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J. Biol. Chem., Vol. 275, Issue 28, 21624-21630, July 14, 2000
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From the
Received for publication, January 18, 2000
MI1IQ is a complex of
calmodulin and an epitope-tagged 85-kDa fragment representing the
amino-terminal catalytic motor domain and the first of 6 calmodulin-binding IQ domains of the mammalian myosin I gene, rat
myr-1 (130-kDa myosin I or MI130). We have
determined the transient kinetic parameters that dictate the ATP
hydrolysis cycle of mammalian myosin I by examining the properties of
MI1IQ. Transient kinetics reveal that the affinity of
MI1IQ for actin is 12 nM. The ATP-induced
dissociation of actin-MI1IQ is biphasic. The fast phase is
dependent upon [ATP], whereas the slow phase is not; both phases show
a Ca2+ sensitivity. The fast phase is eliminated by the
addition of ADP, 10 µM being required for half-saturation
of the effect in the presence of Ca2+ and 3 µM ADP in the absence of Ca2+. The slow phase
shares the same rate constant as ADP release (8 and 3 s Class I myosins in mammals are mechanochemical molecules with an
amino-terminal motor domain containing an ATP and actin-binding region,
a neck region with one or more so-called IQ domains to which calmodulin
binds, and a carboxyl-terminal tail region (1). One member of the class
I myosins, MYR-1, is ubiquitously expressed in mammalian cells.
myr-1 contains up to 6 IQ domains; alternate splice forms
containing 4, 5, or 6 IQ domains exist (2). The 130-kDa myosin I
isolated from rat liver is a myr-1 gene product (3, 4).
Although quantitation of calmodulin has indicated that this preparation
contains 6 mol of calmodulin/130-kDa myosin I heavy chain (4), isoforms
corresponding to the 5 IQ and 4 IQ variants are also expressed in liver
(2) and may be present.
The 130-kDa myosin I translocates actin filaments slowly and in a
Ca2+-sensitive manner (5). At 10 µM free
Ca2+ and above, motility is inhibited. This decrease in
motility can be reversed by the addition of exogenous calmodulin,
indicating that a calcium-induced dissociation of calmodulin might be
responsible for the decrease in motility. Laser trap analyses have
indicated that the 130-kDa myosin I translocates actin in a two-step
process (6). The authors proposed that the two mechanical steps are coupled to Pi and ADP release, respectively. Transient
kinetic analyses have indicated that the mechanical step coupled to ADP release is unlikely to contribute to force generation or to motility, but could be a system for providing a strain-sensitive ADP release mechanism. This, together with the slow ATP-induced dissociation of
actin-myosin I, suggests that this myosin is best suited for maintenance of tension (7).
We have coexpressed in baculovirus calmodulin and a fragment
representing the first 728 amino acids of MYR-1, which codes for the amino-terminal motor domain and 1 IQ domain; we refer to this
complex as MI1IQ 1 (8).
MI1IQ translocates actin filaments in vitro.
Unlike the parent molecule, the rate of actin translocation is not
affected by the Ca2+ concentration over the range of
pCa 4-7 and the rates of movement of MI130 and
MI1IQ are comparable.
The availability of a homogeneous preparation from the baculovirus
expression system has allowed us to explore in detail the kinetics of
this construct representing the motor domain and the first IQ domain.
Our results indicate that the truncated myosin I possesses kinetic
properties indistinguishable from the parent molecule. Thus, the 5 deleted IQ domains and their associated calmodulins play no role in
defining the unloaded properties of the myosin I head. Furthermore, our
results indicate that, when bound to actin, myosin I exists in two
conformations in equilibrium and that ATP can bind to only one of the
two conformations. The equilibrium between the two forms is sensitive
to both Ca2+ and ADP concentrations and we propose that the
conformational change between the two forms may correlate with (i) the
double step observed in laser trap studies with this molecule (6) and
(ii) the ADP-dependent conformational change identified by electron microscopy for a closely related myosin I, brush border myosin
I (9, 10).
