ADP on the fluorescence of pyrene-labeled actin. We first mixed a complex of nucleotide-free myosin IIB·actin with 2 mM ADP; and, as in the case of smooth muscle myosin II, we saw no change in the pyrene fluorescence emission (data not shown). This means that both AM·D1 and AM·D2 are strong actin binding states.
What are the actin affinities of the M·D1 and M·D2 states? To address this question, we mixed myosin IIB and 2 mM ADP with a 57-fold molar excess of pyrene-labeled actin and 2 mM ADP in the stopped-flow spectrometer. Formation of a strongly bound myosin IIB·ADP·actin complex will quench the fluorescence of a pyrenyl probe on actin, as noted above. The resulting fluorescence transient is shown in Fig. 5 (inset) and consists of fast and slow phases, with relative amplitudes of 0.350.4/0.650.60. The rates of both phases depend on actin concentration, but in different ways. The fast phase (closed squares) depends linearly on actin concentration (dashed line), and this dependence defines an apparent second-order rate constant of 1.40 ± 0.04 µM1 s1. This second-order rate constant also describes the kinetics produced by mixing nucleotide-free myosin IIB with pyrene-labeled actin (open triangles), and it is very similar to the second-order rate constant for binding of rigor smooth muscle myosin II to actin (1.24 µM1 s1) (30). Thus, the rapid phase is due to pyrene quenching by one or more strong myosin IIB·ADP binding states.
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By contrast, the slow phase demonstrates a hyperbolic dependence on actin
concentration (open squares, solid line), defining a maximum rate of
6.4 ± 0.6 s1 and an apparent dissociation
constant of 5.9 ± 1.5 µM. These kinetics are to be
expected for a two-step binding reaction, with formation of an initial weak
actin binding complex followed by isomerization to a strong binding complex
that quenches the pyrene fluorescence: A* +
M·D A*M·D
AM·D. Thus, in the absence of actin, myosin
IIB·ADP is a mixture of strong and weak actin binding and strong and
weak ADP binding states. However, actin binding shifts this distribution to
produce a mixture of strong and weak ADP binding states that uniformly bind
strongly to actin. A similar situation was found to apply to smooth muscle
myosin II (26), which could be
explained by Scheme 3, where
Ka, Kb, and
Kc are affinity constants for the various myosin
IIB·ADP states for actin. This scheme allows us to integrate the
fluorescence lifetime and pyrene kinetic studies. Because ADP does not quench
the fluorescence of pyrene-labeled actomyosin IIB, we conclude that the
AM·D1 state is not populated
(e.g. K5' » 1); and therefore,
f2/f1 =
[AM·D2]/[AM·D1] =
K6'. Furthermore, the presence of two phases in the
pyrene fluorescence transient (Fig.
5) implies that, like smooth muscle myosin, an appreciable
population of myosin IIB·ADP is in the
M·D1 state. Consequently,
K5' » K5, which in turn
requires that Ka «
Kb. This would be expected because, by
definition, the actin affinity of the
M·D1 state should be much less
than that of the M·D1 state.
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Kinetics of ADP Binding to and Release from Actomyosin
IIBScheme 3 predicts that ADP dissociation from actomyosin should occur in two phases,
reflecting the AM·D1 AM·D2
AM transitions. This prediction is confirmed in
Fig. 6A. Mixing myosin
IIB·2'-dmD with actin and 4 mM ADP produced a biphasic
fluorescence decrease. The rate of the faster phase shows a hyperbolic
dependence on actin concentration, with an extrapolated value of 8.2 ±
1.9 s1 and an apparent dissociation constant of
10.2 ± 4.9 µM, whereas that of the slower phase shows
little actin concentration dependence (0.64 ± 0.06
s1). The rates of these two phases depend on the
values of k6',
k6', and
k7'. To measure k7', we
examined the kinetics of the fluorescence enhancement produced by mixing
nucleotide-free actomyosin IIB with 2'-dmD. A plot of rate
versus 2'-dmD concentration should be linear, and
Fig. 6B confirms this.
The slope defines an apparent second-order rate constant of 4.90 ± 0.05
µM1 s1; the
y intercept defines a k7' value of 7.5
± 1.4 s1; and the ratio defines a
K7 value of 1.56 µM
(Table I).
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ADP Affinity for Actomyosin IIBWhen interpreted in the
context of Scheme 3, our
results indicate that K6' for actomyosin IIB is
11-fold lower than for smooth muscle actomyosin II
(26). This in turn implies
that the ADP affinity for actomyosin IIB should be correspondingly higher. We
measured the affinity of ADP for actomyosin IIB by incubating a complex of 300
nM nucleotide-free myosin IIB and 300 nM pyrene-labeled
actin with varying concentrations of ADP and mixing in the stopped-flow
spectrometer with 4 mM ATP. The resulting fluorescence decrease
consisted of two phases: a rapid phase (rate of 250350
s1) due to ATP binding to nucleotide-free
actomyosin IIB and a slow phase (rate of 0.71 ± 0.08
s1) due to rate-limiting ADP release followed by
ATP binding and dissociation (Fig.
7A). The total amplitude remained constant over the range
of ADP concentrations tested, implying little ADP-induced dissociation (data
not shown). However, the relative amplitudes of these two phases did vary with
ADP concentration in a reciprocal manner and are plotted in
Fig. 7B.
Scheme 3 predicts that the
fractional amplitude of the fast phase (
) obeys the following
relationship (Equation 1),
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Myosin IIB Duty RatioThe duty ratios of smooth and skeletal
muscle myosin II are in the range of 34% because phosphate release is
at least 10-fold slower than ADP release
(26,
27). However, the slow phase
of the 2'-dmD release transient (Fig.
6A), which is a measure of the rate of the
AM·D1 AM·D2 transition, is
no faster than phosphate release (Fig.
3). We would therefore expect that the duty ratio of myosin IIB
should be appreciably higher than that of muscle myosin II isoforms. We
directly measured the duty ratio as a function of actin concentration by
measuring the fluorescence of a complex of myosin IIB and a 5-fold molar
excess of pyrene-labeled actin in the presence and absence of 4 mM
ATP. We compared these results with those obtained with an equimolar
concentration of pyrene-labeled actin without myosin to calculate the fraction
of myosin strongly bound. Fig.
8 depicts the data, which fit a hyperbola and extrapolate to a
maximum duty ratio of 0.82 ± 0.16.
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DISCUSSION |
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Isomerization of Myosin IIB·ADP StatesAs with smooth muscle myosin II, myosin IIB·ADP is a mixture of several states, both in the presence and absence of actin. Measurements of the fluorescence lifetimes of bound 2'-dmD, of the kinetics of myosin IIB·ADP binding to pyrene-labeled actin, of the kinetics of 2'-dmD binding to and release from actomyosin IIB, of the ADP affinity for actomyosin IIB, and of the myosin IIB duty ratio are all consistent with a general scheme first described in our study of smooth muscle actomyosin (26) and presented in this study as Scheme 3.
Interpretation of the data for actomyosin IIB within the context of this scheme is fairly straightforward because the AM·D1 state is not populated at equilibrium. This allows us to utilize the fluorescence lifetime data to directly calculate a value of K6'. The validity of our model is further supported by our fitting data from Fig. 7B to Equation 1, which provides an independent measure of K6' and one that is remarkably close to that generated from the lifetime data. This analysis allows us to make direct comparisons with smooth muscle actomyosin II. In doing so, it becomes apparent that a major difference between these two contractile systems is in the value of K6', which is 11-fold higher for smooth muscle actomyosin. This difference would favor formation of the AM·D1 state for actomyosin IIB, and it would consequently slow ADP release and enhance relative ADP affinity compared with smooth muscle actomyosin. In fact, the affinity of ADP for actomyosin IIB is higher than for any other myosin isoform studied (Table I). This finding also explains why our measurements of the steady-state ATPase of actomyosin IIB demonstrated no evidence of actin activation (data not shown) because sufficient ADP would be generated within a few turnovers to effectively inhibit the enzyme.