Proteins--
MI1IQ was expressed in baculovirus and
purified from insect lysates as described in the accompanying article
(8). MI130 was prepared from rat liver as described
previously (3). Rabbit skeletal muscle actin was prepared according to
(11) and, in some cases, labeled with pyrene at Cys-374 according to
Ref. 12.
Transient Enzyme Kinetics--
All kinetic experiments were
performed at 19.8 °C in 20 mM MOPS, 100 mM
KCl, 5 mM MgCl2, 1 mM
dithiothreitol, and 1 mM EGTA or 1 mM EGTA and
1.1 mM CaCl2 at pH 7.0 except for the
Ca2+ dependence of the ADP release rate, which used 2 mM EGTA and 2 mM CaEGTA mixed in the
appropriate proportions to give the required pCa (13, 14).
All measurements were performed with a Hi-Tech Scientific SF-61 single
mixing stopped-flow system using a 100-watt xenon/mercury lamp and a
monochromator for wavelength selection. Pyrene fluorescence was excited
at 365 nm and emission detected after passing through a KV 389-nm
cut-off. Tryptophan fluorescence was excited at 297 nm and observed
through a WG 320 filter. The stated concentrations of reactants are
those after mixing in the stopped-flow observation cell. Stopped-flow
data were fitted to exponentials by a non-linear least-squares curve
fit using software provided by Hi-Tech.
The analysis of the titration of MI binding to actin was performed as
described by Kurzawa and Geeves (15), where the amplitude of the
observed transient is assumed to be proportional to the concentration
of actin-myosin complex present before addition of ATP. The data were
fitted to the physically significant root of the following quadratic
equation.
Data Interpretation--
As shown in Scheme 1, part A, we
interpret the kinetics of the interaction of myosin I or its expressed
truncated form with nucleotide (T, ATP; D, ADP) in terms of the model
described by Bagshaw et al. (16) for conventional myosin (M)
where k+i, k
ATP binds rapidly to myosin in a two-step reaction before ATP is
reversibly hydrolyzed on the protein. This results in a conformational change, which limits phosphate release and the faster ADP release. The
ATP-induced dissociation of actin-MI130 or
actin-MI1IQ and the inhibition of the reaction by ADP have
also been interpreted in terms of the models developed for conventional
myosin by Millar and Geeves (17) and Siemankowski and White (18). As
shown in Scheme 1, part B, ATP binds rapidly and reversibly to
actin-myosin and is followed by a rate-limiting isomerization
(k+2) of the complex, which leads to rapid
dissociation of actin. ADP competes with ATP for the nucleotide binding
site. The dissociation constants of actin for myosin, actin for
myosin-ADP, and ADP for actin-myosin are
KA, KDA, and
KAD, respectively.
The transient kinetics of the interaction of MI1IQ
with actin and ATP were examined and compared with our previous
measurements on the native protein, MI130 (7). In all cases
the results were very similar. Fig.
1A shows the fluorescent
transient observed upon addition of 100 µM ATP to a 25 nM complex of MI1IQ and pyrene-labeled actin in
the presence of Ca2+. The transient was well described by a
single exponential with a kobs of 2.4 s Repeating the measurement in the absence of Ca2+ gave
similar results, except that the slow component of the transient
comprised 50% of the total amplitude with kobs
of 3.4 s We had previously observed for MI130 that the fast phase of
the transient was eliminated by incubating the proteins with 20 µM ADP before initiating the reaction by mixing with ATP.