Determining the corresponding equilibrium constants for myosin
IIB·ADP is less straightforward because our data indicate that the
M·D1,
M·D1, and M·D2 states are
all populated at equilibrium. An assumption underlying the formulation of
Scheme 3 is that actin affinity
and nucleotide affinity are partially uncoupled in the presence of ADP, which
means that Kb
Kc. Furthermore, for myosin
IIB·2'-dmD at 20 °C,
f2/f1 =
[M·D2]/([M·D1]
+ [M·D1]) = 0.58. Combining these two constraints
gives us K5 and K6 estimates of 1.5
and 0.5, respectively, consistent as well with the requirement that
K5' » K5.
What is the pathway that actomyosin IIB utilizes in its mechanochemical
cycle? Given that K5' is very high and the
AM·D1 state is not populated in
the steady state, it is reasonable to assume that k5 is
also high. Therefore, the main pathway following rebinding of myosin
IIB·ADP·Pi to actin would be
AM·D·Pi
AM·D1
AM·D1
AM·D2. This in turn
implies that myosin IIB bypasses the
M·D1 state during its normal
mechanochemical cycle. This conclusion is consistent with a recent report that
the lever arm in smooth muscle myosin II·ADP (which we have shown is
>95% in the M·D1 state)
(26) assumes a rigor-like
orientation in the absence of actin
(11). These results predict
that binding of M·D1 to actin
would rotate the lever arm from a rigor position to an "ADP"-like
position, which would oppose the normal physiologic operation of this
motor.
The kinetics of 2'-dmD release from myosin IIB followed a single
exponential process at 20 °C, which could be explained if
K5 and K6 were rapid equilibria
relative to k7. For 2'-dmD release from actomyosin
IIB, two phases were seen. Solution of the rate equations is as described in
our prior study of smooth muscle actomyosin II
(26):
,
where S = k6' +
k6' +
k7' and C =
k6'·k7'. This
solution must be consistent with the value of K6'
derived from lifetime measurements and the value of
k7' derived from the kinetics of 2'-dmD
binding to actomyosin IIB. A reasonable fit that satisfies these requirements
is obtained with k6' = 0.8
s1,
k6' =
4s1, and k7' = 7.0
s1. The corresponding values for smooth muscle actomyosin
are k6' = 30 s1,
k6' = 15
s1, and k7' = 45
s1 (Table
I). Whereas each of these rate constants is considerably lower for
myosin IIB, the greatest decrease is seen in the value of
k6', which assumes a value that is slower than the
rate of phosphate release by other myosin isoforms
(Table I).
Duty Ratio and Rate-limiting StepIf
k6' is the rate-limiting step in the mechanochemical
cycle of actomyosin IIB, then the duty ratio should be appreciably higher than
in other myosin II isoforms. We confirmed this
(Fig. 8) and found that the
duty ratio was in the same range as that seen in some unconventional myosins
such as myosin V. The value of the duty ratio varied with actin concentration,
and its maximum value is related to two kinetic steps, the rate of formation
of a strong binding state and the effective rate of ADP release: duty ratio =
(rate of formation of AM·D1)/(rate of formation of
AM·D1 + rate of loss of
AM·D1) =
k4'/(k4' +
k6'). Inserting the values of
k6'and the duty ratio yields a calculated
k4' value of 3.7 s1,
which confirms that for myosin IIB, the rate-limiting step is not phosphate
release, but rather the AM·D1
AM·D2 transition.
Comparison with Other MyosinsThe enzymology of smooth
muscle myosin II has served as a standard for conventional myosin II behavior.