A similar result was observed here for the expressed myosin I fragment
(Fig. 2A). In this case we
were able to titrate the fast phase of the transient, and a plot of the
amplitude against [ADP] in the presence and absence of
Ca2+ is shown in Fig. 2B. A fit of the amplitude
to a hyperbola gave a best fit of 10 and 3 µM,
respectively, in the presence and absence of Ca2+, and this
is assigned to the affinity of ADP for the A.MI1IQ complex
(KAD). The kobs of
the slow phase remained almost constant over the range of [ADP] used
(8.5 s
Kinetic Analyses of a Truncated Mammalian Myosin I Suggest a
Novel Isomerization Event Preceding Nucleotide Binding*
§,
, and
Department of Biosciences, University of
Kent, Canterbury, Kent CT2 7NJ, United Kingdom and the
¶ Boston Biomedical Research Institute,
Watertown, Massachusetts 02472
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ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 in the presence and absence of
Ca2+, respectively), but cannot be eliminated by decreasing
[ADP]. We interpret these results to suggest that actin-myosin I
exists in two forms in equilibrium, one of which is unable to bind
nucleotide. These results also indicate that the absence of the
COOH-terminal 5 calmodulin binding domains of myr-1 do not
influence the kinetic properties of MI130 and that the
Ca2+ sensitivity of the kinetics are in all likelihood due
to Ca2+ binding to the first IQ domain.
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INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(Eq. 1)
is the fraction of actin with myosin bound, [M] is the
total concentration of myosin added, [A]0 is the
concentration of actin and Kd is the dissociation
constant of myosin for actin.
i are the forward and reverse rate
constants, respectively, and Ki (=
k+i/k
i) represents the equilibrium constant of the ith reaction.
Normal characters are used to indicate reactions involving myosin only, and bold characters refer to reactions in which actin is also present.
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RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 and corresponds to the dissociation of
actin from the complex. At low ATP concentrations (25-200
µM), the reaction was monophasic and
kobs was linearly dependent upon [ATP] with an
apparent second order rate constant,
K1k+2, of 5.3 × 104 M
1
s
1. Above 200 µM ATP, the
reaction became biphasic and could be described by a two exponential
term (Fig. 1B). The slow phase was independent of [ATP]
with a kobs of 8 s
1
and an amplitude of 25-30% of the total transient. The fast phase showed a hyperbolic dependence upon [ATP], and the best fit gave a
maximum observed rate constant of 79 s
1 with
1.5 mM ATP required for half saturation of
kobs (see Fig. 1C). By analogy with
other actomyosin systems, the maximal rate and the ATP concentration at
half the maximal rate were assigned to
k+2, and
1/K1, respectively, the rate
constant for an isomerization of the actin-myosin complex, which limits
the dissociation of actin and the affinity of ATP for the actin-myosin
complex (Table I).

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Fig. 1.
ATP-induced dissociation of pyrene-labeled
actin-MI130. A and B, 25 nM MI130 was incubated with 25 nM
phalloidin-stabilized pyrene-labeled actin and pyrene fluorescence was
measured immediately following addition of ATP in the presence of
Ca2+. A, dissociation of actin-MI130
by 100 µM ATP resulted in an increase in fluorescence and
the best fit to a single exponential is superimposed with
kobs = 2.4 s
1 and an
amplitude of 28.4%. B, increasing ATP to 2.5 mM
gave a biphasic reaction with kobs = 42.3 and
6.4 s
1 with amplitudes of 21.8 and 8.3%,
respectively. C, the dependence of
kobs on [ATP] and Ca2+. The
measurements were repeated in either 1 mM EGTA
(
Ca2+; circles) or 0.1 mM free
Ca2+ (+Ca2+; squares), and
kobs was plotted as a function of [ATP]. The
best fits to kobs = K1k+2[ATP]/(1 + K1[ATP]) for the fast phase
(closed symbols) are superimposed. The data were
well described by hyperbolas with maximal observed rates
(k+2) of 79 s
1
and 36 s
1 and 1.5 mM and 1.2 mM ATP required for half-maximal saturation
(1/K1) with and without
Ca2+, respectively. The values for
kobs for the slow phase (open
symbols) are not fitted.
Transient kinetic analysis of native rat MI130 and expressed
fragment MI1IQ
1 and the best fit to the [ATP]
dependence of the kobs of the fast component
gave k+2 = 36 s
1
and 1/K1 = 1.2 mM for an
apparent second order rate constant (
Ca2+) of 3 × 104 M
1
s
1 (Fig. 1C).
1 in Ca2+; 2.45 s
1,
Ca2+) and was assigned to
k
AD, the rate of ADP dissociation from
A.MI1IQ.ADP. Since KAD = k
AD/k+AD, it is possible to calculate k+AD, the
apparent second order rate constant of ADP binding to
A·MI1IQ. This gave values of 0.8 × 106
M
1 s
1
(+Ca2+) and 1.1 × 106
M
1 s
1
(
Ca2+), i.e. the apparent second order rate
constant of ADP binding is Ca2+ independent and 15-40 fold
faster than the apparent second order rate constant of ATP binding to
A·MI1IQ.

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Fig. 2.
Influence of ADP on the ATP-induced
dissociation of actin-MI130. A, addition of
2.5 mM ATP to 25 nM
pyrene-actin-MI130 in the absence of ADP resulted in a
rapid increase in fluorescence best described by a double exponential
(kobs = 43.8 and 8.5 s
1 and amplitudes of 18.8 and 9.4%,
respectively). In the presence of 20 µM ADP, the change
in fluorescence can be described by a single exponential with a
kobs = 10.3 s
1 and an
amplitude of 19.8%; however, the data can be equally described by a
double exponential with kobs = 37.7 and 8.5 s
1 and amplitudes of 3.8% and 17.3%,
respectively. B, titration of the amplitude of the fast
phase against [ADP] added to the protein before mixing with ATP in
the presence and absence of Ca2+. The data were fitted to a
hyperbola in each case, and the apparent affinity
(KAD) for ADP is 10 µM in
the presence of Ca2+ and 3 µM in its
absence.
To verify that the rate of ADP binding is faster than ATP binding, we
examined the rates of competitive binding of ADP and ATP to
A·MI1IQ. A·MI1IQ at 30 nM was
mixed in the stopped flow apparatus with 1 mM ATP and 0-40
µM ADP. The amplitude of the fast phase decreased,
whereas kobs increased with increasing [ADP]
(data not shown). This demonstrates that ADP effectively competes with
ATP even at less than one-tenth of the concentration. If the rate of
ADP dissociation from the A·M·D complex is much less than the rate
at which ADP and ATP bind to A·M (i.e.
[A·M·D]k
AD
[ATP]K1k+2 + [ADP]k+AD), then
kobs = [ATP]K1k+2 + [ADP]k+AD. At a fixed ATP concentration, kobs increased from 23 to 42 s
1 at 20 µM ADP and further
increased to 73 s
1 at 40 µM.
The data over this limited range are therefore compatible with a value
of k+AD of 0.95-1.25 × 106 M
1
s
1 in agreement with the estimate above.
The slow phase of the ATP-induced dissociation of A·MI1IQ
has a kobs that is very similar to
k
AD, the rate constant of ADP
dissociation from the A·MI1IQ·D complex even in the
absence of added ADP. It is possible, therefore, that the slow phase
represents a fraction of A·MI1IQ that is isolated with
ADP bound. Extensive treatment of the protein with apyrase did not
eliminate the slow phase of the reaction. In contrast, if the protein
was treated with 20 µM ADP such that the fast phase was
eliminated, then treatment with apyrase restored the fast phase but
only to the same extent as in the original measurement. Thus, apyrase
treatment does eliminate the protein bound ADP effectively. We
therefore conclude that the slow phase is not caused by the presence of
ADP bound to the protein.