As summarized in Table I, this
behavior includes rapid induction of the weak binding conformation by ATP,
hydrolysis at 25 s1, rate-limiting phosphate
release, actin-activated ADP release, and a low duty ratio. These are the
characteristics expected of a motor that operates in a large ensemble and that
is designed to produce shortening. By contrast, the physiologic demands on an
unconventional myosin that acts in isolation, such as myosin V, predicts that
it would need a high duty ratio and a mechanism to allosterically regulate the
timing of forward stepping so that the two heads of such a motor would work in
a coordinated fashion. Myosin V appears to have addressed this requirement by
markedly shortening the lifetime of weakly bound states
(Table I). Thus, ATP hydrolysis
and phosphate release are accelerated up to several hundredfold, whereas ADP
release kinetics remain similar to those for smooth muscle myosin II. The net
effect is that ADP release is rate-limiting and the duty cycle is
2025-fold larger.
A variety of inhibition studies have led to the suggestion that myosin IIB generates sustained cortical tension and works in small ensembles of <30 motors per filament. These physiologic demands would be expected to have specific effects on the enzymology of this motor. We would predict that myosin IIB is a relatively slow enzyme because rapid shortening should not be required of a motor designed to produce sustained tension. Furthermore, we would expect a high duty ratio and a strain-dependent release mechanism, which would ensure that heads would remain strongly attached for a relatively long duration and would not release until other heads of the same filament were strongly attached.
The data presented in this study support these predictions. Like its unconventional relative myosin V, myosin IIB has arranged its enzymology to ensure that it has a high duty ratio. As noted above, the value of the duty ratio is determined by the relative rates of phosphate and ADP release. The high duty ratio of myosin V results from its rapid rate of phosphate release and relatively slow (compared with smooth or skeletal muscle myosin II) rate of ADP release (Table I). By contrast, myosin IIB has engineered a high duty ratio in a different manner. Its rate of phosphate release is nearly identical to that for smooth muscle myosin II. However, ADP release is markedly slow because of a rate-limiting isomerization of ADP states (k6'). This produces a slowly cycling motor that resembles myosin V in some aspects and smooth muscle myosin II in others. Furthermore, myosin IIB has accomplished this largely by altering one step: the isomerization of actomyosin IIB·ADP states.
Finally, reducing the value of k6' has the effect of markedly enhancing ADP affinity. Because intracellular ADP concentrations are 1250 µM (28), the myosin IIB motor would be permanently stalled unless an additional mechanism existed to accelerate ADP release. Such a mechanism could involve mechanical strain, which has been used to explain the processive movement of myosin V (23). In myosin V, strain produced by attachment of the lead head to actin has been proposed to accelerate ADP release from the trailing head. We argue that a similar situation applies as well to myosin IIB, although in this case, the strain applied to one head may originate from other heads of other myosin IIB molecules that occupy the same thick filament. Furthermore, although strain may accelerate ADP release from native myosin V, single-headed constructs are still capable of releasing nucleotide (21). Hence, strain modulates the cycle, but is not essential for myosin V to be enzymatically active. By contrast, the very high ADP affinity of actomyosin IIB means that a strain-dependent release mechanism is an absolute requirement for myosin IIB to function as an enzyme and motor.
ConclusionThis study has shown that myosin IIB, a member of the conventional myosin II family, has a number of features that it shares with unconventional myosins such as myosins V and VI. These include a high ADP affinity, a high duty ratio, rate-limiting ADP release, and the need for a strain-dependent mechanism to ensure normal activity. Each of these features would help support the putative role of this motor as a generator of cortical tension and, taken together, demonstrate how the physiologic demands placed on this motor make it unconventionally conventional.
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FOOTNOTES |
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To whom correspondence should be addressed: Dept. of Neurology, University of
Alabama at Birmingham, Birmingham, AL 35294-3293. Tel.: 205-934-1813; Fax:
205-975-7546; E-mail:
stevensr{at}uab.edu.
1 The abbreviations used are: MDCC,
N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin 3-carboxamide;
mant-, N-methylanthraniloyl-; 2'-dmD,
2'-deoxy-N-methylanthraniloyl-ADP.
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REFERENCES |
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