Another possibility is that ATP could be the source of contaminating ADP. ATP normally contains about 1% ADP. If ADP binds to the protein faster than ATP (as shown above), then the contaminant ADP could bind a fraction of the protein to produce the slow phase. The true substrate for myosin is MgATP, and the product is MgADP. Since ATP binds Mg2+ more tightly than ADP, reducing the free Mg2+ concentration to a minimum should reduce the contaminant MgADP concentration; however, under limiting Mg2+ concentrations, the slow phase remained constant (data not shown). Furthermore, addition of up to 5% ADP into the ATP had no effect on the amplitude of the slow phase (see above). These results indicate that the slow phase is not due to ADP contamination in the ATP.
The effect of Ca2+ on the rate of ADP release from
MI130 was measured by determining in buffers containing
fixed amounts of Ca2+, the rate of the ATP-induced
dissociation of pyrene-labeled actin-MI in the presence of saturating
amounts of ADP (Fig. 3). The
kobs was plotted as a function of
[Ca2+], and the line represents the best fit to the Hill
equation with midpoint of 6.8 ± 0.6 µM
Ca2+ and kobs in the presence and
absence of Ca2+ of 8.0 and 1.5 s
1, respectively. The slope of the graph that
defines the Hill coefficient was poorly defined as 3 ± 1.5. These
data indicate that binding of Ca2+ to the myosin I complex
is positively cooperative.
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The binding of ATP to MI1IQ was followed using intrinsic
protein fluorescence and gave results that were indistinguishable from those of MI130 (data not shown), indicating a second order
rate constant for ATP binding of 0.1 × 106
M
1 s
1
that was independent of Ca2+. There was no evidence of a
slow component of the reaction; however, the signal to noise ratio was
very poor in these measurements, and the possibility of a slower
component cannot be eliminated.
The affinity of MI1IQ for actin was measured by monitoring
the amplitude of the ATP-induced dissociation of pyrene-labeled
A·MI1IQ as a function of [MI1IQ] (15).
Using 30 nM pA and 400 µM ATP, the amplitude
of the reaction was measured for MI1IQ from 10 to 150 nM (Fig. 4). Analysis of the
data gave a value of KA of 12 nM, in good agreement with the value obtained for the
native protein by a less direct method (7).
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DISCUSSION |
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Our results demonstrate that MI1IQ is essentially indistinguishable from MI130 in terms of its interaction with actin and nucleotide, indicating that the presence of the 5 additional IQ domains and associated calmodulins in the parent molecule has no effect on these properties (Table I). The truncated myosin I shows the same actin-activated Mg2+-ATPase activity as the parent molecule (8) and the ATP, ADP, and actin binding to A·MI1IQ are identical to the parent. Since the actin and nucleotide binding to MI1IQ are unchanged, the coupling between actin and nucleotide binding is also expected to remain unaffected by the missing IQ domains and their associated calmodulins. In addition, the Ca2+ sensitivity of all of the above properties is identical to that of the parent molecule, MI130. These results demonstrate that the Ca2+ sensitivity of these properties is not a function of the missing calmodulins and must result from Ca2+ binding to the first calmodulin or from a novel independent Ca2+ binding site in the motor domain.
All of the events in the acto-MI ATPase cycle that we have measured are significantly faster than the turnover rate and are therefore not rate-limiting in the ATPase reaction. It is therefore likely that Pi release is rate-limiting, as has been observed for other myosins, and in this case the Pi release must be regulated by Ca2+, as previously observed for scallop muscle myosin II (19). A biphasic fluorescent transient was observed upon introduction of ATP to complexes of pyrene-labeled actin with both MI130 and MI1IQ. The amplitudes and rates of the two phases were similar for the two proteins, and in both cases the rate constant of the slow phase was very similar to the rate constant limiting ADP release from A·MI. To address the possibility that the slow component was due to ADP present either in association with the expressed myosin I or as a trace contaminant in the ATP, several experiments designed to reduce ADP levels were performed. In all cases, the slow phase remained unaltered.
For the native protein we considered the alternative possibility that the slow phase in the ATP dissociation reaction was due to the presence of a fraction of damaged myosin I, e.g. missing one or more of the calmodulins, or that a contaminant myosin I could be present and therefore responsible for the biphasic nature of the ADP-induced dissociation. It seems unlikely, however, that the same contaminant would be present in both myosin I preparations (i.e. the native protein isolated from liver and the expressed fragment from insect cells) since they derive from different cell types and involve different purification schemes. We are therefore forced to conclude that this represents an intrinsic property of both the expressed protein and the parent MI130.
The simplest explanation of the biphasic nature of the ATP-induced
dissociation is that the protein exists in two forms: one to which ATP
can bind readily (at an apparent second order rate constant of 5.3 × 104 M
1
s
1 in the presence of Ca2+) and
the other, which cannot bind ATP without first isomerizing. One
possible model is shown in Fig. 5, where
the nucleotide pocket must open before nucleotide can bind. (In this
model, we have assumed a direct link between the opening of the
nucleotide pocket and an ADP-induced structural change, i.e.
"wagging" of the myosin neck; see below.) The rate constant for the
opening of the pocket is very similar to the net rate constant of ADP
dissociation suggesting a similar process limits ADP release
(k+
= k+
D = 8 s
1,
Fig. 5). From the data presented in Figs. 1 and 2, we can define the
rate and equilibrium constants of each of the transitions shown in Fig.
5. The assignment of the constants is described in the legend to Fig.
5, and the values are listed in Table
II.
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The evidence points toward a significant proportion (~30%) of the
conformation with a closed nucleotide pocket being present in the
absence of nucleotide. By increasing the rate constant of pocket
closing, ADP causes almost all of the myosin I to be in the closed
pocket form. In contrast, the presence of Ca2+ increases
the proportion of the open pocket form by increasing the rate of the
opening of the pocket in both the presence and absence of ADP. In terms
of nucleotide binding, Ca2+ lowers the affinity of A·MI
for ADP by increasing the net rate constant of ADP release
(k+
D). Ca2+ also increases
the rate of ATP-induced dissociation of actin (k+2, Scheme I and Table I) from A·MI,
but does not affect the affinity of ATP for A·MI
(K1). Thus, Ca2+ can stimulate
the detachment of actin from A·MI·D by increasing the rate of ADP
release and the rate of the subsequent dissociation by ATP. Note,
however, that the effects of Ca2+ are small (in all cases,
values do not differ by more than a factor of 3) and therefore probably
do not represent an on/off switch but rather a modulator of activity.
At physiological nucleotide concentrations of 1 mM ATP and
10-50 µM ADP, the net rates of nucleotide binding to
A·MI will be 18 s
1 for ATP and 10-50
s
1 for ADP in the absence of
Ca2+. Thus, the A·MI that has released ADP is as likely
to rebind ADP as to bind ATP. The low efficiency for binding ATP and
detaching from actin supports our proposal that myosin I is designed
for tension maintenance not motility (7). The presence of
Ca2+ increases the net rate of ATP binding
(K1k+2) but does
not alter the rate of ADP rebinding
k
D/KADP.
The possibility that myosin I exists in two conformational states, as
shown by the kinetic evidence presented here, is of great interest
since an isomerization of A·MI·D can be responsible for the second
displacement seen in laser trap assays for MI130, brush
border myosin I (6), a close relative to MI130, and smooth
muscle myosin S1 (smS1) (20). It is also required for the structural
changes or "tail wagging" seen in three-dimensional reconstructions
from electron micrographs for some actomyosins including brush border
myosin I and smS1 (9, 21). If our assignment is correct, then the
identified isomerization provides a direct link between the
accessibility of nucleotide to its binding pocket and the mechanical
transient and lends strong support for the role of nucleotide release
in providing a strain-sensing mechanism (22). We previously argued that
a MI cross-bridge bearing the typical isometric tension could have ADP
release reduced up to 100-fold. In the current model, the strain would
act directly on the isomerization shown in Fig. 5. Any load or strain
on the cross-bridge would inhibit the swing of the tail against the
load. The effect would be to reduce k+
D
and hence K
.
The Ca2+ sensitivity of the A·MI isomerization also establishes a link between the binding of Ca2+ (presumably to the remaining calmodulin), and the biochemical, structural, and mechanical events discussed above. If the protein isomerization is a mechanical sensor that slows down the rates of both ADP release and ATP binding in the presence of strain, then the effect of Ca2+ may be far more dramatic for a head bearing strain. Ca2+ could act on the strained head by binding to calmodulin to alter the elasticity of the calmodulin-IQ complex and so reduce the strain leading to acceleration of the isomerization, ADP release, and cross-bridge detachment. The other 5 calmodulin-IQ domains could also contribute to the Ca2+-assisted release of strain if they are elastically distorted in a strained cross-bridge. All that is required is for the Ca2+-induced change in calmodulin conformation to alter the rest length of each calmodulin-IQ domain such that the strain on a A·MI·D head is modulated. It has previously been noted that Ca2+ binding reduces the affinity of calmodulin for the IQ domain but probably not enough to cause calmodulin dissociation at cellular concentrations of calmodulin.
A remaining question about the A·MI conformation is its relation to
conformational changes in MI alone and to similar structural changes in
other myosins. To date we have no evidence for two conformations of MI
in the absence of actin and the rate of ATP binding is reasonably fast
(0.1 × 106 M
1
s
1); however, the fluorescent signal changes
that monitor ATP binding are very small and the presence of a second
component cannot be eliminated.
smS1 shows a similar mechanical and structural change on binding ADP to
A·M, yet a close examination of earlier data (22) shows no evidence
of a second phase in the ATP-induced dissociation of A·smS1. There
are two possible explanations for the lack of a second component.
Either K
. is much larger (>10), such
that in the absence of ADP the closed conformation is not significantly
occupied, or the rate of pocket opening is very fast
(k+
> 200 s
1), such that the opening can only be
observed at very high ATP concentrations. The first possibility is
compatible with the smS1 data, where electron micrographs indicate an
ADP-induced swing of the myosin neck. The second possibility is
formally equivalent to a strain-dependent transition state
for nucleotide binding and release proposed for skeletal muscle myosin
(23). The proposed model may therefore reflect a property of myosins in
general. In this respect it is noteworthy that the neck of the related molecular motor, kinesin, has recently been proposed to adopt various
nucleotide-dependent conformations when attached to its microtubule track (24). The interpretation of our data and of the
recent kinesin data allows for a strain-sensitive ADP to ATP exchange
mechanism. In the kinesin mechanism, this could provide gating of the
interaction between the two heads of kinesin. In the case of myosin I,
it provides a mechanism for a single-headed molecule to maintain
tension with low ATP turnover. If this myosin I clusters on a membrane
or vesicle as has been proposed for Acanthamoeba myosin I
(25), then a similar gating mechanism could also apply.
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ACKNOWLEDGEMENT |
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We thank Dr. Christine Berger (University of Kent at Canterbury) for assistance with the transient kinetic measurements.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM56130 (to L. M. C.), a grant from the March of Dimes (to L. M. C.), and a Wellcome Trust program grant (to M. A. G.).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. E-mail: m.a.geeves@ ukc.ac.uk.
Present address: Gwathmey, Inc., Cambridge, MA 02138.
Published, JBC Papers in Press, April 25, 2000, DOI 10.1074/jbc.M000342200
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ABBREVIATIONS |
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The abbreviations used are: MI1IQ, epitope-tagged motor domain and first IQ domain of 130-kDa myosin I; MI130, 130-kDa myosin I; A, actin; M, myosin; MI, myosin I; MOPS, 4-morpholinepropanesulfonic acid; D, ADP; T, ATP; S1, subfragment 1; sm, smooth muscle myosin.
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REFERENCES |
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